U.S. patent application number 11/151744 was filed with the patent office on 2006-07-06 for allogeneic and xenogeneic transplantation.
This patent application is currently assigned to The General Hospital Corporation, a Massachusetts Corporation. Invention is credited to David H. Sachs.
Application Number | 20060147428 11/151744 |
Document ID | / |
Family ID | 34812502 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060147428 |
Kind Code |
A1 |
Sachs; David H. |
July 6, 2006 |
Allogeneic and xenogeneic transplantation
Abstract
The invention provides methods for restoring or inducing
imnmunocompetence, the methods including the step of introducing
donor thymic tissue into the recipient. The invention also provides
methods for inducing tolerance in a recipient including introducing
donor thymic tissue into the recipient. The invention further
provides methods of inducing tolerance including administering to
the recipient a short course of help reducing treatment or
administering a short course and methods of prolonging the
acceptance of a graft by administering a short course of an
immunosuppressant.
Inventors: |
Sachs; David H.; (Newton,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
The General Hospital Corporation, a
Massachusetts Corporation
|
Family ID: |
34812502 |
Appl. No.: |
11/151744 |
Filed: |
June 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09710971 |
Nov 9, 2000 |
6911220 |
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11151744 |
Jun 13, 2005 |
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09126704 |
Jul 30, 1998 |
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09710971 |
Nov 9, 2000 |
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08458720 |
Jun 1, 1995 |
5876708 |
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09126704 |
Jul 30, 1998 |
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08266427 |
Jun 27, 1994 |
5614187 |
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08458720 |
Jun 1, 1995 |
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08451210 |
May 26, 1995 |
6296846 |
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08458720 |
Jun 1, 1995 |
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07838595 |
Feb 19, 1992 |
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08451210 |
May 26, 1995 |
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08220371 |
Mar 29, 1994 |
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08458720 |
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PCT/US94/05527 |
May 16, 1994 |
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08458720 |
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08243653 |
May 16, 1994 |
5658564 |
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08458720 |
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08114072 |
Aug 30, 1993 |
5624823 |
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08458720 |
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08150739 |
Nov 10, 1993 |
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08458720 |
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08212228 |
Mar 14, 1994 |
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08458720 |
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PCT/US94/01616 |
Feb 14, 1994 |
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08458720 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 51/1282 20130101;
C07K 14/70539 20130101; C07K 16/2812 20130101; A61K 35/26 20130101;
A61K 48/00 20130101; A61K 2039/505 20130101; A61K 39/001 20130101;
A61K 2035/124 20130101; A61K 2039/5156 20130101; C12N 2510/00
20130101; A61K 35/16 20130101; A61K 2300/00 20130101; A61K 39/39541
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 45/06 20130101; A61K 38/13 20130101; A61K 35/28 20130101; A61K
35/16 20130101; A61K 35/407 20130101; A61K 35/26 20130101; C07K
16/28 20130101; A61K 39/39541 20130101; A61K 35/28 20130101; A61K
35/407 20130101; A61K 38/13 20130101; A61K 2121/00 20130101 |
Class at
Publication: |
424/093.7 |
International
Class: |
A61K 35/407 20060101
A61K035/407 |
Claims
1. A method of restoring or promoting the thymus-dependent ability
for T cell progenitors to develop into mature functional T cells in
a primate recipient which is capable of producing T cell
progenitors but which is thymus-function deficient, the method
comprising introducing into said primate recipient xenogeneic fetal
or neonatal thymus tissue from a donor animal of a different
species, so that recipient T cells can mature in said implanted
donor thymus tissue.
2. The method of claim 1, wherein the donor animal is a swine.
3. The method of claim 2, further comprising implanting swine fetal
liver tissue in said recipient.
4. The method of claim 2, wherein said primate recipient is a
human.
5. The method of claim 2, wherein said donor swine is a miniature
swine.
6. The method of claim 2, wherein said swine thymus tissue is
capable of supporting in the recipient primate clonal deletion or
anergy of thymocytes reactive with donor xenoantigens.
7. The method of claim 2, further comprising introducing swine
hematopoietic cells into said recipient and inactivating NK cells
of said recipient.
8. The method of claim 2, further comprising the step of
inactivating mature CD4.sup.+ T cells in the recipient.
9. A method of inducing tolerance in a recipient primate of a first
species to a graft obtained from a donor animal of a second
discordant species, said method comprising prior or simultaneous
with transplantation of said graft, introducing into said
recipient, thymic tissue from the second species; and implanting
said graft in said recipient.
10. The method of claim 9, wherein the donor animal is a swine.
11. The method of claim 10, wherein the same swine is the donor of
both the graft and the thymic tissue.
12. The method of claim 9, wherein said primate is a human.
13. The method of claim 9, said method further comprising the step
of, prior or simultaneous with transplantation of said graft,
inactivating mature CD4.sup.+ T cells of the recipient.
14. The method of claim 9, further comprising the step of, prior to
thymic tissue transplantation, irradiating the recipient with low
dose whole body irradiation.
15. The method of claim 14, wherein said low dose irradiation is at
least 100 rads and less than 400 rads.
16. The method of claim 9, further comprising the step, prior to
thymic tissue transplantation, adsorbing natural antibodies from
the blood of said recipient.
17. The method of claim 9, wherein said thymic tissue is capable of
supporting, in the recipient primate, clonal deletion or anergy of
thymocytes reactive with donor xenoantigens.
18. The method of claim 9, wherein hematopoietic cells are
administered to said recipient and wherein the method includes
inactivating NK cells of said recipient.
19. The method of claim 9, wherein said primate recipient is a
human and said donor is a miniature swine.
20. A method of providing mature recipient T cells in a primate
recipient which is thymus function deficient, comprising
introducing into the primate recipient swine xenogeneic donor
thymic tissue, so that recipient T cells can mature in said
implanted swine donor thymic tissue.
21. The method of claim 20, wherein said primate is a human and
said donor is a miniature swine.
22. The method of claim 20, wherein said thymus function is
deficient in said recipient due to an immune disorder.
23.-39. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/126,704, filed on Jul. 30, 1998, which is a continuation of U.S.
Ser. No. 08/458,720, filed on Jun. 1, 1995, now U.S. Pat. No.
5,876,708, which is a continuation-in-part of: U.S. Ser. No.
08/266,427, filed on Jun. 27, 1994 now U.S. Pat. No. 5,614,187;
U.S. Ser. No. 08/451,210, filed on May 26, 1995, which is a file
wrapper continuation of U.S. Ser. No. 07/838,595, filed on Feb. 19,
1992, now abandoned; U.S. Ser. No. 08/220,371, filed on Mar. 29,
1994, now abandoned; PCT/US94/05527 filed on May 16, 1994; U.S.
Ser. No. 08/08/243,653, filed on May 16, 1994, now U.S. Pat. No.
5,658,564; U.S. Ser. No. 08/114,072, filed on Aug. 30, 1993, now
U.S. Pat. No. 5,624,823; U.S. Ser. No. 08/150,739, filed on Nov.
10, 1993, now abandoned; U.S. Ser. No. 08/212,228, filed on Mar.
14, 1994, now abandoned; and PCT/US94/01616 filed on Feb. 14, 1994.
All of the above-referenced applications are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to the replacement of thymus function
and to the induction or restoration of immunological tolerance. The
invention further relates to tissue and organ transplantation.
[0003] The thymus is the central organ for the development of
mature, self-tolerant T cells that recognize peptide antigens in
the context of self major histocompatibility (MHC) antigens. The
requirement for self MHC molecules to present antigen is termed MHC
restriction. Athymic individuals do not have an organ in which to
generate normal numbers of MHC restricted T cells and are therefore
immunoincompetent.
SUMMARY OF THE INVENTION
[0004] It has been discovered that host T cells of an athymic T
cell depleted host which has received a thymic graft, e.g., a
xenogeneic thymic graft, can mature in the donor thymic tissue,
e.g., in xenogeneic thymic tissue. Host T cells which mature in the
implanted xenogeneic thymic tissue are immunocompetent.
[0005] Accordingly, the invention features, in one aspect, a method
of restoring or inducing immunocompetence (or restoring or
promoting the thymus-dependent ability for T cell progenitors to
mature or develop into functional mature T cells) in a host or
recipient, e.g., a primate host or recipient, e.g., a human, which
is capable of producing T cell progenitors but which is
thymus-function deficient and thus unable to produce a sufficient
number of mature functional T cells for a normal immune response.
The invention includes the step of introducing into the primate
host, donor thymic tissue, e.g., xenogeneic thymic tissue,
preferably fetal or neonatal thymic tissue, so that host T cells
can mature in the implanted thymic tissue.
[0006] In preferred embodiments the donor of the thymic tissue is a
xenogeneic species and: the thymic xenograft is a discordant
xenograft; the thymic xenograft is a concordant xenograft; the host
is a primate, e.g., a human, and the thymic tissue is swine, e.g.,
miniature swine, thymic tissue, or primate thymic tissue.
[0007] The method can include other steps which facilitate
acceptance of the donor tissue, or otherwise optimize the method.
In preferred embodiments the thymic tissue is xenogeneic and: liver
or spleen tissue, preferably fetal or neonatal liver or spleen
tissue, is implanted with the thymic tissue; donor hematopoietic
cells, e.g., cord blood stem cells or fetal or neonatal liver or
spleen cells, are administered to the recipient, e.g., a suspension
of fetal liver cells is administered intraperitoneally or
intravenously; the recipient is thymectomized, preferably before or
at the time the xenograft thymic tissue is introduced.
[0008] In other preferred embodiments the method includes:
(preferably prior to or at the time of introducing the thymic
tissue into the recipient) depleting, inactivating or inhibiting
recipient natural killer (NK) cells, e.g., by introducing into the
recipient an antibody capable of binding to NK cells of the
recipient, to prevent NK mediated rejection of the thymic tissue;
(preferably prior to or at the time of introducing the thymic
tissue into the recipient) depleting, inactivating or inhibiting
host T cell function, e.g., by introducing into the recipient an
antibody capable of binding to T cells of the recipient;
(preferably prior to or at the time of introducing the thymic
tissue into the recipient) depleting, inactivating or inhibiting
host CD4.sup.+ cell function, e.g., by introducing into the
recipient an antibody capable of binding to CD4, or CD4.sup.+ cells
of the recipient.
[0009] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, or the administration of a hematopoietic
stem cell inactivating or depleting antibody, to deplete or
partially deplete the bone marrow of the recipient (preferably
prior to thymic tissue transplantation).
[0010] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the host
with, e.g., about 700 rads of thymic irradiation, administering to
the recipient one or more doses of an anti T cell antibody, e.g.,
an anti-CD4 and/or an anti-CD8 monoclonal antibody, or
administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0011] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of: the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
host's blood, e.g., by contacting the host's blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0012] In preferred embodiments the host or recipient is a
post-natal individual, e.g., an adult, or a child.
[0013] In preferred embodiments the method further includes the
step of identifying a host or recipient which is capable of
producing T cell progenitors but which is thymus-function deficient
and thus unable to produce a sufficient number of mature functional
T cells for a normal immune response.
[0014] In other preferred embodiments, a graft which is obtained
from a different organ than is the thymic tissue is implanted in
the recipient; and the recipient does not receive hematopoietic
stem cells, e.g., bone marrow cells, from the donor or the donor
species.
[0015] Other methods can be combined with the methods disclosed
herein to promote the acceptance of the graft by the recipient. For
example, tolerance to the donor tissue can also be induced by
inserting a nucleic acid which expresses a donor antigen, e.g., a
donor MHC gene, into a cell of the recipient, e.g., a hematopoietic
stem cell, and introducing the genetically engineered cell into the
recipient. For example, human recipient stem cells can be
engineered to express a swine MHC gene, e.g., a swine class I or
class II MHC gene, or both a class I and a class II gene, and the
cells implanted in a human recipient who will receive swine thymic
tissue. When inserted into a recipient primate, e.g., a human,
expression of the donor MHC gene results in tolerance to subsequent
exposure to donor antigen, and can thus induce tolerance to thymic
tissue from the donor. These methods, and other methods which can
be combined with the methods disclosed herein, are discussed in
Sachs, U.S. Ser. No. 08/126, 122, filed Sep. 23, 1993, hereby
incorporated by reference and in Sachs, U.S. Ser. No. 08/129,608,
filed Sep. 29, 1993, hereby incorporated by reference.
[0016] Methods of inducing tolerance, e.g., by the implantation of
hematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes,
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, hereby incorporated
by reference, can also be combined with the methods disclosed
herein.
[0017] Other methods of inducing tolerance may also be used to
promote acceptance of the donor tissue. For example, suppression of
T cell help, which can be induced, e.g., by the administration of a
short course of high dose immunosuppressant, e.g., cyclosporine,
has been found to induce tolerance. In these methods, T cell help
is suppressed for a comparatively short period just subsequent to
implantation of a graft, and does not require or include chronic
immunosuppression. These methods, as well as other methods which
can be combined with the methods disclosed herein, are described in
Sachs, U.S. Ser. No. 08/220,371, filed Mar. 29, 1994, hereby
incorporated by reference.
[0018] Other methods of promoting tolerance or promoting the
acceptance of grafts, e.g., by altering levels of cytokine
activity, are disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S.
Ser. No. 08/114,072, filed Aug. 30, 1993, hereby incorporated by
reference.
[0019] It has also been discovered that xenogeneic thymic tissue
can be used to induce tolerance to a xenogeneic graft in a
recipient.
[0020] Accordingly, in another aspect, the invention features, a
method of inducing tolerance in a recipient mammal, e.g., a
primate, e.g., a human, of a first species to a graft obtained from
a mammal of a second species, e.g., a discordant species. The
method includes: prior to or simultaneous with transplantation of
the graft, introducing into the recipient mammal thymic tissue,
e.g., thymic epithelium, preferably fetal or neonatal thymic
tissue, of the second species; and (optionally) implanting the
graft in the recipient. The thymic tissue prepares the recipient
for the graft that follows, by inducing immunological tolerance at
the T-cell level.
[0021] In preferred embodiments: the thymic xenograft is a
discordant xenograft; the thymic xenograft is a concordant
xenograft; the recipient is a human and the thymic tissue is swine,
e.g., miniature swine, thymic tissue, or primate thymic tissue.
[0022] Preferred embodiments include other steps to promote
acceptance of the graft thymus and the induction of immunological
tolerance or to otherwise optimize the procedure. In preferred
embodiments: liver or spleen tissue, preferably fetal or neonatal
liver or spleen tissue, is implanted with the thymic tissue; donor
hematopoietic cells, e.g., cord blood stem cells or fetal or
neonatal liver or spleen cells, are administered to the recipient,
e.g., a suspension of fetal liver cells administered
intraperitoneally or intravenously; the recipient is thymectomized,
preferably before or at the time the xenograft thymic tissue is
introduced.
[0023] In other preferred embodiments the method includes
(preferably prior to or at the time of introducing the thymic
tissue or stem cells into the recipient) depleting, inactivating or
inhibiting recipient NK cells, e.g., by introducing into the
recipient an antibody capable of binding to natural killer (NK)
cells of the recipient, to prevent NK mediated rejection of the
thymic tissue; (preferably prior to or at the time of introducing
the thymic tissue into the recipient) depleting, inactivating or
inhibiting recipient T cells, e.g., by introducing into the
recipient an antibody capable of binding to T cells of the
recipient; (preferably prior to or at the time of introducing the
thymic tissue or stem cells into the recipient) depleting,
inactivating or inhibiting host CD4.sup.+ cell function, e.g., by
introducing into the recipient an antibody capable of binding to
CD4, or CD4.sup.+ cells of the recipient. An anti-mature T cell
antibody which lyses T cells as well as NK cells can be
administered. Lysing T cells is advantageous for both thymic tissue
and xenograft survival. Anti-T cell antibodies are present, along
with anti-NK antibodies, in anti-thymocyte anti-serum. Repeated
doses of anti-NK or anti-T cell antibody may be preferable.
Monoclonal preparations can be used in the methods of the
invention.
[0024] Other preferred embodiments include those in which: the
recipient does not receive hematopoietic cells from the donor or
the donor species: the same mammal of the second species is the
donor of both the graft and the thymic tissue; the donor mammal is
a swine, e.g., a miniature swine; an anti-human thymocyte
polyclonal anti-serum, obtained, e.g., from a horse or pig is
administered to the recipient.
[0025] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, the administration of a hematopoietic stem
cell inactivating or depleting antibody, to deplete or partially
deplete the bone marrow of the recipient.
[0026] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the
recipient with, e.g., about 700 rads of thymic irradiation,
administering to the recipient one or more doses of an anti T cell
antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody,
or administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0027] In preferred embodiments the host or recipient is a
post-natal individual, e.g., an adult, or a child.
[0028] In preferred embodiments the method further includes the
step of identifying a host or recipient which is in need of a
graft.
[0029] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of: the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
recipient's blood, e.g., by contacting the hosts blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0030] Other methods can be combined with the methods disclosed
herein to promote the acceptance of the graft by the recipient. For
example, tolerance to the xenogeneic thymic tissue can also be
induced by inserting a nucleic acid which expresses a donor
antigen, e.g., a donor MHC gene, into a cell of the recipient,
e.g., a hematopoietic stem cell, and introducing the genetically
engineered cell into the recipient. For example, human recipient
stem cells can be engineered to express a swine MHC gene, e.g., a
swine class I or class II MHC gene, or both a class I and class II
gene, and the cells implanted in a human recipient who will receive
swine thymic tissue. When inserted into a recipient primate, e.g.,
a human, expression of the donor MHC gene results in tolerance to
subsequent exposure to donor antigen, and can thus induce tolerance
to thymic tissue from the donor. These methods, and other methods
which can be combined with the methods disclosed herein, are
discussed in Sachs, U.S. Ser. No. 08/126,122, filed Sep. 23, 1993,
and in Sachs, U.S. Ser. No. 08/129,608, filed Sep. 29, 1993.
[0031] Methods of inducing tolerance, e.g., by the implantation of
hematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes,
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined
with the methods disclosed herein.
[0032] Other methods of inducing tolerance may also be used to
promote acceptance of the xenogeneic thymic tissue. For example,
suppression of T cell help, which can be induced, e.g., by the
administration of a short course of high dose immunosuppressant,
e.g., cyclosporine, has been found to induce tolerance. In these
methods, T cell help is suppressed for a comparatively short period
just subsequent to implantation of a graft, and does not require or
include chronic immunosuppression. These methods, as well as other
methods which can be combined with the methods disclosed herein,
are described in Sachs, U.S. Ser. No. 08/220,371, filed Mar. 29,
1994.
[0033] Other methods of promoting tolerance or promoting the
acceptance of grafts, e.g., by altering levels of cytokine
activity, are disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S.
Ser. No. 08/114,072 filed Aug. 30, 1993.
[0034] In another aspect, the invention features, a method of
restoring or inducing immunocompetence in a recipient, e.g., a
primate recipient, e.g., a human, at risk for an acquired immune
disorder, (e.g., a human at risk for AIDS), which is capable of
producing T cell progenitors but which is thymus-function deficient
and thus unable to produce a sufficient number of mature functional
T cells to provide a normal immune response. The invention includes
the steps of introducing into the primate recipient, donor thymic
tissue, e.g., xenogeneic thymic tissue, so that recipient T cells
can mature in the implanted donor thymic tissue. The thymic tissue
is preferably fetal or neonatal thymic tissue.
[0035] In preferred embodiments the thymic tissue is xenogeneic
and: the thymic xenograft is a discordant xenograft; the thymic
xenograft is a concordant xenograft; the recipient is a human and
the thymic tissue is vertebrate, e.g., swine, e.g., miniature
swine, thymic tissue, or primate thymic tissue.
[0036] Acceptance of a graft, especially a xenogeneic graft, will
depend on the stage of the immune disorder. Generally, the more
advanced the disorder the more compromised the recipient immune
system and the easier it is to induce acceptance of donor thymic
tissue. In some cases, the tolerizing effect of the graft itself
will be sufficient to provide for acceptance of xenogeneic thymus.
In other cases, additional measures will be needed. Thus, the
method can include other steps which facilitate acceptance of the
donor tissue or otherwise optimize the method. In preferred
embodiments: liver or spleen tissue, preferably fetal or neonatal
liver or spleen tissue, is implanted with the thymic tissue; donor
hematopoietic cells, e.g., cord blood stem cells or fetal or
neonatal liver or spleen cells, are administered to the recipient,
e.g., a suspension of fetal liver cells is administered
intraperitoneally or intravenously; the recipient is thymectomized,
preferably before or at the time the xenograft thymic tissue is
introduced.
[0037] In preferred embodiments: the method includes (preferably
prior to or at the time of introducing the thymic tissue into the
recipient) depleting, inactivating or inhibiting recipient NK
cells, e.g., by introducing into the recipient an antibody capable
of binding to natural killer (NK) cells of the recipient, to
prevent NK mediated rejection of the thymic tissue; the method
includes (preferably prior to or at the time of introducing the
thymic tissue into the recipient), depleting, inactivating or
inhibiting recipient T cells, e.g., by introducing into the
recipient an antibody capable of binding to T cells of the
recipient; (preferably prior to or at the time of introducing the
thymic tissue into the recipient) depleting, inactivating or
inhibiting host CD4.sup.+ cell function, e.g., by introducing into
the recipient an antibody capable of binding to CD4, or CD4.sup.+
cells of the recipient.
[0038] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, the administration of a hematopoietic stem
cell inactivating or depleting antibody, to deplete or partially
deplete the bone marrow of the recipient.
[0039] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the
recipient mammal with, e.g., about 700 rads of thymic irradiation,
administering to the recipient one or more doses of an anti T cell
antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody,
or administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0040] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of: the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
recipient's blood, e.g., by contacting the hosts blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0041] In preferred embodiments the host or recipient is a
post-natal individual, e.g., an adult, or a child.
[0042] In preferred embodiments the method further includes the
step of identifying a host or recipient which is at risk for an
acquired immune disorder, (e.g., a human at risk for AIDS), which
is capable of producing T cell progenitors but which is
thymus-function deficient and thus unable to produce a sufficient
number of mature functional T cells to provide a normal immune
response.
[0043] Other methods can be combined with the methods disclosed
herein to promote the acceptance of the thymic graft by the
recipient. For example, tolerance to donor tissue can also be
induced by inserting a nucleic acid which expresses a donor
antigen, e.g., a donor MHC gene, into a cell of the recipient,
e.g., a hematopoietic stem cell, and introducing the genetically
engineered cell into the recipient. For example, human recipient
stem cells can be engineered to express a swine MHC gene, e.g., a
swine class I or class II MHC gene, or both a class I and a class
II MHC gene, and the cells implanted in a human recipient who will
receive swine thymic tissue. When inserted into a recipient
primate, e.g., a human, expression of the donor MHC gene results in
tolerance to subsequent exposure to donor antigen, and can thus
induce tolerance to thymic tissue from the donor. These methods,
and other methods which can be combined with the methods disclosed
herein, are discussed in Sachs, U.S. Ser. No. 08/126, 122, filed
Sep. 23, 1993, hereby incorporated by reference and in Sachs, U.S.
Ser. No. 08/220,371, filed Mar. 29, 1994.
[0044] Methods of inducing tolerance, e.g., by the implantation of
hematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes,
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined
with the methods disclosed herein.
[0045] Other methods of inducing tolerance may also be used to
promote acceptance of the donor thymic tissue. For example,
suppression of T cell help, which can be induced, e.g., by the
administration of a short course of high dose immunosuppressant,
e.g., cyclosporine, has been found to induce tolerance. In these
methods, T cell help is suppressed for a comparatively short period
just subsequent to implantation of a graft, and does not require or
include chronic immunosuppression. These methods, as well as other
methods which can be combined with the methods disclosed herein,
are described in Sachs, U.S. Ser. No. 08/220,371, filed Mar. 29,
1994.
[0046] Other methods of promoting tolerance or promoting the
acceptance of grafts, e.g., by altering levels of cytokine
activity, are disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S.
Ser. No. 08/114,072, filed Aug. 30, 1993.
[0047] In another aspect, the invention features, a method of
restoring or inducing immunocompetence in a recipient, e.g., a
primate recipient, e.g., a human, at risk for an acquired immune
disorder, (e.g., a human at risk for AIDS) which is unable to
produce a normal or sufficient number of mature functional T cells
to provide normal immune function. The invention includes the steps
of introducing into the primate recipient, donor hematopoietic stem
cells, so that donor T cells can mature in the recipient
thymus.
[0048] In preferred embodiments the donor stem cells are from a
xenogeneic donor and: the xenograft hematopoietic stem cells are
from a discordant species; the hematopoietic stem cells are from a
concordant species; the recipient is a human and the hematopoietic
stem cells are vertebrate, e.g., swine, e.g., miniature swine,
hematopoietic stem cells, or primate hematopoietic stem cells.
[0049] Acceptance of the donor cells will depend on the stage of
the immune disorder. Generally, the more advanced the disorder the
more compromised the recipient immune system and the easier it is
to induce acceptance of donor, especially xenogeneic donor, tissue.
In some cases, the tolerizing effect of the stem cells themselves
will be sufficient to provide for acceptance. In other cases,
additional measures will be needed. Thus, the method can include
other steps which facilitate acceptance of the donor cells or
otherwise optimize the method. In preferred embodiments: liver or
spleen tissue, preferably fetal or neonatal liver or spleen tissue,
is implanted with the donor hematopoietic cells, e.g., cord blood
stem cells or fetal or neonatal liver or spleen cells, are
administered to the recipient, e.g., a suspension of fetal liver
cells is administered intraperitoneally or intravenously.
[0050] In preferred embodiments: the method includes, (preferably
prior to or at the time of introducing the donor cells into the
recipient) depleting, inactivating or inhibiting recipient NK
cells, e.g., by introducing into the recipient an antibody capable
of binding to natural killer (NK) cells of the recipient, to
prevent NK mediated rejection of the thymic tissue; the method
includes (preferably prior to or at the time of introducing the
thymic tissue into the recipient), depleting, inactivating or
inhibiting recipient T cells, e.g., by introducing into the
recipient an antibody capable of binding to T cells of the
recipient; (preferably prior to or at the time of introducing the
thymic tissue into the recipient) depleting, inactivating or
inhibiting host CD4.sup.+ cell function, e.g., by introducing into
the recipient an antibody capable of binding to CD4, or CD4.sup.+
cells of the recipient.
[0051] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, the administration of a hematopoietic stem
cell inactivating or depleting antibody, to deplete or partially
deplete the bone marrow of the recipient.
[0052] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the
recipient mamma i with, e.g., about 700 rads of thymic irradiation,
administering to the recipient one or more doses of an anti T cell
antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody,
or administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0053] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of: the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
recipient's blood, e.g., by contacting the hosts blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0054] In preferred embodiments the host or recipient is a
post-natal individual, e.g., an adult, or a child.
[0055] In preferred embodiments the method further includes the
step of identifying a host or recipient which is at risk for an
acquired immune disorder, (e.g., a human at risk for AIDS) and
which is unable to produce a normal or sufficient number of mature
functional T cells to provide normal immune function.
[0056] Other methods can be combined with the methods disclosed
herein to promote the acceptance of the transplanted stem cells by
the recipient. For example, tolerance to donor tissue can be
induced by inserting a nucleic acid which expresses a donor
antigen, e.g., a donor MHC gene, into a cell of the recipient,
e.g., a hematopoietic stem cell, and introducing the genetically
engineered cell into the recipient. For example, human recipient
stem cells can be engineered to express a swine MHC gene, e.g., a
swine class I or class II MHC gene, or both a class I and a class
II gene, and the cells implanted in a human recipient who will
receive swine thymic tissue. When inserted into a recipient
primate, e.g., a human, expression of the donor MHC gene results in
tolerance to subsequent exposure to donor antigen, and can thus
induce tolerance to tissue from the donor. These methods, and other
methods which can be combined with the methods disclosed herein,
are discussed in Sachs, U.S. Ser. No. 08/126, 122, filed Sep. 23,
1993, and in Sachs, U.S. Ser. No. 08/220,371, filed Mar. 29,
1994.
[0057] Methods of inducing tolerance, e.g., by the implantation of
hematopoietic stem cells, disclosed in Sachs, Cosimi and Sykes,
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined
with the methods disclosed herein.
[0058] Other methods of inducing tolerance can be combined with the
methods disclosed herein to promote acceptance of donor tissue. For
example, suppression of T cell help, which can be induced, e.g., by
the administration of a short course of high dose
immunosuppressant, e.g., cyclosporine, has been found to induce
tolerance. In these methods, T cell help is suppressed for a
comparatively short period just subsequent to implantation of a
graft, and does not require or include chronic immunosuppression.
These methods, as well as other methods which can be combined with
the methods disclosed herein, are described in Sachs, U.S. Ser. No.
08/220,371, filed Mar. 29, 1994.
[0059] Other methods of promoting tolerance or promoting the
acceptance of grafts, e.g., by altering levels of cytokine
activity, are disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S.
Ser. No. 08/114,072, filed Aug. 30, 1993.
[0060] In another aspect, the invention features, a method of
restoring or inducing immunocompetence in a recipient, e.g., a
primate recipient, e.g., a human, at risk for an acquired immune
disorder, (e.g., a human at risk for AIDS), which is
thymus-function deficient and thus unable to produce a normal
number of mature functional T cells or a sufficient number of
mature functional T cells for a normal immune response. The
invention includes the steps of introducing into the primate
recipient, donor thymic tissue, preferably, xenogeneic thymic
tissue, and donor hematopoietic stem cells, preferably xenogeneic
hematopoietic stem cells, so that donor T cells can mature in the
implanted donor thymic tissue. The thymic tissue is preferably
fetal or neonatal thymic tissue.
[0061] In preferred embodiments the thymic graft is a xenograft
and: the thymic xenograft is a discordant xenograft; the thymic
xenograft is a concordant xenograft; the recipient is a human and
the thymic tissue is vertebrate, e.g., swine, e.g., immature swine,
thymic tissue, or primate thymic tissue.
[0062] In preferred embodiments: the xenograft hematopoietic stem
cells are from a discordant species; the hematopoietic stem cells
are a concordant species; the recipient is a human and the
hematopoietic stem cells are vertebrate, e.g., swine, e.g.,
miniature swine, hematopoietic stem cells, or primate hematopoietic
stem cells.
[0063] In preferred embodiments the donor of the thymic graft and
the donor of the stem cells are: the same organism; from the same
species; syngeneic; matched at least one MHC locus; matched at
least one class I MHC locus; matched at least one class II MHC
locus; sufficiently MHC matched that one will not reject a graft
from the other; miniature swine from a herd which is completely or
partially inbred.
[0064] Acceptance of donor tissue, especially xenogeneic tissue,
will depend on the stage of the immune disorder. Generally, the
more advanced the disorder the more compromised the recipient
immune system and the easier it is to induce acceptance of donor
tissue. In some cases, the tolerizing effect of the donor tissue
itself will be sufficient to provide for acceptance. In other
cases, additional measures will be needed. Thus, the method can
include other steps which facilitate acceptance of donor tissue or
otherwise optimize the method. In preferred embodiments: liver or
spleen tissue, preferably fetal or neonatal liver or spleen tissue,
is implanted with the thymic tissue; donor hematopoietic cells,
e.g., cord blood cells or fetal or neonatal liver or spleen cells,
are administered to the recipient, e.g., a suspension of fetal
liver cells is administered intraperitoneally or intravenously; the
recipient is thymectomized, preferably before or at the time the
xenograft thymic tissue is introduced.
[0065] In preferred embodiments: the method includes, (preferably
prior to or at the time of introducing the thymic tissue or stem
cells into the recipient) depleting, inactivating or inhibiting
recipient NK cells, e.g., by introducing into the recipient an
antibody capable of binding to natural killer (NK) cells of the
recipient, to prevent NK mediated rejection of the thymic tissue;
the method includes, (preferably prior to or at the time of
introducing the thymic tissue or stem cells into the recipient)
depleting, inactivating or inhibiting recipient T cells, e.g., by
introducing into the recipient an antibody capable of binding to T
cells of the recipient mammal; (preferably prior to or at the time
of introducing the thymic tissue or stem cells into the recipient)
depleting, inactivating or inhibiting host CD4.sup.+ cell function,
e.g., by introducing into the recipient an antibody capable of
binding to CD4, or CD4.sup.+ cells of the recipient.
[0066] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, the administration of a hematopoietic stem
cell inactivating or depleting antibody, to deplete or partially
deplete the bone marrow of the recipient.
[0067] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the
recipient mammal with, e.g., about 700 rads of thymic irradiation,
administering to the recipient one or more doses of an anti T cell
antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody,
or administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0068] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
recipient's blood, e.g., by contacting the hosts blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0069] In preferred embodiments the host or recipient is a
post-natal individual, e.g., an adult, or a child.
[0070] In preferred embodiments the method further includes the
step of identifying a host or recipient which is at risk for an
acquired immune disorder, (e.g., a human at risk for AIDS), and
which is thymus-function deficient and thus unable to produce a
normal number of mature functional T cells or a sufficient number
of mature functional T cells for a normal immune response.
[0071] Other methods can be combined with the methods disclosed
herein to promote the acceptance of donor tissue by the recipient.
For example, tolerance to donor tissue can be induced by inserting
a nucleic acid which expresses a donor antigen, e.g., a donor MHC
gene, into a cell of the recipient, e.g., a hematopoietic stem
cell, and introducing the genetically engineered cell into the
recipient. For example, human recipient stem cells can be
engineered to express a swine MHC gene, e.g. a swine class I or
class II MHC gene, or both a class I and a class II gene, and the
cells implanted in a human recipient who will receive swine thymic
tissue. When inserted into a recipient primate, e.g., a human,
expression of the donor MHC gene results in tolerance to subsequent
exposure to donor antigen, and can thus induce tolerance to tissue
from the donor. These methods and other methods which can be
combined with the methods disclosed herein are discussed in Sachs,
U.S. Ser. No. 08/126, 122, filed Sep. 23, 1993, and in Sachs, U.S.
Ser. No. 08/220,371, filed Mar. 29, 1994.
[0072] Methods of inducing tolerance, e.g., by the implantation of
hematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes,
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined
with the methods disclosed herein.
[0073] Other methods of inducing tolerance can be combined with the
methods disclosed herein to promote acceptance of donor tissue. For
example, suppression of T cell help, which can be induced, e.g., by
the administration of a short course of high dose
immunosuppressant, e.g., cyclosporine, has been found to induce
tolerance. In these methods, T cell help is suppressed for a
comparatively short period just subsequent to implantation of a
graft, and does not require or include chronic immunosuppression.
These methods, as well as other methods which can be combined with
the methods disclosed herein, are described in Sachs, U.S. Ser. No.
08/220,371, filed Mar. 29, 1994.
[0074] Other methods of promoting tolerance or promoting the
acceptance of donor tissue, e.g., by altering levels of cytokine
activity, or inhibiting Graft-versus-recipient-disease, are
disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S. Ser. No.
08/114,072, filed Aug. 30, 1993. It has also been discovered that
hematopoietic cells can be use to induce tolerance to a graft.
[0075] Accordingly, in another aspect, the invention features, a
method of inducing immunological tolerance in a recipient mammal,
e.g., a primate, e.g., a human, of a first species to a graft
obtained from a donor mammal of a second species, e.g., a
discordant species e.g., a discordant primate species. The method
includes: prior to or simultaneous with transplantation of the
graft, introducing into the recipient mammal hematopoietic stem
cells, e.g., bone marrow cells, or fetal liver or spleen cells, of
the second species; (preferably, the hematopoietic stem cells home
to a site in the recipient mammal); optionally, (preferably prior
to introducing the hematopoietic stem cells into the recipient
mammal), depleting, inactivating or inhibiting recipient NK cells,
e.g., by introducing into the recipient mammal an antibody capable
of binding to natural killer (NK) cells of the recipient mammal, to
prevent NK mediated rejection of the hematopoietic cells; and
(optionally) implanting the graft in the recipient. As will be
explained in more detail below, the hematopoietic cells prepare the
recipient for the graft that follows, by inducing tolerance at both
the B-cell and T-cell levels. Preferably, hematopoietic cells are
fetal liver or spleen, or bone marrow cells, including immature
cells (i.e., undifferentiated hematopoietic stem cells; these
desired cells can be separated out of the bone marrow prior to
administration), or a complex bone marrow sample including such
cells can be used.
[0076] One source of anti-NK antibody is anti-human thymocyte
polyclonal anti-serum. A second, anti-mature T cell antibody can be
administered as well, which lyses T cells as well as NK cells.
Lysing T cells is advantageous for both bone marrow and xenograft
survival. Anti-T cell antibodies are present, along with anti-NK
antibodies, in anti-thymocyte anti-serum. Repeated doses of anti-NK
or anti-T cell antibody may be preferable. Monoclonal preparations
can be used in the methods of the invention.
[0077] Preferred embodiments include: the step of introducing into
the recipient mammal, donor species-specific stromal tissue,
preferably hematopoietic stromal tissue, e.g., fetal liver or
thymus; and the step of prior to hematopoietic stem cell
transplantation, introducing into the recipient mammal an antibody
capable of binding to mature T cells of the recipient mammal.
[0078] Preferred embodiments include those in which: the stromal
tissue is introduced simultaneously with, or prior to, the
hematopoietic stem cells; the hematopoietic stem cells are
introduced simultaneously with, or prior to, the antibody; the
stromal tissue is introduced simultaneously with, or prior to, the
hematopoietic stem cells, and the hematopoietic stem cells are
introduced simultaneously with, or prior to, the antibody.
[0079] Preferred embodiments include those in which: the same
mammal of the second species is the donor of both the graft and the
hematopoietic cells; the donor mammal is a swine, e.g., a miniature
swine; the introduction is by intravenous injection; and an
anti-human thymocyte polyclonal anti-serum; obtained, e.g. from a
horse or pig is administered.
[0080] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, the administration of a hematopoietic stem
cell inactivating or depleting antibody, to deplete or partially
deplete the bone marrow of the recipient.
[0081] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the
recipient mammal with, e.g., about 700 rads of thymic irradiation,
administering to the recipient one or more doses of an anti T cell
antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody,
or administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0082] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of: the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
recipient's blood, e.g., by contacting the hosts blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0083] Preferred embodiments include: (preferably prior to
hematopoietic stem cell transplantation) depleting, inactivating,
or inhibiting recipient T cells, e.g., by introducing into the
recipient an antibody capable of binding to mature T cells of the
recipient.
[0084] Preferably the graft is obtained from a different organ than
the hematopoietic stem cells.
[0085] Preferred embodiments include those in which: the primate is
a cynomolgus monkey; the primate is a human; the stromal tissue is
fetal or neonatal liver; the stromal tissue is fetal or neonatal
thymus; the mammal is a swine; e.g., a miniature swine; the graft
is a liver; the graft is a kidney.
[0086] Other methods can be combined with the methods disclosed
herein to promote the acceptance of the graft by the recipient. For
example, tolerance to the xenogeneic thymic tissue can also be
induced by inserting a nucleic acid which expresses a donor
antigen, e.g., a donor MHC gene, into a cell of the recipient,
e.g., a hematopoietic stem cell, and introducing the genetically
engineered cell into the recipient. For example, human recipient
stem cells can be engineered to express a swine class I or class II
MHC gene, or both a class I and II gene, and the cells implanted in
a human recipient who will receive swine thymic tissue. When
inserted into a recipient primate, e.g., a human, expression of the
donor MHC gene results in tolerance to subsequent exposure to donor
antigen, and can thus induce tolerance to thymic tissue from the
donor. These methods, and other methods which can be combined with
the methods disclosed herein, are discussed in Sachs, U.S. Ser. No.
08/126, 122, filed Sep. 23, 1993, and in Sachs, U.S. Ser. No.
08/220,371, filed Mar. 29, 1994.
[0087] Methods of inducing tolerance, e.g., by the implantation of
hematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes,
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined
with the methods disclosed herein.
[0088] Other methods of inducing tolerance may also be used to
promote acceptance of the xenogeneic thymic tissue. For example,
suppression of T cell help, which can be induced, e.g., by the
administration of a short course of high dose immunosuppressant,
e.g., cyclosporine, has been found to induce tolerance. In these
methods, T cell help is suppressed for a comparatively short period
just subsequent to implantation of a graft, and does not require or
include chronic immunosuppression. These methods, as well as other
methods which can be combined with the methods disclosed herein,
are described in Sachs, U.S. Ser. No. 08/220,371, filed Mar. 29,
1994, hereby. incorporated by reference.
[0089] Other methods of promoting tolerance or promoting the
acceptance of grafts, e.g., by altering levels of cytokine
activity, are disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S.
Ser. No. 08/114,072, filed Aug. 30, 1993.
[0090] In another aspect, the invention features a method of
inducing immunological tolerance in a recipient mammal, e.g., a
primate, e.g., a human to a graft obtained from a donor mammal of
the same species. The method includes the following: (preferably
prior to or simultaneous with transplantation of the graft)
introducing into the recipient mammal hematopoietic stem cells,
e.g., bone marrow cells or fetal liver or spleen cells, obtained
from a mammal (preferably, the hematopoietic stem cells home to a
site in the recipient mammal); and, preferably, introducing the
graft into the recipient.
[0091] Preferred embodiments include: the step of introducing into
the recipient mammal, donor species-specific stromal tissue,
preferably hematopoietic stromal tissue, e.g., fetal liver or
thymus; and prior to hematopoietic stem cell transplantation,
depleting, inactivating or inhibiting recipient T cells, e.g., by
introducing into the recipient mammal an antibody capable of
binding to mature T cells of the recipient mammal.
[0092] Other preferred embodiments include the step of (preferably
prior to thymic tissue or hematopoietic stem cell transplantation)
creating hematopoietic space, e.g., by one or more of: irradiating
the recipient mammal with low dose, e.g., between about 100 and 400
rads, whole body irradiation, the administration of a
myelosuppressive drug, the administration of a hematopoietic stem
cell inactivating or depleting antibody, to deplete or partially
deplete the bone marrow of the recipient.
[0093] Other preferred embodiments include (preferably prior to
thymic tissue or hematopoietic stem cell transplantation)
inactivating thymic T cells by one or more of: irradiating the
recipient mammal with, e.g., about 700 rads of thymic irradiation,
administering to the recipient one or more doses of an anti T cell
antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody,
or administering to the recipient a short course of an
immunosuppressant, as is described in U.S. Ser. No. 08/220,371 and
further described below.
[0094] Other preferred embodiments include depleting or otherwise
inactivating natural antibodies, e.g., by one or more of: the
administration of a drug which depletes or inactivates natural
antibodies, e.g., deoxyspergualin; the administration of an
anti-IgM antibody; or the adsorption of natural antibodies from the
recipient's blood, e.g., by contacting the hosts blood with donor
antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or
a liver, from the donor species.
[0095] In other preferred embodiments; the method includes:
(preferably prior to or at the time of introducing the thymic
tissue into the recipient) depleting, inactivating or inhibiting
recipient natural killer (NK) cells, e.g., by introducing into the
recipient an antibody capable of binding to NK cells of the
recipient, to prevent NK mediated rejection of the thymic tissue;
(preferably prior to or at the time of introducing the thymic
tissue into the recipient) depleting, inactivating or inhibiting
host T cell function, e.g., by introducing into the recipient an
antibody capable of binding to T cells of the recipient.
[0096] Other methods can be combined with-the methods disclosed
herein to promote tolerance to a graft. Methods of inducing
tolerance, e.g., by the implantation of hematopoietic stem cells,
disclosed in Sachs, Cosimi, and Sykes, U.S. Ser. No. 07/838,595,
filed Feb. 19, 1992, can also be combined with the methods
disclosed herein.
[0097] Other methods of inducing tolerance may also be used to
promote acceptance of donor tissue. For example, suppression of T
cell help, which can be induced, e.g., by the administration of a
short course of high dose immunosuppressant, e.g., cyclosporine,
has been found to induce tolerance. In these methods, T cell help
is suppressed for a comparatively short period just subsequent to
implantation of a graft, and does not require or include chronic
immunosuppression. These methods, as well as other methods which
can be combined with the methods disclosed herein, are described in
Sachs, U.S. Ser. No. 08/220,371, filed Mar. 29, 1994.
[0098] Other methods of promoting tolerance or promoting the
acceptance of grafts, e.g., by altering levels of cytokine
activity, are disclosed in Sachs, LeGuern, Sykes, and Blancho, U.S.
Ser. No. 08/114,072, filed Aug. 30, 1993.
[0099] The invention further provides several methods of inducing
tolerance to foreign antigens, e.g., to antigens on allogeneic or
xenogeneic tissue or organ grafts. These methods can be used
individually or in combination with one another. For example, it
has been found that the short-term administration of a help
reducing agent, e.g., a short high dose course of cyclosporine A
(CsA), can significantly prolong graft acceptance. The short term
help reduction-methods of the invention can be combined with one or
more other methods for prolonging graft acceptance. For example, a
short course of high dose cyclosporine treatment to induce
tolerance to unmatched donor class I and other minor unmatched
donor antigens can be combined with implantation of retrovirally
transformed bone marrow cells to induce tolerance to unmatched
donor class II. A short course of high dose cyclosporine
administered to induce tolerance to unmatched donor class I and
other minor antigens can also be combined with implantation of
donor bone marrow cells to induce tolerance to unmatched donor
class II.
[0100] Accordingly, the invention features, in one aspect, a method
of inducing tolerance in a recipient mammal, e.g., a primate, e.g.,
a human, to an allograft from a donor primate including: implanting
the graft in the recipient; and administering to the recipient a
short course of help reducing treatment, e.g., a short course of
high dose cyclosporine. The short course of help reducing treatment
is generally administered at about the time the graft is introduced
into the recipient.
[0101] Preferably, the recipient is mismatched at a first locus
which affects graft rejection, e.g., an MHC class I or II locus, or
a minor antigen locus, and matched, or tolerant of a mismatch, at a
second locus which affects graft rejection, e.g., an MHC class I or
II locus, or a minor antigen locus. Matching at the second locus
can be achieved by selection of a recipient or donor of the
appropriate genotype. The recipient can be rendered tolerant of a
mismatch at the second locus by any method of tolerance induction,
e.g., by administering donor bone marrow tissue to the recipient to
induce tolerance to donor antigens expressed on the donor bone
marrow, by expressing an MHC antigen of the donor from a stem cell
of the recipient to induce tolerance to the donor antigen, or by
altering the immunological properties of the graft, e.g., by
masking, cleaving, or otherwise modifying cell surface molecules on
the graft. In preferred embodiments, any of the methods which can
be used to match or induce tolerance to the second locus can be
used to match or induce tolerance to a third locus which affects
graft rejection, e.g., an MHC class I or II locus, or a minor
antigen locus.
[0102] In preferred embodiments, the recipient and donor are
matched at a class II locus and the short course of help reducing
treatment induces tolerance to unmatched class I and/or minor
antigens on the graft. In preferred embodiments, tolerance to a
class II antigen is induced by a method other than a short course
of a help reducing treatment, and the short course of help reducing
treatment induces tolerance to unmatched class I and minor antigens
on the graft.
[0103] In preferred embodiments, the duration of the short course
of help reducing treatment is approximately equal to or is less
than the period required for mature T cells of the recipient
species to initiate rejection of an antigen after first being
stimulated by the antigen (in humans this is usually 8-12 days,
preferably about 10 days); in more preferred embodiments, the
duration is approximately equal to or is less than two, three,
four, five, or ten times the period required for mature T cells of
the recipient to initiate rejection of an antigen after first being
stimulated by the antigen.
[0104] In other preferred embodiments, the short course of help
reducing treatment is administered in the absence of a treatment
which stimulates the release of a cytokine by mature T cells in the
recipient, e.g. in the absence of a steroid drug in a sufficient
concentration to counteract the desired effect of the help reducing
treatment, e.g., in the absence of Prednisone
(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentration
which stimulates the release of a cytokine by mature T cells in the
recipient. In preferred embodiments, the short course of help
reducing treatment is administered in the absence of a steroid
drug, e.g., in the absence of Prednisone.
[0105] In preferred embodiments: the help reducing treatment is
begun before or at about the time the graft is introduced; the
short course is perioperative, or the short course is
postoperative; or the donor and recipient are class I matched.
[0106] Methods of inducing tolerance by a short-term administration
of a help reducing agent, e.g., a short high dose course of
cyclosporine A (CsA), can be combined with other methods for
inducing tolerance, e.g., methods for the implantation of
transduced bone marrow cells to induce tolerance to an antigen,
e.g., the methods described in U.S. Ser. No. 008/126,122, filed on
Sep. 23, 1993.
[0107] Accordingly, in another aspect, the invention features a
method of inducing tolerance in a recipient mammal, e.g., a
primate, e.g., a human, of a first species to a graft from a
mammal, e.g., a swine, e.g., a miniature swine, of a second
species, which graft preferably expresses a major
histocompatibility complex (MHC) antigen. The method includes
inserting DNA encoding an MHC antigen of the second species into a
hematopoietic stem cell, e.g., a bone marrow hematopoietic stem
cell, of the recipient mammal; allowing the MHC antigen encoding
DNA to be expressed in the recipient; preferably, implanting the
graft in the recipient; and, preferably, administering to the
recipient a short course of help reducing treatment, e.g., a short
course of high dose cyclosporine treatment. The short course of
help reducing treatment is generally administered at about the time
the graft is introduced into the recipient.
[0108] In preferred embodiments, the short course of help reducing
treatment induces tolerance to unmatched class I and/or minor
antigens on a graft which is introduced into the recipient
subsequent to expression of the MHC antigen.
[0109] In preferred embodiments, the duration of the short course
of help reducing treatment is approximately equal to or is less
than the period required for mature T cells of the recipient
species to initiate rejection of an antigen after first being
stimulated by the antigen; in more preferred embodiments, the
duration is approximately equal to or is less than two, three,
four, five, or ten times the period required for a mature T cell of
the recipient species to initiate rejection of an antigen after
first being stimulated by the antigen.
[0110] In other preferred embodiments, the short course of help
reducing treatment is administered in the absence of a treatment
which stimulates the release of a cytokine by mature T cells in the
recipient, e.g., in the absence of a steroid drug in a sufficient
concentration to counteract the desired effect of the help reducing
treatment, e.g., in the absence of Prednisone
(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentration
which stimulates the release of a cytokine by mature T cells in the
recipient. In preferred embodiments, the short course of help
reducing treatment is administered in the absence of a steroid
drug, e.g., in the absence of Prednisone.
[0111] In preferred embodiments: the help reducing treatment is
begun before or at about the time the graft is introduced; the
short course is perioperative; or the short course is
postoperative.
[0112] Preferred embodiments include those in which: the cell is
removed from the recipient mammal prior to the DNA insertion and
returned to the recipient mammal after the DNA insertion; the DNA
is obtained from the individual mammal from which the graft is
obtained; the DNA is obtained from an individual mammal which is
syngeneic with the individual mammal from which the graft is
obtained; the DNA is obtained from an individual mammal which is
MHC matched, and preferably identical, with the individual mammal
from which the graft is obtained; the DNA includes an MHC class I
gene; the DNA includes an MHC class II gene; the DNA is inserted
into the cell by transduction, e.g., by a retrovirus, e.g., by a
Moloney-based retrovirus; and the DNA is expressed in bone marrow
cells and/or peripheral blood cells of the recipient for at least
14, preferably 30, more preferably 60, and most preferably 120
days, after the DNA is introduced into the recipient.
[0113] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, creating hematopoietic
space, e.g., by irradiating the recipient mammal with low dose,
e.g., between about 100 and 400 rads, whole body irradiation to
deplete or partially deplete the bone marrow of the recipient;
inactivating thymic T cells by one or more of: prior to
hematopoietic stem cell transplantation, irradiating the recipient
mammal with, e.g., about 700 rads of thymic irradiation, or
administering to the recipient a short course of an
immunosuppressant, as is described herein.
[0114] Other preferred embodiments include: the step of, prior to
implantation of a graft, depleting natural antibodies from the
blood of the recipient mammal, e.g., by hemoperfusing an organ,
e.g., a liver or a kidney, obtained from a mammal of the second
species. (In organ hemoperfusion antibodies in the blood bind to
antigens on the cell surfaces of the organ and are thus removed
from the blood.)
[0115] In other preferred embodiments: the method further includes,
prior to hematopoietic stem cell transplantation, introducing into
the recipient an antibody capable of binding to mature T cells of
said recipient mammal.
[0116] Other preferred embodiments further include the step of
introducing into the recipient a graft obtained from the donor,
e.g., a liver or a kidney.
[0117] In another aspect, the invention features a method of
inducing tolerance in a recipient mammal, preferably a primate,
e.g., a human, to a graft obtained from a donor of the same
species, which graft preferably expresses an MHC antigen. The
method includes: inserting DNA encoding an MHC antigen of the donor
into a hematopoietic stem cell, e.g., bone marrow hematopoietic
stem cell, of the recipient; allowing the MHC antigen encoding DNA
to be expressed in the recipient; preferably, implanting the graft
in the recipient; and, preferably, administering to the recipient a
short course of help reducing treatment, e.g., a short course of
high dose cyclosporine. The short course of help reducing treatment
is generally administered at about the time the graft is introduced
into the recipient.
[0118] In preferred embodiments, the short course of help reducing
treatment induces tolerance to unmatched class I and/or minor
antigens on a graft which is introduced into the recipient
subsequent to expression of the MHC antigen.
[0119] In preferred embodiments, the duration of the short course
of help reducing treatment is approximately equal to or is less
than the period required for mature T cells of the recipient
species to initiate rejection of an antigen after first being
stimulated by the antigen; in more preferred embodiments, the
duration is approximately equal to or is less than two, three,
four, five, or ten times the period required for a mature T cell of
the recipient species to initiate rejection of an antigen after
first being stimulated by the antigen.
[0120] In other preferred embodiments, the short course of help
reducing treatment is administered in the absence of a treatment
which stimulates the release of a cytokine by mature T cells in the
recipient, e.g., in the absence of a steroid drug in a sufficient
concentration to counteract the desired effect of the help reducing
treatment, e.g., in the absence of Prednisone
(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentration
which stimulates the release of a cytokine by mature T cells in the
recipient. In preferred embodiments, the short course of help
reducing treatment is administered in the absence of a steroid
drug, e.g., in the absence of Prednisone
[0121] In preferred embodiments: the help reducing treatment is
begun before or at about the time the graft is introduced; the
short course is perioperative, or the short course is
postoperative; or the donor and recipient are class I matched.
[0122] Preferred embodiments include those in which: the cell is
removed from the recipient prior to the DNA insertion and returned
to the recipient after the DNA insertion; the DNA includes a MHC
class I gene; the DNA includes a MHC class II gene; the DNA is
inserted into the cell by transduction, e.g. by a retrovirus, e.g.,
by a Moloney-based retrovirus; and the DNA is expressed in bone
marrow cells and/or peripheral blood cells of the recipient at
least 14, preferably 30, more preferably 60, and most preferably
120 days, after the DNA is introduced into the recipient.
[0123] In other preferred embodiments: the method further includes,
prior to hematopoietic stem cell transplantation, introducing into
the recipient an antibody capable of binding to mature T cells of
said recipient mammal.
[0124] In preferred embodiments the graft is a liver or a
kidney.
[0125] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, creating hematopoietic
space, e.g., by irradiating the recipient mammal with low dose,
e.g., between about 100 and 400 rads, whole body irradiation to
deplete or partially deplete the bone marrow of the recipient;
inactivating thymic T cells by one or more of: prior to
hematopoietic stem cell transplantation, irradiating the recipient
mammal with, e.g., about 700 rads of thymic irradiation, or
administering to the recipient a short course of an
immunosuppressant, as is described herein.
[0126] Other preferred embodiments include: the step of, prior to
implantation of a graft, depleting natural antibodies from the
blood of the recipient mammal, e.g., by hemoperfusing an organ,
e.g., a liver or a kidney, obtained from a mammal of the second
species. (In organ hemoperfusion antibodies in the blood bind to
antigens on the cell surfaces of the organ and are thus removed
from the blood.)
[0127] Methods of inducing tolerance with a short-term
administration of a help reducing agent, e.g., a short high dose
course of cyclosporine A (CsA), can be combined with other methods
for inducing tolerance, e.g., methods of inducing tolerance which
use the implantation of donor stem cells to induce tolerance to an
antigen, e.g., the methods described in U.S. Ser. No. 07/838,595,
filed Feb. 19, 1992.
[0128] Accordingly, in another aspect, the invention features a
method of inducing tolerance in a recipient mammal of a first
species, e.g., a primate, e.g., a human, to a graft obtained from a
mammal of a second, preferably discordant species, e.g., a swine,
e.g., a miniature swine, or a discordant primate species. The
method includes: preferably prior to or simultaneous with
transplantation of the graft, introducing, e.g., by intravenous
injection, into the recipient mammal, hematopoietic stem cells,
e.g., bone marrow cells or fetal liver or spleen cells, of the
second species (preferably the hematopoietic stem cells home to a
site in the recipient mammal); (optionally) inactivating the
natural killer cells of the recipient mammal, e.g., by prior to
introducing the hematopoietic stem cells into the recipient mammal,
introducing into the recipient mammal an antibody capable of
binding to natural killer cells of said recipient mammal;
preferably, implanting the graft in the recipient; and, preferably,
administering to the recipient a short course of help reducing
treatment, e.g., a short course of high dose cyclosporine. The
short course of help reducing treatment is generally administered
at the time at the graft is introduced into the recipient.
[0129] In preferred embodiments, the short course of help reducing
treatment induces tolerance to unmatched class I and/or minor
antigens on the graft which is introduced into the recipient.
[0130] In preferred embodiments, the duration of the short course
of help reducing treatment is approximately equal to or is less
than the period required for mature T cells of the recipient
species to initiate rejection of an antigen after first being
stimulated by the antigen; in more preferred embodiments, the
duration is approximately equal to or is less than two, three,
four, five, or ten times, the period required for a mature T cell
of the recipient species to initiate rejection of an antigen after
first being stimulated by the antigen.
[0131] In other preferred embodiments, the short course of help
reducing treatment is administered in the absence of a treatment
which stimulates the release of a cytokine by mature T cells in the
recipient, e.g., in the absence of a steroid drug in a sufficient
concentration to counteract the desired effect of the help reducing
treatment, e.g., in the absence of Prednisone
(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentration
which stimulates the release of a cytokine by mature T cells in the
recipient. In preferred embodiments, the short course of help
reducing treatment is administered in the absence of a steroid
drug, e.g., in the absence of Prednisone.
[0132] In preferred embodiments: the help reducing treatment is
begun before or at about the time the graft is introduced; or the
short course is perioperative, the short course is
postoperative.
[0133] As will be explained in more detail below, the hematopoietic
cells prepare the recipient for the graft that follows, by inducing
tolerance at both the B-cell and T-cell levels. Preferably,
hematopoietic cells are fetal liver or spleen, or bone marrow
cells, including immature cells (i.e., undifferentiated
hematopoietic stem cells; these desired cells can be separated out
of the bone marrow prior to administration), or a complex bone
marrow sample including such cells can be used.
[0134] One source of anti-NK antibody is anti-human thymocyte
polyclonal anti-serum. As is discussed below, preferably, a second
anti-mature T cell antibody can be administered as well, which
lyses T cells as well as NK cells. Lysing T cells is advantageous
for both bone marrow and xenograft survival. Anti-T cell antibodies
are present, along with anti-NK antibodies, in anti-thymocyte
anti-serum. Repeated doses of anti-NK or anti-T cell antibody may
be preferable. Monoclonal preparations can be used in the methods
of the invention.
[0135] Other preferred embodiments include: the step of introducing
into the recipient mammal, donor species-specific stromal tissue,
preferably hematopoietic stromal tissue, e.g., fetal liver or
thymus. In preferred embodiments: the stromal tissue is introduced
simultaneously with, or prior to, the hematopoietic stem cells; the
hematopoietic stem cells are introduced simultaneously with, or
prior to, the antibody.
[0136] Other preferred embodiments include those in which: the same
mammal of the second species is the donor of one or both the graft
and the hematopoietic cells; and the antibody is an anti-human
thymocyte polyclonal anti-serum, obtained, e.g., from a horse or
pig.
[0137] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, creating hematopoietic
space, e.g., by irradiating the recipient mammal with low dose,
e.g., between about 100 and 400 rads, whole body irradiation to
deplete or partially deplete the bone marrow of the recipient;
inactivating thymic T cells by one or more of: prior to
hematopoietic stem cell transplantation, irradiating the recipient
mammal with, e.g., about 700 rads of thymic irradiation, or
administering to the recipient a short course of an
immunosuppressant, as is described herein.
[0138] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, depleting natural
antibodies from the blood of the recipient mammal, e.g., by
hemoperfusing an organ, e.g., a liver or a kidney, obtained from a
mammal of the second species. (In organ hemoperfusion antibodies in
the blood bind to antigens on the cell surfaces of the organ and
are thus removed from the blood.)
[0139] In other preferred embodiments: the method further includes,
prior to hematopoietic stem cell transplantation, introducing into
the recipient an antibody capable of binding to mature T cells of
said recipient mammal.
[0140] In other preferred embodiments: the method further includes
inactivating T cells of the recipient, e.g., by, prior to
introducing the hematopoietic stem cells into the recipient,
introducing into the recipient an antibody capable of binding to T
cells of the recipient.
[0141] In preferred embodiments, the method includes the step of
introducing into the recipient a graft obtained from the donor
which is obtained from a different organ than the hematopoietic
stem cells, e.g., a liver or a kidney.
[0142] In another aspect, the invention features a method of
inducing tolerance in a recipient mammal, preferably a primate,
e.g., a human, to a graft obtained from a donor, e.g., of the same
species. The method includes: preferably prior to or simultaneous
with transplantation of the graft, introducing, e.g., by
intravenous injection, into the recipient, hematopoietic stem
cells, e.g., bone marrow cells or fetal liver or spleen cells, of a
mammal, preferably the donor (preferably the hematopoietic stem
cells home to a site in the recipient); (optionally), inactivating
T cells of the recipient, e.g., by, prior to introducing the
hematopoietic stem cells into the recipient, introducing into the
recipient an antibody capable of binding to T cells of the
recipient; preferably, implanting the graft in the recipient; and,
preferably, administering to the recipient a short course of help
reducing treatment, e.g., a short course of high dose cyclosporine.
The short course of help reducing treatment is generally
administered at the time the graft is introduced into the
recipient.
[0143] In preferred embodiments, the short course of help reducing
treatment induces tolerance to unmatched class I and minor antigens
on the graft which is introduced into the recipient.
[0144] In preferred embodiments, the duration of the short course
of help reducing treatment is approximately equal to or is less
than the period required for mature T cells of the recipient
species to initiate rejection of an antigen after first being
stimulated by the antigen; in more preferred embodiments, the
duration is approximately equal to or is less than two, three,
four, five, or ten times the period required for a mature T cell of
the recipient species to initiate rejection of an antigen after
first being stimulated by the antigen.
[0145] In other preferred embodiments, the short course of help
reducing treatment is administered in the absence of a treatment
which stimulates the release of a cytokine by mature T cells in the
recipient, e.g., in the absence of a steroid drug in a sufficient
concentration to counteract the desired effect of the help reducing
treatment, e.g., in the absence of Prednisone
(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentration
which stimulates the release of a cytokine by mature T cells in the
recipient. In preferred embodiments, the short course of help
reducing treatment is administered in the absence of a steroid
drug, e.g., in the absence of Prednisone
[0146] In preferred embodiments: the help reducing treatment is
begun before or at about the time the graft is introduced; the
short course is perioperative, the short course is postoperative;
the donor and recipient are class I matched.
[0147] In preferred embodiments, the hematopoietic stem cells are
introduced simultaneously with, or prior to administration of the
antibody; the antibody is an antihuman thymocyte polyclonal
anti-serum; and the anti-serum is obtained from a horse or pig.
[0148] Other preferred embodiments include: the further step of,
prior to hematopoietic stem cell transplantation, inactivating or
depleting NK cells of the recipient, e.g., by introducing into the
recipient mammal an antibody capable of binding to NK cells of the
recipient mammal; and those in which the same individual is the
donor of both the graft and the bone marrow.
[0149] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, creating hematopoietic
space, e.g., by irradiating the recipient mammal with low dose,
e.g., between about 100 and 400 rads, whole body irradiation to
deplete or partially deplete the bone marrow of the recipient;
inactivating thymic T cells by one or more of, prior to
hematopoietic stem cell transplantation, irradiating the recipient
mammal with, e.g., about 700 rads of thymic irradiation, or
administering to the recipient a short course of an
immunosuppressant, as is described herein.
[0150] Other preferred embodiments include: the further step of,
prior to bone marrow transplantation, adsorbing natural antibodies
from the blood of the recipient by hemoperfusing an organ, e.g.,
the liver, or a kidney, obtained from the donor.
[0151] Preferred embodiments include: the step of introducing into
the recipient mammal, donor species specific stromal tissue,
preferably hematopoietic stromal tissue, e.g., fetal liver or
thymus.
[0152] In preferred embodiments, the method includes the step of
introducing into the recipient, a graft which is obtained from a
different organ than the hematopoietic stem cells, e.g., a liver or
a kidney.
[0153] Methods of inducing tolerance with short-term administration
of a help reducing agent, e.g., a short high dose course of
cyclosporine A (CsA), can be combined with yet other methods for
inducing tolerance, e.g., with: methods which use the implantation
of a xenogeneic thymic graft to induce tolerance, e.g., the methods
described in U.S. Ser. No. 08/163, 912 filed on Dec. 7, 1993;
methods of increasing the level of the activity of a tolerance
promoting or GVHD inhibiting cytokine or decreasing the level of
activity of a tolerance inhibiting or GVHD promoting cytokine,
e.g., the methods described in U.S. Ser. No. 08/114,072, filed Aug.
30, 1993; methods of using cord blood cells to induce tolerance,
e.g., the methods described in U.S. Ser. No. 08/150,739 filed Nov.
10, 1993; and the methods for inducing tolerance disclosed in Sykes
and Sachs, PCT/US94/01616, filed Feb. 14, 1994.
[0154] It has also been discovered that a short course of an
immunosuppressant, e.g., cyclosporine, can be used to diminish or
inhibit T cell activity which would otherwise promote the rejection
of an allograft or xenograft.
[0155] Accordingly, in another aspect, the invention features a
method of diminishing or inhibiting T cell activity, preferably the
activity of thymic or lymph node T cells, in a recipient mammal,
e.g., a primate, e.g., a human, which receives a graft from a donor
mammal. The method includes, inducing tolerance to the graft;
administering to the recipient a short course of an
immunosuppressive agent, e.g., cyclosporine, sufficient to
inactivate T cells, preferably thymic or lymph node T cells; and
preferably transplanting the graft into the recipient.
[0156] Tolerance to the graft can be induced by any method, e.g.,
by any of the methods discussed herein. For example, tolerance can
be induced by the administration of donor allogeneic or xenogeneic
hematopoietic stem cells, the administration of genetically
engineered autologous stem cells, by the administration of a short
course of a help reducing agent, or by altering the immunological
properties of the graft, e.g., by masking, cleaving, or otherwise
modifying cell surface molecules of the graft.
[0157] In preferred embodiments the duration of the short course of
immunosuppressive agent is: approximately equal to 30 days;
approximately equal to or less than 8-12 days, preferably about 10
days; approximately equal to or less than two, three, four, five,
or ten times the 8-12 or 10 day period.
[0158] In preferred embodiments: the short course is begun before
or at about the time the treatment to induce tolerance is begun,
e.g., at about the time, xenogeneic, allogeneic, genetically
engineered syngeneic, or genetically engineered autologous stem
cells are introduced into the recipient; the short course begins on
the day the treatment to induce tolerance is begun, e.g., on the
day, xenogeneic, allogeneic, genetically engineered syngeneic, or
genetically engineered autologous stem cells are introduced into
the recipient; the short course begins within 1, 2, 4, 6, 8, or 10
days before or after the treatment to induce tolerance is begun,
e.g., within 1, 2, 4, 6, 8, or 10 days before or after xenogeneic,
allogeneic, genetically engineered syngeneic, or genetically
engineered autologous stem cells are introduced into the
recipient.
[0159] In other preferred embodiments: the short course of an
immunosuppressive is administered in conjunction with an anti-T
cell antibody; the short course of an immunosuppressive is
sufficient to inactivate T cells, e.g., thymic or lymph node T
cells, which would not be inactivated by antibody-based
inactivation of T cells, e.g., inactivation by intravenous
administrations of ATG antibody, or similar, preparations.
[0160] In preferred embodiments: the recipient mammal is other than
a mouse or rat.
[0161] Methods of inactivating T cells, preferably thymic or lymph
node T cells, of the invention can be combined with methods of
inducing tolerance in which the inactivation of T cells is
desirable. The anti-T cell methods of the invention can be used in
place of, or in addition to, methods for the inactivation of T
cells called for, or useful in such methods of inducing tolerance.
For example, anti-thymic or lymph node T cell methods of the
invention can be used with methods for the implantation of
transduced bone marrow cells to induce tolerance to an antigen,
e.g., the methods described in U.S. Ser. No. 008/126,122, filed on
Sep. 23, 1993.
[0162] Accordingly, in another aspect, the invention features a
method of promoting, in a recipient mammal of a first species, the
acceptance of a graft from a donor mammal of a second species,
which graft, preferably, expresses a major histocompatibility
complex (MHC) antigen. The method includes inserting DNA encoding
an MHC antigen of the second species into a hematopoietic stem
cell, e.g., a bone marrow hematopoietic stem cell, of the recipient
mammal; allowing the MHC antigen encoding DNA to be expressed in
the recipient; and, preferably, administering to the recipient a
short course of an immunosuppressive agent, e.g., a short course of
cyclosporine treatment, sufficient to inactivate recipient T cells,
preferably thymic or lymph node T cells. (Thymic or lymph node T
cells might otherwise inhibit the survival of the graft or
engineered cells.)
[0163] In preferred embodiments, the duration of the short course
of immunosuppressive agent is: approximately equal to 30 days;
approximately equal to or less than 8-12 days, preferably about 10
days; approximately equal to or less than two, three, four, five,
or ten times the 8-12 or 10 day period.
[0164] In preferred embodiments: the recipient mammal is a primate,
e.g., a human, and the donor mammal is a swine, e.g., a miniature
swine.
[0165] In preferred embodiments: the short course is begun before
or at about the time genetically engineered stem cells are
introduced into the recipient; the short course begins on the day
the genetically engineered stem cells are introduced into the
recipient; the short course begins within 1, 2, 4, 6, 8, or 10 days
before or after the genetically engineered stem cells are
introduced into the recipient.
[0166] In other preferred embodiments: the short course of an
immunosuppressive agent is administered in conjunction with an
anti-T cell antibody; the short course of immunosuppressive is
sufficient to inactivate T cells, e.g., thymic or lymph node T
cells, which would not be inactivated by antibody-based
inactivation of T cells, e.g., inactivation by intravenous
administrations of ATG, or similar, antibody preparations.
[0167] Preferred embodiments include those in which: the cell is
removed from the recipient mammal prior to the DNA insertion and
returned to the recipient mammal after the DNA insertion; the DNA
is obtained from the individual mammal from which the graft is
obtained; the DNA is obtained from an individual mammal which is
syngeneic with the individual mammal from which the graft is
obtained; the DNA is obtained from an individual mammal which is
MHC matched, and preferably identical, with the individual mammal
from which the graft is obtained; the DNA includes an MHC class I
gene; the DNA includes an MHC class II gene; the DNA is inserted
into the cell by transduction, e.g., by a retrovirus, e.g., by a
Moloney-based retrovirus; and the DNA is expressed in bone marrow
cells and/or peripheral blood cells of the recipient for at least
14, preferably 30, more preferably 60, and most preferably 120
days, after the DNA is introduced into the recipient.
[0168] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell implantation, creating hematopoietic space
in the recipient so as to promote engraftment and survival of the
implanted stem cells, e.g., by irradiating the recipient mammal
with low dose, e.g., between about 100 and 400 rads, whole body
irradiation to deplete or partially deplete the bone marrow of the
recipient.
[0169] In preferred embodiments, the method further includes the
administration of thymic irradiation to the recipient, e.g., 700
rads of thymic irradiation.
[0170] Other preferred embodiments include: the step of depleting
natural antibodies from the blood of the recipient mammal, e.g., by
hemoperfusing an organ, e.g., a liver or a kidney, obtained from a
mammal of the second species. (In organ hemoperfusion antibodies in
the blood bind to antigens on the cell surfaces of the organ and
are thus removed from the blood.)
[0171] Other preferred embodiments further include the step of
introducing into the recipient a graft obtained from the donor,
e.g., a liver or a kidney.
[0172] In another aspect, the invention features a method of
promoting, in a recipient mammal, preferably a primate, e.g., a
human, acceptance of a graft obtained from a donor of the same
species, which graft expresses an MHC antigen. The method includes:
inserting DNA encoding an MHC antigen of the donor into a
hematopoietic stem cell, e.g., a bone marrow hematopoietic stem
cell, of the recipient; allowing the MHC antigen encoding DNA to be
expressed in the recipient; and, preferably, administering to the
recipient a short course of an immunosuppressive agent, e.g., a
short course of cyclosporine treatment, sufficient to inactivate
recipient T cells, preferably thymic or lymph node T cells. (Thymic
or lymph node T cells might otherwise inhibit the survival of the
graft or engineered cells.)
[0173] In preferred embodiments, the duration of the short course
of immunosuppressive agent is: approximately equal to 30 days;
approximately equal to or less than 8-12 days, preferably about 10
days; approximately equal to or less than two, three, four, five,
or ten times the 8-12 or 10 day period.
[0174] In preferred embodiments: the short course is begun before
or at about the time genetically engineered stem cells are
introduced into the recipient; the short course begins on the day
the genetically engineered stem cells are introduced into the
recipient; the short course begins within 1, 2, 4, 6, 8, or 10 days
before or after the genetically engineered stem cells are
introduced into the recipient.
[0175] In other preferred embodiments: the short course of an
immunosuppressive agent is administered in conjunction with an
anti-T cell antibody; the short course of immunosuppressive is
sufficient to inactivate T cells, e.g., thymic or lymph node T
cells, which would not be inactivated by antibody-based
inactivation of T cells, e.g., inactivation by intravenous
administrations of ATG, or similar, antibody preparations.
[0176] Preferred embodiments include those in which: the cell is
removed from the recipient prior to the DNA insertion and returned
to the recipient after the DNA insertion; the DNA includes a MHC
class I gene; the DNA includes a MHC class II gene; the DNA is
inserted into the cell by transduction, e.g. by a retrovirus, e.g.,
by a Moloney-based retrovirus; and the DNA is expressed in bone
marrow cells and/or peripheral blood cells of the recipient at
least 14, preferably 30, more preferably 60, and most preferably
120 days, after the DNA is introduced into the recipient.
[0177] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell implantation, creating hematopoietic space
in the recipient so as to promote engraftment and survival of the
implanted stem cells, e.g., by irradiating the recipient mammal
with low dose, e.g., between about 100 and 400 rads, whole body
irradiation to deplete or partially deplete the bone marrow of the
recipient.
[0178] In preferred embodiments, the method further includes the
administration of thymic irradiation to the recipient, e.g., 700
rads of thymic irradiation.
[0179] Other preferred embodiments include: the step of depleting
natural antibodies from the blood of the recipient mammal, e.g., by
hemoperfusing an organ, e.g., a liver or a kidney, obtained from a
mammal of the second species. (In organ hemoperfusion antibodies in
the blood bind to antigens on the cell surfaces of the organ and
are thus removed from the blood.)
[0180] Other preferred embodiments further include the step of
introducing into the recipient a graft obtained from the donor,
e.g., a liver or a kidney.
[0181] Methods of inactivating T cells, preferably thymic or lymph
node T cells, of the invention can be combined with methods of
inducing tolerance which use the implantation of donor stem cells
to induce tolerance to an antigen, e.g., the methods described in
U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, hereby incorporated
by reference.
[0182] Accordingly, in another aspect, the invention features a
method of promoting, in a recipient mammal of a first species,
e.g., a primate, e.g., a human, acceptance of a graft obtained from
a mammal of a second, preferably discordant species, e.g., a swine,
e.g., a miniature swine, or a discordant primate species. The
method includes: introducing, e.g., by intravenous injection, into
the recipient mammal, hematopoietic stem cells, e.g., bone marrow
cells or fetal liver or spleen cells, of the second species
(preferably the hematopoietic stem cells home to a site in the
recipient mammal); (optionally) inactivating natural killer cells
of the recipient mammal, e.g., by, prior to introducing the
hematopoietic stem cells into the recipient mammal, introducing
into the recipient mammal an antibody capable of binding to natural
killer cells of said recipient mammal; (optionally) inactivating T
cells of the recipient mammal, e.g., by, prior to introducing the
hematopoietic stem cells into the recipient mammal, introducing
into the recipient mammal an antibody capable of binding to T cells
of the recipient mammal; and, preferably, administering to the
recipient a short course of an immunosuppressive agent, e.g., a
short course of cyclosporine treatment, sufficient to inactivate
recipient T cells, preferably thymic or lymph node T cells. (Thymic
or lymph node T cells might otherwise inhibit the engraftment or
survival of the engineered cells.)
[0183] In preferred embodiments, the duration of the short course
of immunosuppressive agent is: approximately equal to 30 days;
approximately equal to or less than 8-12 days, preferably about 10
days; approximately equal to or less than two, three, four, five,
or ten times the 8-12 or 10 day period mentioned above.
[0184] In preferred embodiments: the short course is begun before
or at about the time stem cells are introduced into the recipient;
the short course begins on the day the stem cells are introduced
into the recipient; the short course begins within 1, 2, 4, 6, 8,
or 10 days before or after the stem cells are introduced into the
recipient.
[0185] In other preferred embodiments: the short course of an
immunosuppressive agent is administered in conjunction with one or
both of an anti-T cell antibody, or thymic irradiation, e.g., 700
rads of thymic irradiation; the short course of immunosuppressive
is sufficient to inactivate T cells, e.g., thymic or lymph node T
cells, which would not be inactivated by antibody-based
inactivation of T cells, e.g., inactivation by intravenous
administrations of ATG antibody preparations.
[0186] As will be explained in more detail below, the hematopoietic
cells prepare the recipient for the graft that follows, by inducing
tolerance at both the B-cell and T-cell levels. Preferably,
hematopoietic cells are fetal liver or spleen, or bone marrow
cells, including immature cells (i.e., undifferentiated
hematopoietic stem cells; these desired cells can be separated out
of the bone marrow prior to administration), or a complex bone
marrow sample including such cells can be used.
[0187] One source of anti-NK antibody is anti-human thymocyte
polyclonal anti-serum. As is discussed below, preferably, a second
anti-mature T cell antibody can be administered as well, which
lyses T cells as well as NK cells. Lysing T cells is advantageous
for both bone marrow and xenograft survival. Anti-T cell antibodies
are present, along with anti-NK antibodies, in anti-thymocyte
anti-serum. Repeated doses of anti-NK or anti-T cell antibody may
be preferable. Monoclonal preparations can be used in the methods
of the invention.
[0188] Other preferred embodiments include: the step of introducing
into the recipient mammal, donor species-specific stromal tissue,
preferably hematopoietic stromal tissue, e.g., fetal liver or
thymus. In preferred embodiments: the stromal tissue is introduced
simultaneously with, or prior to, the hematopoietic stem cells; the
hematopoietic stem cells are introduced simultaneously with, or
prior to, an anti-NK or T cell antibody.
[0189] Other preferred embodiments include those in which: the same
mammal of the second species is the donor of one or both the graft
and the hematopoietic cells; and the anti-T or anti-NK cell
antibody is an anti-human thymocyte polyclonal anti-serum,
obtained, e.g., from a horse or pig.
[0190] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, creating hematopoietic
space in the recipient so as to promote engraftment and survival of
the implanted stem cells, e.g., by irradiating the recipient mammal
with low dose, e.g., between about 100 and 400 rads, whole body
irradiation to deplete or partially deplete the bone marrow of the
recipient.
[0191] In preferred embodiments, the method further includes the
administration of thymic irradiation to the recipient, e.g., 300 to
700 rads of thymic irradiation.
[0192] Other preferred embodiments include: the step of prior to
hematopoietic stem cell transplantation, depleting natural
antibodies from the blood of the recipient mammal, e.g., by
hemoperfusing an organ, e.g., a liver or a kidney, obtained from a
mammal of the second species. (In organ hemoperfusion antibodies in
the blood bind to antigens on the cell surfaces of the organ and
are thus removed from the blood.)
[0193] Other preferred embodiments further include the step of
introducing into the recipient a graft obtained from the donor,
e.g., a graft which is obtained from a different organ than the
hematopoietic stem cells, e.g., a liver or a kidney.
[0194] In preferred embodiments the stem cells are introduced into
the recipient prior to or simultaneous with transplantation of the
graft.
[0195] In another aspect, the invention features a method of
promoting, in a recipient mammal, preferably a primate, e.g., a
human, acceptance of a graft obtained from a donor of the same
species. The method includes: introducing, e.g., by intravenous
injection into the recipient, hematopoietic stem cells, e.g. bone
marrow cells or fetal liver or spleen cells, of a mammal,
preferably the donor (preferably the hematopoietic stem cells home
to a site in the recipient); (optionally) inactivating T cells of
the recipient, e.g., by, prior to introducing the hematopoietic
stem cells into the recipient, introducing into the recipient an
antibody capable of binding to T cells of the recipient; and,
preferably, administering to the recipient a short course of an
immunosuppressive agent, e.g., a short course of cyclosporine
treatment, sufficient to inactivate recipient T cells, preferably
thymic or lymph node T cells. (Thymic or lymph node T cells might
otherwise inhibit the engraftment or survival of the engineered
cells.)
[0196] In preferred embodiments, the duration of the short course
of immunosuppressive agent is: approximately equal to 30 days;
approximately equal to or less than 8-12 days, preferably about 10
days; approximately equal to or less than two, three, four, five,
or ten times the 8-12 or 10 day period mentioned above.
[0197] In preferred embodiments: the short course is begun before
or at about the time stem cells are introduced into the recipient;
the short course begins on the day the stem cells are introduced
into the recipient; the short course begins within 1, 2, 4, 6, 8,
or 10 days before or after the stem cells are introduced into the
recipient.
[0198] In other preferred embodiments: the short course of an
immunosuppressive agent is administered in conjunction with one or
both of an anti-T cell antibody, or thymic irradiation, e.g., 700
rads of thymic irradiation; the short course of immunosuppressive
is sufficient to inactivate T cells, e.g., thymic or lymph node T
cells, which would not be inactivated by antibody-based
inactivation of T cells, e.g., inactivation by intravenous
administrations of ATG antibody preparations.
[0199] In preferred embodiments, the anti-T cell or NK cell
antibody is an antihuman thymocyte polyclonal anti-serum; and the
anti-serum is obtained from a horse or pig.
[0200] Other preferred embodiments include: the further step of,
prior to hematopoietic stem cell transplantation, inactivating
recipient NK cells, e.g., by introducing into the recipient mammal
an antibody capable of binding to NK cells of the recipient mammal;
and those in which the same individual is the donor of both the
graft and the bone marrow.
[0201] Other preferred embodiments include: the step of, prior to
hematopoietic stem cell transplantation, creating hematopoietic
space in the recipient so as to promote engraftment and survival of
the implanted stem cells, e.g., by irradiating the recipient mammal
with low dose, e.g., between about 100 and 400 rads, whole body
irradiation to deplete or partially deplete the bone marrow of the
recipient.
[0202] In preferred embodiments the method further includes
administering thymic irradiation to the recipient, e.g., 700 rads
of thymic irradiation.
[0203] Other preferred embodiments include: the further step of,
prior to bone marrow transplantation, adsorbing natural antibodies
from the blood of the recipient by hemoperfusing an organ, e.g.,
the liver, or a kidney, obtained from the donor.
[0204] Preferred embodiments include: the step of introducing into
the recipient mammal, donor species specific stromal tissue,
preferably hematopoietic stromal tissue, e.g., fetal liver or
thymus.
[0205] Other preferred embodiments further include the step of
introducing into the recipient, a graft obtained from the donor,
e.g., a graft which is obtained from a different organ than the
hematopoietic stem cells, e.g., a liver or a kidney.
[0206] In preferred embodiments, the stem cells are introduced into
the recipient prior to or simultaneous with transplantation of the
graft.
[0207] Methods of inactivating T cells, preferably thymic or lymph
node T cells, of the invention can be used with yet other methods
of inducing tolerance in which the inactivation of thymic or lymph
node T cells is desirable. For example, anti-thymic or lymph node T
cell methods of the invention can be used with: methods which use
the implantation of a xenogeneic thymic graft to induce tolerance,
e.g., the methods described in U.S. Ser. No. 08/163, 912 filed on
Dec. 7, 1993; methods of increasing the level of the activity of a
tolerance promoting or GVHD inhibiting cytokine or decreasing the
level of activity of a tolerance inhibiting or GVHD promoting
cytokine, e.g., the methods described in U.S. Ser. No. 08/114,072,
filed Aug. 30, 1993; methods of using cord blood cells to induce
tolerance, e.g., the methods described in U.S. Ser. No. 08/150,739;
and the methods for inducing tolerance disclosed in Sykes and
Sachs, PCT/US94/01616, filed Feb. 14, 1994.
[0208] "Thymus-function deficient", as used herein, refers to a
condition in which the ability of an individual's thymus to support
the maturation of T cells is impaired as compared with a normal
individual. Thymus deficient conditions include those in which the
thymus or thymus function is essentially absent.
[0209] "Tolerance", as used herein, refers to the inhibition of a
graft recipient's ability to mount an immune response, e.g., to a
donor antigen, which would otherwise occur, e.g., in response to
the introduction of a non self MHC antigen into the recipient.
Tolerance can involve humoral, cellular, or both humoral and
cellular responses. The concept of tolerance includes both complete
and partial tolerance. In other words, as used herein, tolerance
include any degree of inhibition of a graft recipient's ability to
mount an immune response, e.g., to a donor antigen.
[0210] "A discordant species combination", as used herein, refers
to two species in which hyperacute rejection occurs when vascular
organs are grafted. Generally, discordant species are from
different orders, while non-discordant species are from the same
order. For example, rats and mice are non-discordant species, i.e.
their MHC antigens are substantially similar, and they are members
of the same order, rodentia.
[0211] "Hematopoietic stem cell", as used herein, refers to a cell
that is capable of developing into mature myeloid and/or lymphoid
cells. Preferably, a hematopoietic stem cell is capable of the
long-term repopulation of the myeloid and/or lymphoid lineages.
Stem cells derived from the cord blood of the recipient or the
donor can be used in methods of the invention. See U.S. Pat. No.
5,192,553, hereby incorporated by reference, and U.S. Pat. No.
5,004,681, hereby incorporated by reference.
[0212] "Miniature swine", as used herein, refers to completely or
partially inbred miniature swine.
[0213] "Graft", as used herein, refers to a body part, organ,
tissue, cells, or portions thereof
[0214] "Stromal tissue", as used herein, refers to the supporting
tissue or matrix of an organ, as distinguished from its functional
elements or parenchyma.
[0215] Restoring, inducing, or promoting immunocompetence, as used
herein, means one or both of: (1) increasing the number of mature
functional T cells in the recipient (over what would be seen in the
absence of treatment with a method of the invention) by either or
both, increasing the number of recipient-mature functional T cells
or by providing mature functional donor-T cells, which have matured
in the recipient; or (2) improving the immune-responsiveness of the
recipient, e.g., as is measured by the ability to mount a skin
response to a recall antigen, or improving the responsiveness of a
of a T cell of the recipient, e.g., as measured by an in vitro
test, e.g., by the improvement of a proliferative response to an
antigen, e.g., the response to tetanus antigen or to an
alloantigen.
[0216] A mature functional T cell, as used herein, is a T cell (of
recipient or donor origin) which responds to microbial antigens and
tolerant to recipient and donor tissue.
[0217] "Lymph node or thymic T cell", as used herein, refers to T
cells which are resistant to inactivation by traditional methods of
T cell inactivation, e.g., inactivation by a single intravenous
administration of anti-T cell antibodies, e.g., anti-bodies, e.g.,
ATG preparation.
[0218] Restoring or inducing the thymus-dependent ability for T
cell progenitors to mature into mature T cells, as used herein,
means either or both, increasing the number of functional mature T
cells of recipient origin in a recipient, or providing mature
functional donor T cells to a recipient, by providing donor thymic
tissue in which T cells can mature. The increase can be partial,
e.g., an increase which does not bring the level of mature
functional T cells up to a level which results in an essentially
normal immune response or partial, e.g., an increase which falls
short of bringing the recipient's level of mature functional T
cells up to a level which results in an essentially normal immune
response.
[0219] Methods of the invention will allow the induction of
immunocompetence in patients suffering from an immunodeficiency,
e.g., a T cell deficiency, e.g., a thymic based immunodeficiency,
e.g., a congenital immunodeficiency due to thymic aplasia or
dysfunction, an acquired immune disorder, e.g., AIDS,
immunoincompetence resulting form a neoplastic disease, or
immunoincompetence resulting from a medical procedure, e.g.,
chemotherapy or radiation treatment. An acquired immune deficiency
is one which is due primarily to other than genetic defects.
[0220] At risk for AIDS, as used herein, refers to being HIV
positive or having AIDS.
[0221] "An immunosuppressive agent capable of inactivating thymic
or lymph node T cells", as used herein, is an agent, e.g., a
chemical agent, e.g., a drug, which, when administered at an
appropriate dosage, results in the inactivation of thymic or lymph
node T cells. Examples of such agents are cyclosporine, FK-506, and
rapamycin. Anti-T cell antibodies, because they are comparatively
less effective at inactivating thymic or lymph node T cells, are
not preferred for use as agents. An agent should be administered in
sufficient dose to result in significant inactivation of thymic or
lymph node T cells which are not inactivated by administration of
an anti-T cell antibody, e.g., an anti-ATG preparation. Putative
agents, and useful concentrations thereof, can be prescreened by in
vitro or in vivo tests, e.g., by administering the putative agent
to a test animal, removing a sample of thymus or lymph node tissue,
and testing for the presence of active T cells in an in vitro or in
vivo assay. Such prescreened putative agents can then be further
tested in transplant assays.
[0222] "Short course of a immunosuppressive agent", as used herein,
means a transitory non-chronic course of treatment. The treatment
should begin before or at about the time the treatment to induce
tolerance is begun, e.g., at about the time, xenogeneic,
allogeneic, genetically engineered syngeneic, or genetically
engineered autologous stem cells are introduced into the recipient.
e.g., the short course can begin on the day the treatment to induce
tolerance is begun, e.g., on the day, xenogeneic, allogeneic,
genetically engineered syngeneic, or genetically engineered
autologous stem cells are introduced into the recipient or the
short course can begin within 1, 2, 4, 6, 8, or 10 days before or
after the treatment to induce tolerance is begun, e.g., within 1,
2, 4, 6, 8, or 10 days before or after xenogeneic, allogeneic,
genetically engineered syngeneic, or genetically engineered
autologous stem cells are introduced into the recipient. The short
course can last for: a period equal to or less than about 8-12
days, preferably about 10 days, or a time which is approximately
equal to or is less than two, three, four, five, or ten times the
8-12 or 10 day period. Optimally, the short course lasts about 30
days. The dosage should be sufficient to maintain a blood level
sufficient to inactivate thymic or lymph node T cells. A dosage of
approximately 15 mg/kg/day has been found to be effective in
primates.
[0223] "Help reduction", as used herein, means the reduction of T
cell help by the inhibition of the release of at least one
cytokine, e.g., any of IL-2, IL-4, IL-6, gamma interferon, or TNF,
from T cells of the recipient at the time of the first exposure to
an antigen to which tolerance is desired. The inhibition induced in
a recipient's T cell secretion of a cytokine must be sufficient
such that the recipient is tolerized to an antigen which is
administered during the reduction of help. Although not being bound
by theory, it is believed that the level of reduction is one which
substantially eliminates the initial burst of IL-2 which
accompanies the first recognition of a foreign antigen but which
does not eliminate all mature T cells, which cells may be important
in educating and producing tolerance.
[0224] "A help reducing agent", as used herein, is an agent, e.g.,
an immunosuppressive drug, which results in the reduction of
cytokine release. Examples of help reducing agents are
cyclosporine, FK-506, and rapamycin. Anti-T cell antibodies,
because they can eliminate T cells, are not preferred for use as
help reducing agents. A help reducing agent must be administered in
sufficient dose to give the level of inhibition of cytokine release
which will result in tolerance. The help reducing agent should be
administered in the absence of treatments which promote cytokine,
e.g., IL-2, release. Putative agents help reducing agents can be
prescreened by in vitro or in vivo tests, e.g., by contacting the
putative agent with T cells and determining the ability of the
treated T cells to release a cytokine, e.g., IL-2. The inhibition
of cytokine release is indicative of the putative agent's efficacy
as a help reducing agent. Such prescreened putative agents can then
be further tested in a kidney transplant assay. In a kidney
transplant assay a putative help reducing agent is tested for
efficacy by administering the putative agent to a recipient monkey
and then implanting a kidney from a class II matched class I and
minor antigen mismatched donor monkey into the recipient. Tolerance
to the donor kidney (as indicated by prolonged acceptance of the
graft) is indicative that the putative agent is, at the dosage
tested, a help reducing agent.
[0225] "Short course of a help reducing agent", as used herein,
means a transitory non-chronic course of treatment. The treatment
should begin before or at about the time of transplantation of the
graft. Alternatively, the treatment can begin before or at about
the time of the recipient's first exposure to donor antigens.
Optimally, the treatment lasts for a time which is approximately
equal to or less than the period required for mature T cells of the
recipient species to initiate rejection of an antigen after first
being stimulated by the antigen. The duration of the treatment can
be extended to a time approximately equal to or less than two,
three, four, five, or ten times, the period required for a mature T
cell of the recipient species to initiate rejection of an antigen
after first being stimulated by the antigen. The duration will
usually be at least equal to the time required for mature T cells
of the recipient species to initiate rejection of an antigen after
first being stimulated by the antigen. In pigs and monkeys, about
12 days of treatment is sufficient. Experiments with cyclosporine A
(10 mg/kg) in pigs show that 6 days is not sufficient. Other
experiments in monkeys show that IL-2 administered on day 8, 9, or
10 of cyclosporine A treatment will result in rejection of the
transplanted tissue. Thus, 8, 9, or 10 days is probably not
sufficient in pigs. In monkeys, a dose of 10 mg/kg cyclosporine
with a blood level of about 500-1,000 ng/ml is sufficient to induce
tolerance to class II matched class I and minor antigen mismatched
kidneys. The same blood level, 500-1,000 ng/ml, is sufficient to
induce tolerance in pigs. Long-term administration of 5 mg/kg
prevents rejection (by long term immune suppression) but does not
result in tolerance.
[0226] The help suppressing methods of the invention avoid the
undesirable side effects of long-term or chronic administration of
the broad spectrum immune suppressants often used in
transplantation. Long-term or chronic administration of drugs such
as Prednisone, Imuran, CyA, and, most recently FK506, have all had
an important impact on the field of transplantation. However, all
of these drugs cause nonspecific suppression of the immune system
which must be titrated sufficiently to avoid rejection while not
completely eliminating immune function. Patients who must stay on
chronic immunosuppressive therapy for the remainder of their lives
face major complications arising from too much or too little
immunosuppression, causing infection and rejection, respectively.
The help suppressing methods of the invention are based on the
administration of a transitory short term high dose course of a
help reducing treatment.
[0227] "MHC antigen", as used herein, refers to a protein product
of one or more MHC genes; the term includes fragments or analogs of
products of MHC genes which can evoke an immune response in a
recipient organism. Examples of MHC antigens include the products
(and fragments or analogs thereof) of the human MHC genes, i.e.,
the HLA genes. MHC antigens in swine, e.g., miniature swine,
include the products (and fragments and analogs thereof) of the SLA
genes, e.g., the DRB gene.
[0228] Recipient thymic or lymph node T cells are responsible for
significant resistance to implanted grafts, e.g., to transplanted
hematopoietic cells or transplanted organs. It has been found that
the usual methods of T cell depletion or inactivation, e.g., the
administration of anti-T cell antibodies, often fall short of an
optimum level of T cell depletion or inactivation. In particular,
such methods fail to provide optimum levels of depletion or
inactivation of thymic or lymph node T cells. Methods of the
invention in which a short course of an immunosuppressant, e.g.,
cyclosporine, capable of inactivating recipient thymic or lymph
node T cells is administered to the recipient, result in more
thorough inactivation of thymic or lymph node T cells and thus in
improved acceptance of graft tissue.
[0229] The retroviral methods of the invention allow the
reconstitution of a graft recipient's bone marrow with transgenic
autologous bone marrow cells expressing allogeneic or xenogeneic
MHC genes. Expression of the transgenic MHC genes confers tolerance
to grafts which exhibit the products of these or closely related
MHC genes. Thus, these methods provide for the induction of
specific transplantation tolerance by somatic transfer of MHC
genes. Retroviral methods of the invention avoid the undesirable
side effects of broad spectrum immune suppressants which are often
used in transplantation.
[0230] Tolerance to transplantation antigens can be achieved
through induction of lymphohematopoietic chimerism by bone marrow
transplantation (BMT). BMT across MHC barriers presents two major
risks: if mature T cells are not removed from the marrow inoculum
the recipient may develop severe graft versus host disease (GVHD);
removal of these cells often leads to failure of engraftment.
Retroviral methods of the invention, which induce specific
tolerance by reconstitution of the recipient's bone marrow with
autologous (as opposed to allogeneic or heterologous) bone marrow
cells, allow tolerance to be conferred with minimal risk of GVHD
and with minimal need to remove T cells from the marrow
inoculum.
[0231] Retroviral methods of the invention can be combined with the
help suppression and T cell inactivation methods of the invention
to prolong graft acceptance.
[0232] Hematopoietic cell transplant methods of the invention avoid
the undesirable side effects of broad spectrum immune suppressants
which are often used in transplantation. Hematopoietic cell
transplant methods of the invention can be combined with the help
suppression and T cell elimination methods of the invention to
prolong graft acceptance.
[0233] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0234] FIG. 1A is a graph showing growth of fetal pig THY/LIV graft
vs. time after transplantation in the presence of mature mouse T
cells in the periphery.
[0235] FIG. 1B is a dot plot analysis of live peripheral white
blood cells of a representative animal 16 weeks post-transplant.
Upper left quadrant 0.5%; upper right quadrant 12.1%.
[0236] FIG. 2 is a graph of mouse anti-pig mixed lymphocyte
reactions (MLR's) performed to determine whether or not mouse T
cells which matured in pig thymus grafts were tolerant to pig
antigens.
[0237] FIG. 3 is a diagram of the GS4.5 retroviral construct.
[0238] FIG. 4 is a diagram of the GS4.5 proviral genome and the
expected transcripts.
[0239] FIGS. 5a and 5b are representations of flow cytometry
profile of transduced cells.
[0240] FIG. 6 is a diagram of the transduction assay.
[0241] FIG. 7 is a diagram of genetic maps of the C57BL/10,
B10.AKM, and B10.MBR strains.
[0242] FIG. 8 is a diagram of the FACS profile of spleen cells from
a recipient of transduced bone marrow.
[0243] FIGS. 9a and 9b are graphs of survival versus time in skin
graft experiments.
[0244] FIGS. 10a-d are diagrams of FACS analysis of thymocytes from
graft rejecters, and controls.
[0245] FIG. 11 is a diagram of the N2-B19-H2b vector.
DETAILED DESCRIPTION
Maturation of Host T Cells in a Xenogeneic Thymus and Induction of
Tolerance to a Xenograft by Xenogeneic Thymic Tissue
[0246] The following procedure was designed to promote the
acceptance of a xenograft thymus by a host and thusly to either or
both: 1, lengthen the time an implanted organ (a xenograft)
survives in a xenogeneic host prior to rejection; and 2, provide
xenogeneic thymic tissue in which host T cells can mature.
[0247] In the case of an organ transplant, the organ can be any
organ, e.g., a liver, e.g., a kidney, e.g., a heart. The two main
strategies are elimination of natural antibodies and
transplantation of thymic tissue to induce tolerance.
[0248] Preparation of the recipient for either organ
transplantation or thymus replacement includes any or all of the
following steps. Preferably they are carried out in the following
sequence.
[0249] First, a preparation of horse anti-human thymocyte globulin
(ATG) is intravenously injected into the recipient. The antibody
preparation eliminates mature T cells and natural killer cells. If
not eliminated, mature T cells might promote rejection of both the
thymic transplant and, after sensitization, the xenograft organ.
The ATG preparation also eliminates natural killer (NK) cells. NK
cells probably have no effect on an implanted organ, but might act
immediately to reject the newly introduced thymic tissue.
Anti-human ATG obtained from any mammalian host can also be used,
e.g., ATG produced in pigs, although thus far preparations of pig
ATG have been of lower titer than horse-derived ATG. ATG is
superior to anti-NK monoclonal antibodies, as the latter are
generally not lytic to all host NK cells, while the polyclonal
mixture in ATG is capable of lysing all host NK cells. Anti-NK
monoclonal antibodies can, however, be used. In a relatively
severely immunocompromised individual this step may not be
necessary. As host (or donor) T cells mature in the xenogeneic
thymus they will be tolerant of the xenogeneic thymic tissue.
Alternatively, as the host immune system is progressively restored,
it may be desirable to treat the host to induce tolerance to the
xenogeneic thymic tissue.
[0250] Optimally, the recipient can be thymectomized. In
thymectomized recipients, recipient T cells do not have an
opportunity to differentiate in the recipient thymus, but must
differentiate in the donor thymus. In some cases it may be
necessary to splenectomize the recipient in order to avoid
anemia.
[0251] Second, the recipient can be administered low dose
radiation. Although this step is thought to be beneficial in bone
marrow transplantation (by creating hematopoietic space for newly
injected bone marrow cells), it is of less importance in thymic
grafts which are not accompanied by bone marrow transplantation.
However, a sublethal dose e.g., a dose about equal to 100, or more
than 100 and less than about 400, rads, whole body radiation, plus
700 rads of local thymic radiation, can be used.
[0252] Third, natural antibodies can be adsorbed from the
recipient's blood. (This is of more importance in organ grafts but
can be used in thymus replacement procedures as well.) Antibody
removal can be accomplished by exposing the recipient's blood to
donor or donor species antigens, e.g., by hemoperfusion of a liver
of the donor species to adsorb recipient-natural antibodies.
Pre-formed natural antibodies (nAb) are the primary agents of graft
rejection. Natural antibodies bind to xenogeneic endothelial cells
and are primarily of the IgM class. These antibodies are
independent of any known previous exposure to antigens of the
xenogeneic donor. B cells that produce these natural antibodies
tend to be T cell-independent, and are normally tolerized to self
antigen by exposure to these antigens during development. The
mechanism by which newly developing B cells are tolerized is
unknown. The liver is a more effective adsorber of natural
antibodies than the kidney. Again, this step may not be required,
at least initially, in a relatively severely immunocompromised
patient.
[0253] Donor thymic tissue, preferably fetal or neonatal thymic
tissue is implanted in the recipient. Fetal or neonatal liver or
spleen tissue can be included.
[0254] While any of these procedures may aid the survival of
implanted thymic tissue or another xenogeneic organ, best results
are achieved when all steps are used in combination.
[0255] Methods of the invention can be used to confer tolerance to
allogeneic grafts, e.g., wherein both the graft donor and the
recipient are humans, and to xenogeneic grafts, e.g., wherein the
graft donor is a nonhuman animal, e.g., a swine, e.g., a miniature
swine, and the graft recipient is a primate, e.g., a human.
[0256] The donor of the implant and the individual that supplies
the tolerance-inducing thymic graft should be the same individual
or should be as closely related as possible. For example, it is
preferable to derive implant tissue from a colony of donors that is
highly or completely inbred. The donor of the organ used for
perfusion need not be closely related to the donor of the implant
or thymic tissue.
Xenograft Thymic Tissue Transplantation: Detailed Protocol
[0257] Immunocompetent C57BL/10 (B10) mice were used to test the
ability of pig thymus to induce specific tolerance to discordant
pig antigens. B10 mice were treated with a non-myeloablative
conditioning regimen which has previously shown to permit induction
of tolerance to rat xeno-antigens in mice, see e.g., Sharabi et
al., 1990, J. Exp. Med. 172:195-202. Euthymic or thymectomized
(ATX) mice received depleting doses of anti-T cell and anti-NK cell
mAbs, 7 Gy mediastinal irradiation and 3 Gy whole body irradiation
(WBI), and then received fetal swine thymus/liver (THY/LIV)
transplants under the kidney capsule followed by administration of
10.sup.8 fetal liver cells (FLC) i.p. Mice either received no
further anti-T cell and anti-NK cell mAb treatments after 0 to 6
weeks post-tx, or were maintained on chronic mAb treatment for the
duration of the experiment.
[0258] Swine THY/LIV grafts grew initially in treated euthymic
mice, but stopped growing after T cell and natural killer (NK)
cell-depleting monoclonal antibodies (mAbs) were discontinued, and
these mice developed anti-pig IgG response. When euthymic mice were
maintained on chronic mAb treatment, the grafts enlarged markedly
and no anti-pig IgG response was observed. Pig thymopoiesis was
supported for at least 32 weeks during chronic mAb administration,
although no pig T cells were detected in the periphery by flow
cytometry (FCM). Percentages of intra-graft CD4.sup.+/CD8.sup.-,
CD4.sup.-/CD8.sup.+, CD4.sup.+/CD8.sup.+, and CD4.sup.-/CD8.sup.-
pig thymocyte subsets were similar to those in normal pig
thymus.
[0259] In contrast, swine THY/LIV grafts grew markedly in adult
thymectomized mice (ATX-THY/LIV) which received only a short (less
than 6 weeks) course of mAb treatment post-transplant (post-tx).
FCM analysis of peripheral WBC in these mice 6 weeks after
discontinuing mAb treatment revealed the presence of mature
(.alpha..beta.-TCR.sup.hi) mouse T cells. Unlike T cells in
euthymic grafted mice, these cells were tolerant to pig antigens,
as evidenced by the growth of swine THY/LIV grafts, (FIG. 1), and
the absence of anti-pig IgG antibody responses. The majority (more
than 90%) of the .alpha..beta.-TCR.sup.hi T cells were
CD4.sup.+/CD8.sup.-. FMC analyses 13 to 26 weeks post-tx
demonstrated normal mouse thymocyte subsets in swine thymi. For
example, 7.9% CD4.sup.+/CD8.sup.-, 2.9% CD4.sup.-/CD8.sup.+, 85.5%
CD4.sup.+/CD8.sup.+, 3.7% CD4.sup.-/CD8.sup.- and 11.6%
.alpha..beta.-TCR.sup.hi thymocytes were found in a swine THY/LIV
graft by FCM 17 weeks post-tx compared to 3.5% CD4.sup.+/CD8.sup.-,
3.2% CD4.sup.-/CD8.sup.+, 87.8% CD4.sup.+/CD8.sup.+, 5.5%
CD4.sup.-/CD8.sup.- and 10.0% .alpha..beta.-TCR.sup.hi thymocytes
in a normal B10 thymus. Fetal swine liver grafted without a thymus
fragment did not grow in control nAb-treated ATX-B10 mice and
.alpha..beta.-TCR.sup.hi T cells did not appear in the periphery.
Thus, the pig thymus was required for the development of mature
mouse T cells.
[0260] Mouse anti-pig mixed lymphocyte reactions (MLR's) were
performed to determine whether or not mouse T cells which matured
in pig thymus grafts were tolerant to pig antigens. ATX-THY/LIV B10
mice (H-2.sup.b) mounted no anti-B10 or anti-pig responses, but
demonstrated normal allo-responses against a fully MHC-mismatched
allogeneic stimulator, B10.BR (H-2.sup.k) (FIG. 2).
[0261] In order to determine if host bone marrow-derived cells were
participating in negative selection of the developing mouse
thymocytes, fetal pig THY/LIV grafts were transplanted into both
I-E.sup.+ (BALB/c nude) and I-E.sup.- (ATX B10) recipients.
I-E.sup.+ mice delete V.sub..beta.11 T cells because of
presentation in the thymus of an endogenous superantigen in
association with I-E, whereas I-E.sup.- mice do not delete this T
cell family. The percentages of V.sub..beta.11 T cells were
therefore compared between I-E.sup.+ and I-E.sup.- recipients of
fetal pig thymus grafts in which murine T cells developed in pig
thymi. ATX B10 recipients were treated as described above. BALB/c
nude mice were depleted of NK cells and irradiated with 3 Gy WBI
prior to transplant. These mice also developed large numbers of
mature CD4.sup.+ T cells that migrated to the periphery. Complete
deletion of V.sub..beta.11 T cells was observed in the periphery of
BALB/c nude recipients of fetal swine thymus grafts (Table I),
indicating that mouse I-E also participated in negative selection
of mouse T cells developing in pig thymi. Negative selection is
most likely carried out by murine Ia.sup.+dendritic cells which
were detected predominantly in the cortico-medullary junction of
swine thymus grafts by immunoperoxidase staining. In the ATX B10
recipients of swine THY/LIV grafts, reduction in the percentage of
V.sub..beta.11 T cells was observed compared to normal B10 mice
(mean 2.8% of T cells.+-.0.8 S.D., normal B10 5.2%, p<0.005)
suggesting that the pig SLA DR class II, which shares significant
homology with mouse I-E class II, may participate in negative
selection of mouse T cells developing in the pig thymus graft
(Table I). TABLE-US-00001 TABLE I N Strain Thy/Liv Graft %
V.sub..beta.8.1/8.2 % V.sub..beta.11 4 Normal C57BL/10 - 16.3 .+-.
2.2 5.2 .+-. 0.5 4 Normal BALB/c - 20.8 .+-. 0.3 0.2 .+-. 0.1 4
C57BL/10 + 16.7 .+-. 3.0 2.8 .+-. 0.8 5 BALB/c nude + 20.0 .+-. 3.7
0.4 .+-. 0.3
Table I. Clonal deletion in mice transplanted with swine THY/LIV
grafts. B10 recipients were treated as described below. Normal
percentages of T cells staining with V.sub..beta.8.1/8.2
demonstrates normal positive selection in grafted mice of a
V.sub..beta.T cell family which is not deleted in I-E.sup.+ or
I-E.sup.- mice. BALB/c nude mice were depleted of NK cells using
rabbit anti-Asialo-Gm1 serum, and were given 3 Gy WBI, and fetal
swine THY/LIV grafts implants under the kidney capsule, followed by
injection of 10.sup.8 FLC i.p. on Day 0. ACK-lysed splenocytes or
peripheral white blood cells (red blood cells were removed by
hypotonic shock) were collected 13 to 19 weeks post-tx and analyzed
by FCM for V.sub..beta.11 T cell deletion. Murine F.sub.cR's were
blocked using rat anti-mouse F.sub.cR mAb, 2.4G2, and then cells
were stained with either fluoresceinated hamster anti-mouse
V.sub..beta.8.1/8.2 TCR (Pharmingen) or rat anti-mouse
V.sub..beta.11 TCR (Pharmingen) (green fluorescence) followed by
phycoerythrin-conjugated rat anti-mouse CD4 and CD8 mAbs
(Pharmingen) (orange fluorescence) and analyzed by two color FCM as
described below. Fluoresceinated murine mAb HOPCI, with no known
reactivity to mouse cells, or rat anti-mouse IgG1 (Zymed
Laboratories, Inc.) was used as the negative control mAb in the
green fluorescence. Phycoerythrin-conjugated mAb Leu4
(Becton-Dickinson), was used as the negative control mAb in the
orange fluorescence. Approximately 5,000 gated CD4.sup.+ and
CD8.sup.+ cells were usually collected for analysis of V.sub..beta.
families. Non-viable cells were excluded using the vital nucleic
acid stain, propidium iodide. Percentages of positive cells were
determined as described below. Results are presented as the
mean.+-.SD of results obtained for individual mice. p
value<0.005 for % V.sub..beta.11 in normal B10 mice compared to
THY/LIV-grafted B10 mice. p value is >0.20 for % V.sub..beta.11
in normal BALB/c mnice compared to THY/LIV-grafted BALB/c nude
mice.
[0262] These studies demonstrate that discordant xenogeneic thymic
stroma is capable of supporting mouse thymopoiesis and that
CD4.sup.+/CD8.sup.-/.alpha..beta.-TCR.sup.hi T cells which are
released into the periphery are phenotypically normal, functional
and tolerant to donor xeno-antigens, and to host antigens. The lack
of CD4.sup.-/CD8.sup.+/.alpha..beta.-TCR.sup.hi repopulation in the
periphery may be due to failure of mouse CD8 to interact with pig
MHC class I molecules, as has been demonstrated for mouse
anti-human responses, thereby preventing positive selection of
CD8.sup.+ thymocytes by swine thymic epithelium. Since human
CD8.sup.+ T cells are able to interact with pig MHC class I
directly, human CD8.sup.+ T cells should mature effectively in
swine fetal thymus grafts.
[0263] Presumably, tolerance to pig antigens is not induced in
euthymic mice which receive swine THY/LIV grafts because mouse T
cell progenitors mature in the host thymus, which lacks the pig
cells necessary to tolerize developing mouse thymocytes. The
non-myeloablative conditioning regimen used in this study permits
engraftment of rat marrow and induction of donor-specific tolerance
in murine recipients. Tolerance is thought to be induced in this
model by rat dendritic cells detected at the cortico-medullary
junction of the thymus of chimeric animals. In the present study,
failure of pig hematopoietic stem cells, present in the FLC
suspension administered on Day 0, to migrate to the mouse thymus
may be due to failure of homing and differentiation, possibly
reflecting species specificity of cytokines and adhesion molecules.
In ATX recipients, on the other hand, mouse T cell progenitors home
to the pig thymus graft and are tolerized pig antigens. No mouse TE
is present in ATX hosts, but mouse dendritic cells are detectable
in THY/LIV grafts and probably mediate the observed clonal deletion
of cells reactive to host antigen. Although the decreased
percentage of V.sub..beta.11 T cells in ATX B10 recipients of swine
THY/LIV grafts suggests clonal deletion by swine cells, it is
possible that there is a defect in the positive selection of this
V.sub..beta. family on swine thymic stroma. However, the normal
percentages of V.sub..beta.8.1/8.2 T cells in both ATX B10 and
BALB/c nude recipients of swine THY/LIV grafts compared to those in
normal B10 and BALB/c mice suggests that no defect in positive
selection is present. The observed tolerance could be explained if
the swine thymic stroma either clonally deletes or anergizes
developing mouse thymocytes reactive to donor xeno-antigens.
[0264] Survival of swine THY/LIV grafts in euthymic and
thymectomized B10 mice depleted of T cells and NK cells, was
determined as follows. 6-12 week old euthymic or ATX C57BL/10 (B10)
mice received i.p. injections of mAbs GK1.5 (anti-mouse CD4), 2.43
(anti-mouse CD8), 30-H12 (anti-mouse Thy1.2) and PK136 (anti-mouse
NK1.1) in depleting doses, as described in Sharabi et al., on days
-6 and -1 prior to transplantation. On either day -1 or day 0, 7 Gy
localized thymic irradiation and 3 Gy whole body irradiation were
administered to recipients, and second trimester (gestational day
36-72) fetal thymic and liver fragments, approximately 1 mm.sup.3
in size, were transplanted under the kidney capsule via a midline
laparotomy incision. (Thymic irradiation was not found to be
necessary for mouse T cells to mature in pig thymus grafts in
subsequent experiments, and was therefore eliminated from the
conditioning regimen.) After the abdomen was closed in two layers,
10.sup.8 fetal liver cells (FLC) in suspension were injected i.p.
Recipients were treated on a weekly basis post-tx with depleting
doses of the same four mAbs for a period of 0-6 weeks. No
difference in murine CD4.sup.+ T cell reconstitution or tolerance
to pig antigens was observed in mice which were treated with no mAb
post-tx compared to those which received 6 weeks mAb treatment
post-tx. Some groups of control mice were maintained on chronic mAb
treatment until the time of sacrifice.
[0265] As described above, an increase in fetal pig THY/LIV graft
size was observed upon exploratory laparotomy performed at 5 and 19
weeks post-tx, despite the presence of mature
CD4.sup.+/.alpha..beta.TCR.sup.hi T cells in the peripheral blood
(shown 16 weeks after mAbs were discontinued). Growth of fetal pig
THY/LIV graft in the presence of mature mouse T cells in the
periphery was studied as follows. Peripheral WBC contained 12.1%
CD4.sup.+/.alpha..beta.-TCR.sup.hi T cells and 0.5%
CD8.sup.+/.alpha..beta.-TRC.sup.+ T cells. Control ATX mice which
received fetal swine liver grafts without a thymus fragment did not
maintain their grafts, and developed less than 5%
.alpha..beta.-TRC.sup.+ T cells in the periphery. ATX mice were
conditioned as described above. Exploratory laparotomies were
performed at 5-6 and 15-19 weeks post-transplant to measure graft
size. Mice were tail bled at regular intervals post-tx to obtain
peripheral WBC which were prepared by hypotonic shock to remove red
blood cells. Cells were stained with a fluoresceinated rat
anti-mouse CD4 mAb (Pharmigen) (green fluorescence) versus
biotinylated hamster anti-mouse .alpha..beta.TRC mAb (Pharmigen)
plus phycoerythrin streptavidin (orange fluorescence) and analyzed
by two-color flow cytometry (FCM) using either a FACScan or FACSort
flow cytometer (Becton-Dickinson). Murine mAb HOPC1, with no known
reactivity to pig or mouse cells, was used as the negative control
mAb in both the green and orange fluorescence. Percentages of
positive cells were determined by subtracting the percentage of
cells staining with the control mAb HOPC1 from the percentage of
cells staining with the anti-mouse mAbs. A dot plot analysis of
live peripheral white blood cells of a representative animal 16
weeks post-tx (16 weeks after mAbs were discontinued) is shown in
FIG. 2. Overall, 57% (27/47) of ATX mice treated with this protocol
maintained swine grafts and reconstituted their CD4.sup.+ T cell
compartment. In recent experiments this result was achieved in 90%
(9/10) of mice treated with this regimen.
[0266] As described above, ATX-THY/LIV B10 (H-2.sup.b) mice
demonstrated specific unresponsiveness to pig antigens while
maintaining normal allo-responsiveness to a fully MHC-mismatched
B10.BR (H-2.sup.k) stimulator. Specific unresponsiveness of B10
mice transplanted with fetal pig THY/LIV grafts to pig antigens in
mixed lymphocyte reaction (MLR) was determined as follows. Control
ATX-B10 mice which received a swine liver graft without a thymus
fragment (ATX-LIV) mounted no responses to any stimulator,
demonstrating the importance of the pig thymus graft in the
development of functional mouse T cells. Positive control anti-pig
MLR was from a mouse immunized with a swine skin graft, since mice
do not mount primary anti-pig responses. Sterile splenocyte
suspensions from normal B10 (right diagonal bar), normal B10
grafted with GG (SLA-I.sup.c/SLA-II.sup.d) pig skin 12 weeks
earlier (GG'-B10 solid bar), normal B10BR (crosshatched bar), and
thymectomized B10 mice conditioned with the non-myeloablative
regimen described above and transplanted with either a fetal pig
(SLA-I.sup.d/SLA-II.sup.d) THY/LIV graft (ATX-THY/LIV stippled
bar), or a fetal pig liver graft only (ATX-LIV left diagonal bar)
were ACK-lysed, washed and reconstituted in RPMI medium
supplemented with 15% CPSR-2 (controlled processed serum
replacement, Sigma), 4% nutrient mixture (L-glutamine, nonessential
amino acids, sodium pyruvate and penicillin/streptomycin), 1% HEPES
buffer and 10.sup.-5M 2-me. Swine PBL were prepared by
centrifugation over a Ficoll-Hypaque layer. 4.times.10.sup.5
responders were incubated with either 4.times.10.sup.5 murine
stimulators (3 Gy) or 1.times.10.sup.5 swine stimulators (3 Gy) in
a total volume of 0.2 ml of media at 37 .quadrature.C for 4 days in
5% CO.sub.2. Cultures were pulsed with 1 .mu.Ci .sup.3H on the
third day, harvested on the fourth day with a Tomtec automated
harvester and counted on a Pharmacia LKB liquid scintillation
counter. MLR's for all mice tested (N=3) were set up in duplicate
and pulsed on Days 3 and 4 and harvested on Days 4 and 5 with
similar results.
[0267] Thus, if murine T cells are permitted to develop in a mouse
thymus, they are not tolerized to pig antigens, and they reject pig
thymus/liver grafts. (Thus, if the recipient has significant thymic
function thymectomy is indicated.) If mouse T cells are continually
depleted by mAb, swine thymopoiesis occurs in the swine
thymus/liver graft. If a mouse is thymectomized but not chronically
treated with anti-T cell mAb's, mouse thymopoiesis occurs in the
pig thymus, and these cells are tolerized to pig antigens.
Host T Cells which Mature in a Xenogeneic Thymus are Functional
[0268] B10.BR (full MHC mismatch to B10) and C3H.SW (minor antigen
mismatch only) skin grafts (1 mouse) were rejected by mouse T cells
which had matured in a pig thymic graft, thus demonstrating their
immunocompetence and ability to recognize minor antigens in a host
MHC-restricted fashion. The grafts were full thickness tail skin
grafts on the upper thorax with a skin bridge separating them.
These results show that swine fetal thymic tissue can be used
clinically to induce a state of specific xenograft tolerance while
ensuring immunocompetence in thymectomized recipients.
Alternative Preparative Regimens
[0269] As is stated above, the depletion of NK cells, whole body
irradiation, and thymic irradiation, can in some cases be dispensed
with. As is shown by the experiments summarized below, inactivation
or depletion of CD4.sup.+ cells, e.g., by the administration of an
anti-CD4 antibody, is sufficient to allow growth of xenogeneic
thymic tissue and maturation of host T cells in the xenogeneic
thymic tissue. (The antibodies needed may differ depending on the
species combination. E.g., in the case of a human recipient and a
pig donor, because human CD8 will likely interact with pig class I
molecules, it may also be necessary to administer anti-CD8
antibodies.)
[0270] As is shown by the data in Table II below, graft growth and
host T cell development (as measured by the presence of peripheral
T cells 9 and 10 weeks post THY/LIV transplant) was seen in ATX
mice treated with anti-CD4 antibodies and whole body irradiation.
B10 mice received 3 Gy whole body irradiation and fetal pig THY/LIV
graft and 10.sup.8 fetal liver cells in experiments essentially
similar to those described in the previous section except that
anti-CD4, anti-THY1.2, and anti NK cell antibodies were not
administered. TABLE-US-00002 TABLE II THY/LIV graft growth and T
cell maturation does not require extensive antibody treatment.
GRAFT % CD4.sup.+/% CD8.sup.+ Animal # TREATMENT L/R T CELLS (WBC)
639/40 NO ATX/.alpha.CD4/8/ 17.7/4.6 THY1.2/NK1.1 643/44 NO
ATX/.alpha.CD4/8/ 9.4/2.6 THY1.2/NK1.1 601/02 ATX +/ +/++ 11.1/0.8
.alpha.CD4/8/THY1.2/NK1.1 603/04 ATX +/ +/+ 1.7/1.2
.alpha.CD4/8/THY1.2/NK1.1 605/06 ATX +/ -/- 0.6/0.3
.alpha.CD4/8/THY1.2/NK1.1 607/08 ATX +/ ++/++ 0.9/0.6
.alpha.CD4/8/THY1.2/NK1.1 609/10 ATX +/ ++/+ 1.8/0.5
.alpha.CD4/8/THY1.2/NK1.1 611/12 ATX +/ ++/++ 3.4/1.6
.alpha.CD4/8/THY1.2/NK1.1 615/16 ATX + .alpha.CD4/8 +/++ 8.9/0.7
617/18 '' +/- 2.1/0.3 619/20 '' ++/++ 20.2/1.5 621/22 '' +/-
1.1/0.1 623/24 '' ++/++ 6.8/0.3 625/26 ATX + .alpha.CD4 ++/-
7.2/4.0 627/28 '' -/+ 1.0/5.1 629/30 '' ++/++ 10.1/3.7 631/32 ''
++/+ 3.7/3.5 633/34 '' -/- 0.4/3.7 635/36 '' ++/++ 28.0/4.2 ATX =
thymectomy; ++ = large, bulky graft with vascularization; + =
moderate sized graft; - = poor graft (thin, poor tissue);
.alpha.CD4/8/THY1.2/NK1.1 indicates the administration of the
described antibody.
[0271] As is shown by the data in Table III below, graft growth was
seen in ATX mice treated with monoclonal antibodies but given no
irradiation. In these experiments, B10 mice were given anti-CD4,
CD8, THY1.2, and NK1.1 monoclonal antibodies. TABLE-US-00003 TABLE
III THY/LIV graft growth does not require host irradiation. GRAFT
Animal # TREATMENT L/R 560/61 ATX + mAb's + 3 Gy WBI ++/++ 562/63
'' +/++ 564/65 '' -/- 566/67 '' -/+ 574/75 '' -/- 576/77 '' -/-
578/79 '' -/- 582/83 '' +/- 552/53 ATX + mAb's (no WBI) +/- 554/55
'' ++/- 556/57 '' ++/+ 568/69 '' ++/++ 570/71 '' ++/++
[0272] As is shown by the data in Table IV below, host T cell
development (as measured by the presence of peripheral T cells 7
weeks post THY/LIV transplant) was seen in ATX mice given no
irradiation and treated only with anti-CD4 antibodies.
TABLE-US-00004 TABLE IV Rapid T cell recovery in THY/LIV graft
recipients with thymectomy and anti CD4 antibodies alone. Mean %
(SD) peripheral blood Number of T cells at 7 weeks animals in
post-transplant group TREATMENT CD4.sup.+ CD8.sup.+ 3 NO ATX 12.9
(1.7) 2.7 (0.1) .alpha.CD4/8/THY1.2/NK1.1 + 3 Gy WBI 8 ATX 3.4
(2.4) 0.7 (0.5) .alpha.CD4/8/THY1.2/NK1.1 + 3 Gy WBI 7 ATX 19.5
(3.4) 7.1 (1.8) .alpha.CD4/
Xenogeneic Thymic Tissue and Stem Cell Transplantation
[0273] The following procedure introduces donor thymic tissue and
donor stem cells into the recipient and thus can be used to restore
or induce immune function or to lengthen the time an implanted
organ (a xenograft) survives in a xenogeneic host prior to
rejection.
[0274] In the case of an organ graft, the organ can be any organ,
e.g., a liver, e.g., a kidney, e.g., a heart. The two main
strategies are elimination of natural antibodies by organ
perfusion, and transplantation of tolerance-inducing bone
marrow.
[0275] Preparation of the recipient includes any or all of the
following steps. Preferably they are carried out in the following
sequence.
[0276] Elimination of NK and T cells. First, a preparation of horse
anti-human thymocyte globulin (ATG) is intravenously injected into
the recipient. The antibody preparation eliminates mature T cells
and natural killer cells. If not eliminated, mature T cells would
promote rejection of both the bone marrow transplant and, after
sensitization, the xenograft itself of equal importance, the ATG
preparation also eliminates natural killer (NK) cells. NK cells
probably have no effect on the implanted organ, but would act
immediately to reject the newly introduced bone marrow. Anti-human
ATG obtained from any mama host can also be used, e.g., ATG
produced in pigs, although thus far preparations of pig ATG have
been of lower titer than horse-derived ATG. ATG is superior to
anti-NK monoclonal antibodies, as the latter are generally not
lytic to all host NK cells, while the polyclonal mixture in ATG is
capable of lysing all host NK cells. Anti-NK monoclonal antibodies
can, however, be used.
[0277] Thymic tissue transplant. In cases where the procedure is to
restore or induce immunocompetence donor thymic tissue (preferably
fetal or neonatal thymic tissue) is implanted in the recipient so
that donor T cells (and recipient T cells if the are present and
functional) can mature. Fetal or neonatal liver or spleen tissue
can be implanted with the thymic tissue.
[0278] The presence of donor antigen in the thymus during the time
when host T cells are regenerating post-transplant is critical for
tolerizing host T cells. If donor hematopoietic stem cells are not
able to become established in the host thymus and induce tolerance
before host T cells regenerate repeated doses of anti-recipient T
cell antibodies may be necessary throughout the non-myeloablative
regimen. Continuous depletion of host T cells may be required for
several weeks. Alternatively, e.g., if this approach is not
successful, and tolerance (as measured by donor skin graft
acceptance, specific cellular hyporesponsiveness in vitro, and
humoral tolerance) is not induced in these animals, the approach
can be modified to include host thymectomy. In thymectomized
recipients, host T cells do not have an opportunity to
differentiate in a host thymus, but must differentiate in the donor
thymus. Immunocompetence can be measured by the ability to reject a
non-donor type allogeneic donor skin graft, and to survive in a
pathogen-containing environment.
[0279] It may also be necessary or desirable to splenectomize the
recipient in order to avoid anemia.
[0280] Creation of hematopoietic space. The recipient is
administered low dose radiation in order to make room for newly
injected bone marrow cells. A sublethal dose e.g., a dose about
equal to 100, or more than 100 and less than about 400, rads, whole
body radiation, plus 700 rads of local thymic radiation, has been
found effective for this purpose.
[0281] Natural antibody elimination. Natural antibodies are
adsorbed from the recipient's blood by hemoperfusion of a liver of
the donor species. Pre-formed natural antibodies-(nAb) are the
primary agents of graft rejection. Natural antibodies bind to
xenogeneic endothelial cells and are primarily of the IgM class.
These antibodies are independent of any known previous exposure to
antigens of the xenogeneic donor. B cells that produce these
natural antibodies tend to be T cell-independent, and are normally
tolerized to self antigen by exposure to these antigens during
development. The mechanism by which newly developing B cells are
tolerized is unknown. The liver is a more effective adsorber of
natural antibodies than the kidney.
[0282] Implantation of donor stromal tissue. The next step in the
non-myeloablative procedure is to implant donor stromal tissue,
preferably obtained from fetal liver, thymus, and/or fetal spleen,
into the recipient, preferably in the kidney capsule. Stem cell
engraftment and hematopoiesis across disparate species barriers is
enhanced by providing a hematopoietic stromal environment from the
donor species. The stromal matrix supplies species-specific factors
that are required for interactions between hematopoietic cells and
their stromal environment, such as hematopoietic growth factors,
adhesion molecules, and their ligands.
[0283] Each organ includes an organ specific stromal matrix that
can support differentiation of the respective undifferentiated stem
cells implanted into the host. Although adult thymus may be used,
fetal tissue obtained sufficiently early in gestation is preferred
because it is free from mature T lymphocytes which can cause GVHD.
Fetal tissues also tend to survive better than adult tissues when
transplanted. As an added precaution against GVHD, thymic stromal
tissue can be irradiated prior to transplantation, e.g., irradiated
at 1000 rads. As an alternative or an adjunct to implantation,
fetal liver cells can be administered in fluid suspension.
[0284] Finally, bone marrow cells (BMC), or another source of
hematopoietic stem cells, e.g., a fetal liver suspension, or cord
blood stem cells, of the donor are injected into the recipient.
Donor stem cells home to appropriate sites of the recipient and
grow contiguously with remaining host cells and proliferate,
forming a chimeric lymphohematopoietic population. By this process,
newly forming B cells (and the antibodies they produce) are exposed
to donor antigens, so that the transplant will be recognized as
self. Tolerance to the donor is also observed at the T cell level
in animals in which hematopoietic stem cell, e.g., BMC, engraftment
has been achieved. When an organ graft is placed in such a
recipient several months after bone marrow chimerism has been
induced, natural antibody against the donor will have disappeared,
and the graft should be accepted by both the humoral and the
cellular arms of the immune system. This approach has the added
advantage of permitting organ transplantation to be performed
sufficiently long following transplant of hematopoietic cells,
e.g., BMT, e.g., a fetal liver suspension, that normal health and
immunocompetence will have been restored at the time of organ
transplantation. The use of xenogeneic donors allows the
possibility of using bone marrow cells and organs from the same
animal, or from genetically matched animals. As liver is the major
site of hematopoiesis in the fetus, fetal liver can also serve as
an alternative to bone marrow as a source of hematopoietic stem
cells.
[0285] While any of these procedures may aid the survival of an
implanted organ, best results are achieved when all steps are used
in combination. Methods of the invention can be used to confer
tolerance to allogeneic grafts, e.g., wherein both the graft donor
and the recipient are humans, and to xenogeneic grafts, e.g.,
wherein the graft donor is a nonhuman animal, e.g., a swine, e.g.,
a miniature swine, and the graft recipient is a primate, e.g., a
human.
[0286] In the case of xenogeneic grafts, the donor of the implant
and the individual that supplies either the tolerance-inducing
hematopoietic cells or the liver to be perfused should be the same
individual or should be as closely related as possible. For
example, it is preferable to derive implant tissue from a colony of
donors that is highly or completely inbred.
Detailed Protocol
[0287] In the following protocol for preparing a cynomolgus monkey
for receipt of a kidney from a miniature swine donor, zero time is
defined as the moment that the arterial and venous cannulas of the
recipient are connected to the liver to be perfused.
[0288] On day -1 a commercial preparation (Upjohn) of horse
anti-human anti-thymocyte globulin (ATG) is injected into the
recipient. ATG eliminates mature T cells and natural killer cells
that would otherwise cause rejection of the bone marrow cells used
to induce tolerance. The recipient is anesthetized, an IV catheter
is inserted into the recipient, and 6 ml of heparinized whole blood
are removed before injection. The ATG preparation is then injected
(50 mg/kg) intravenously. Six ml samples of heparinized whole blood
are drawn for testing at time points of 30 min., 24 hrs and 48 hrs.
Blood samples are analyzed for the effect of antibody treatment on
natural killer cell activity (testing on K562 targets) and by FACS
analysis for lymphocyte subpopulations, including CD4, CD8, CD3,
CD11b, and CD16. Preliminary data from both assays indicate that
both groups of cells are eliminated by the administration of ATG.
If mature T cells and NK cells are not eliminated, ATG can be
re-administered at later times in the procedure, both before and
after organ transplantation.
[0289] Sublethal irradiation is administered to the recipient
between days -1 and -8. Irradiation is necessary to eliminate
enough of the recipient's endogenous BMC to stimulate hematopoiesis
of the newly introduced foreign BMC. Sublethal total body
irradiation is sufficient to permit engraftment with minimal toxic
effects to the recipient. Whole body radiation (150 Rads) was
administered to cynomolgus monkey recipients from a bilateral
(TRBC) cobalt teletherapy unit at 10 Rads/min. Local irradiation of
the thymus (700 Rads) was also employed in order to facilitate
engraftment.
[0290] Natural antibodies are a primary cause of organ rejection.
To remove natural antibodies from the recipient's circulation prior
to transplantation, on day 0 an operative adsorption of natural
antibodies (nAB) is performed, using a miniature swine liver, as
follows. At -90 minutes the swine donor is anesthetized, and the
liver prepared for removal by standard operative procedures. At -60
minutes the recipient monkey is anesthetized. A peripheral IV
catheter is inserted, and a 6 ml sample of whole blood is drawn.
Through mid-line incision, the abdominal aorta and the vena cava
are isolated. Silastic cannulas containing side ports for blood
sampling are inserted into the blood vessels.
[0291] At -30 minutes the liver is perfused in situ until it turns
pale, and then removed from the swine donor and placed into cold
Ringers Lactate. The liver is kept cold until just prior to
reperfusion in the monkey. A liver biopsy is taken. At -10 minutes
the liver is perfused with warm albumin solution until the liver is
warm (37 degrees).
[0292] At 0 time the arterial and venous cannulas of the recipient
are connected to the portal vein and vena cava of the donor liver
and perfusion is begun. Liver biopsies are taken at 30 minutes and
60 minutes, respectively. Samples of recipient blood are also drawn
for serum at 30 minutes and 60 minutes respectively. At 60 minutes
the liver is disconnected from the cannulas and the recipient's
large blood vessels are repaired. The liver, having served its
function of adsorbing harmful natural antibodies from the recipient
monkey, is discarded. Additional blood samples for serum are drawn
from the recipient at 2, 24, and 48 hours. When this procedure was
performed on two sequential perfusions of swine livers, the second
liver showed no evidence of mild ischemic changes during perfusion.
At the end of a 30 minute perfusion the second liver looked grossly
normal and appeared to be functioning, as evidenced by a darkening
of the venous outflow blood compared to the arterial inflow blood
in the two adjacent cannulas. Tissue sections from the livers were
normal, but immunofluorescent stains showed IgM on endothelial
cells. Serum samples showed a decrease in natural antibodies.
[0293] To promote long-term survival of the implanted organ through
T-cell and B-cell mediated tolerance, donor bone marrow cells are
administered to the recipient to form chimeric bone marrow. The
presence of donor antigens in the bone marrow allows newly
developing B cells, and newly sensitized T cells, to recognize
antigens of the donor as self, and thereby induces tolerance for
the implanted organ from the donor. To stabilize the donor BMC,
donor stromal tissue, in the form of tissue slices of fetal liver,
thymus, and/or fetal spleen are transplanted under the kidney
capsule of the recipient. Stromal tissue is preferably implanted
simultaneously with, or prior to, administration of hematopoietic
stem cells, e.g., BMC, or a fetal liver cell suspension.
[0294] To follow chimerism, two color flow cytometry can be used.
This assay uses monoclonal antibodies to distinguish between donor
class I major histocompatibility antigens and leukocyte common
antigens versus recipient class I major histocompatibility
antigens.
[0295] BMC can in turn be injected either simultaneously with, or
preceding, organ transplant. Bone marrow is harvested and injected
intravenously (7.5.times.10.sup.8/kg) as previously described
(Pennington et al., 1988, Transplantation 45:21-26). Should natural
antibodies be found to recur before tolerance is induced, and
should these antibodies cause damage to the graft, the protocol can
be modified to permit sufficient time following BMT for humoral
tolerance to be established prior to organ grafting.
[0296] The approaches described above are designed to
synergistically prevent the problem of transplant rejection. When a
kidney is implanted into a cynomolgus monkey following liver
adsorption of natural antibodies, without use of bone marrow
transplantation to induce tolerance, renal functions continued for
1-2 days before rejection of the kidney. When four steps of the
procedure were performed (adsorption of natural antibodies by liver
perfusion, administration of ATG, sublethal irradiation and bone
marrow infusion, followed by implant of a porcine kidney into a
primate recipient), the kidney survived 7 days before rejection.
Despite rejection of the transplanted organ, the recipient remained
healthy.
[0297] When swine fetal liver and thymic stromal tissue were
implanted under the kidney capsule of two sublethally irradiated
SCID mice, 25-50% of peripheral blood leukocytes were of donor
lineage two weeks post-transplantation. A significant degree of
chimerism was not detected in a third animal receiving fetal liver
without thymus. These procedures did not employ any chemical
immunosuppressants.
Other Embodiments
[0298] Other embodiments are within the following claims.
[0299] For example, implanted grafts may consist of organs such as
liver, kidney, heart; body parts such as bone or skeletal matrix;
tissue such as skin, intestines, endocrine glands; or progenitor
stem cells of various types.
[0300] The methods of the invention may be employed with other
mammalian recipients (e.g., rhesus monkeys, humans) and may use
other mammalian donors (e.g., primates, swine, sheep, dogs).
[0301] The methods of the invention may be employed in combination,
as described, or in part.
[0302] The method of introducing bone marrow cells may be altered,
particularly by (1) increasing the time interval between injecting
hematopoietic stem cells and implanting the graft; (2) increasing
or decreasing the amount of hematopoietic stem cells injected; (3)
varying the number of hematopoietic stem cell injections; (4)
varying the method of delivery of hematopoietic stem cells; (5)
varying the tissue source of hematopoietic stem cells, e.g., a
fetal liver cell suspension may be used; or (6) varying the donor
source of hematopoietic stem cells. Although hematopoietic stem
cells derived from the graft donor are preferable, hematopoietic
stem cells may be obtained from other individuals or species, or
from genetically-engineered completely or partially inbred donor
strains, or from in vitro cell culture.
[0303] Methods of preparing the recipient for transplant of
hematopoietic stem cells may be varied. For instance, the recipient
may undergo a splenectomy or a thymectomy. The latter would
preferably by administered prior to the non-myeloablative regimen,
e.g., at day -14.
[0304] Hemoperfusion of natural antibodies may: (1) make use of
other vascular organs, e.g., liver, kidney, intestines; (2) make
use of multiple sequential organs; (3) make use of varying the
length of time each organ is perfused; (4) make use of varying the
donor of the perfused organ. Irradiation of the recipient may make
use of: (1) of varying the adsorbed dose of whole body radiation
below the sublethal range; (2) of targeting different body parts
(e.g., thymus, spleen); (3) varying the rate of irradiation (e.g.,
10 rads/min, 15 rads/min); or (4) varying the time interval between
irradiation and transplant of hematopoietic stem cells; any time
interval between 1 and 14 days can be used, and certain advantages
may flow from use of a time interval of 4-7 days. Antibodies
introduced prior to hematopoietic cell transplant may be varied by:
(1) using monoclonal antibodies to T cell subsets or NK cells
(e.g., anti-NKH1.sub.A, as described by U.S. Pat. No. 4,772,552 to
Hercend, et al., hereby incorporated by reference); (2) preparing
anti-human ATG in other mammalian hosts (e.g., monkey, pig, rabbit,
dog); or (3) using anti-monkey ATG prepared in any of the above
mentioned hosts.
[0305] As an alternative or adjunct to hemoperfusion, host
antibodies can be depleted by administration of an excess of
hematopoietic cells.
[0306] Stromal tissue introduced prior to hematopoietic cell
transplant, e.g., BMT, may be varied by: (1) administering the
fetal liver and thymus tissue as a fluid cell suspension; (2)
administering fetal liver or thymus stromal tissue but not both;
(3) placing a stromal implant into other encapsulated,
well-vascularized sites; or (4) using adult thymus or fetal spleen
as a source of stromal tissue.
[0307] As is discussed herein, it is often desirable to expose a
graft recipient to irradiation in order to promote the development
of mixed chimerism. The inventor has discovered that it is possible
to induce mixed chimerism with less radiation toxicity by
fractionating the radiation dose, i.e., by delivering the radiation
in two or more exposures or sessions. Accordingly, in any method of
the invention calling for the irradiation of a recipient, e.g., a
primate, e.g., a human, recipient, of a xenograft or allograft, the
radiation can either be delivered in a single exposure, or more
preferably, can be fractionated into two or more exposures or
sessions. The sum of the fractionated dosages is preferably equal,
e.g., in rads or Gy, to the radiation dosage which can result in
mixed chimerism when given in a single exposure. The fractions are
preferably approximately equal in dosage. For example, a single
dose of 700 rads can be replaced with, e.g., two fractions of 350
rads, or seven fractions of 100 rads. Hyperfractionation of the
radiation dose can also be used in methods of the invention. The
fractions can be delivered on the same day, or can be separated by
intervals of one, two, three, four, five, or more days. Whole body
irradiation, thymic irradiation, or both, can be fractionated.
[0308] Methods of the invention can include recipient
splenectomy.
[0309] As is discussed herein, hemoperfusion, e.g., hemoperfusion
with a donor organ, can be used to deplete the host of natural
antibodies. Other methods for depleting or otherwise inactivating
natural antibodies can be used with any of the methods described
herein. For example, drugs which deplete or inactivate natural
antibodies, e.g., deoxyspergualin (DSG) (Bristol), or anti-IgM
antibodies, can be administered to the recipient of an allograft or
a xenograft. One or more of, DSG (or similar drugs), anti-IgM
antibodies, and hemoperfusion, can be used to deplete or otherwise
inactivate recipient natural antibodies in methods of the
invention. DSG at a concentration of 6 mg/kg/day, i.v., has been
found useful in suppressing natural antibody function in pig to
cynomolgus kidney transplants.
[0310] Some of the methods described herein use irradiation to
create hematopoietic space, and thereby prepare a recipient for the
administration of allogeneic, xenogeneic, syngeneic, or genetically
engineered autologous, stem cells. In any of the methods described
herein, particularly primate or clinical methods, it is preferable
to create hematopoietic space for the administration of such cells
by non-lethal means, e.g., by administering sub-lethal doses of
irradiation, bone marrow depleting drugs, or antibodies. The use of
sublethal levels of bone marrow depletion allows the generation of
mixed chimerism in the recipient. Mixed chimerism is generally
preferable to total or lethal ablation of the recipient bone marrow
followed by complete reconstitution of the recipient with
administered stem cells.
[0311] Xenogeneic thymic tissue is easier to obtain and in general
is less likely to harbor human pathogens. Thus, xenogeneic thymic
tissue is preferred in methods for restoring or inducing
immunocompetence. Allogeneic thymic tissue can however be used in
these methods.
[0312] Some of the methods described herein include the
administration of thymic irradiation, e.g., to inactivate host
thymic-T cells or to otherwise diminish the host's thymic-T cell
mediated responses to donor antigens. It has been discovered that
the thymic irradiation called for in allogeneic or xenogeneic
methods of the invention can be supplemented with, or replaced by,
other treatments which diminish (e.g., by depleting thymic-T cells
and/or down modulating one or more of the T cell receptor (TCR),
CD4 co-receptor, or CD8 co-receptor) the host's thymus function,
e.g., the host's thymic-T cell mediated response. For example,
thymic irradiation can be supplemented with, or replaced by, anti-T
cell antibodies (e.g., anti-CD4 and/or anti-CD8 monoclonal
antibodies) administered a sufficient number of times, in
sufficient dosage, for a sufficient period of time, to diminish the
host's thymic-T cell mediated response.
[0313] For best results, anti-T cell antibodies should be
administered repeatedly. E.g., anti-T cell antibodies can be
administered one, two, three, or more times prior to donor thymus
or bone marrow transplantation. Typically, a pre-thymus or bone
marrow transplantation dose of antibodies will be given to the
patient about 5 days prior to thymus or bone marrow
transplantation. Additional, earlier doses 6, 7, or 8 days prior to
thymus or bone marrow transplantation can also be given. It may be
desirable to administer a first treatment then to repeat pre-thymus
or bone marrow administrations every 1-5 days until the patient
shows excess antibodies in the serum and about 99% depletion of
peripheral T cells and then to perform the bone marrow
transplantation. Anti-T cell antibodies can also be administered
one, two, three, or more times after thymus or donor bone marrow
transplantation. Typically, a post-thymus or bone marrow transplant
treatment will be given about 2-14 days after bone marrow
transplantation. The post thymus or bone marrow administration can
be repeated as many times as needed. If more than one
administration is given the administrations can be spaced about 1
week apart. Additional doses can be given if the patient appears to
undergo early or unwanted T cell recovery. Preferably, anti-T cell
antibodies are administered at least once (and preferably two,
three, or more times) prior to donor thymus or bone marrow
transplantation and at least once (and preferably two, three, or
more times) after donor thymus or bone marrow transplantation.
[0314] It has also been discovered that much or all of the
preparative regimen, if called for, can be delivered or
administered to a recipient, e.g., an allograft or xenograft
recipient, within a few days, preferably within 72, 48, or 24
hours, of transplantation of tolerizing stem cells and/or the
graft. This is particularly useful in the case of humans receiving
grafts from cadavers. Accordingly, in any of the methods of the
invention calling for the administration of treatments prior to the
transplant of stem cells and/or a graft, e.g., treatments to
inactivate or deplete host antibodies, treatments to inactivate
host T cells or NK cells, or irradiation, the treatment(s) can be
administered, within a few days, preferably within 72, 48, or 24
hours, of transplantation of the stem cells and/or the graft. In
particular, primate, e.g., human, recipients of allografts can be
given any or all of treatments to inactivate or deplete host
antibodies, treatments to inactivate host T cells or NK cells, or
irradiation, within a few days, preferably within 72, 48, or 24
hours, of transplantation of stem cells and/or the graft. For
example, treatment to deplete recipient T cells and/or NK cells,
e.g., administration of ATG, can be given on day -2, -1, and 0, and
WBI, thymic irradiation, and stem cell, e.g., bone marrow stem
cells, administered on day 0. (The graft, e.g., a renal allograft,
is transplanted on day 0).
[0315] As described in PCT/US94/01616, hereby incorporated by
reference, it has been discovered that there is a permissible time
period ("window") for hematopoietic stem cell engraftment following
the creation of space (e.g., by whole body irradiation) for the
donor hematopoietic stem cells in a recipient. It has further been
discovered that space created for hematopoietic stem cell
engraftment can be monitored over time by monitoring peripheral
white blood cell levels in a recipient. The myelosuppressive
treatment sufficient to create hematopoietic space generally
results in a reduction in white blood cell (WBC) levels (as
revealed, e.g., by WBC counts) and the WBC reduction serves as a
marker for the presence of hematopoietic space. The marker is a
conservative one since WBC counts may recover at a time when space
is still present in an animal.
[0316] Accordingly, in any method which involves hematopoietic stem
cell transplantation, and thus also requires the creation of
hematopoietic space in a recipient, transplantation can be
performed during the permissible window for engraftment following
creation of space for the hematopoietic stem cells. Likewise, in
any method in which space is created for exogenously administered
hematopoietic stem cells, white blood cell levels can be followed
to monitor space for the donor hematopoietic stem cells (i.e., to
assess the permissible window for engraftment). Examples of
procedures involving hematopoietic stem cell transplantation
include: 1) conditioning of a recipient for an allo- or xenograft
in which hematopoietic stem cell transplantation is performed in
conjunction with transplantation of another allo- or xenograft; 2)
treatment of various hematopoietic disorders, including leukemias,
lymphomas and other hematopoietic malignancies and genetic
hematopoietic disorders (e.g., adenosine deaminase deficiency, bare
lymphocyte syndrome and other congenital immunodeficiency diseases)
in which hematopoietic stem cell transplantation is performed
therapeutically; and 3) transplantation of genetically modified
hematopoietic stem cells (e.g., genetically modified autologous
hematopoietic stem cells) to deliver a gene product to a recipient
(e.g., as gene therapy).
[0317] Accordingly, methods of the invention include a method of
determining if a myelosuppressive or hematopoietic-space inducing
treatment is sufficient to create hematopoietic space. The method
includes administering a myelosuppressive treatment to a recipient,
and determining the level of white blood cells in the recipient,
e.g., by determining the WBC count of the recipient, a depression
in the level of white blood cells being indicative of the presence
or induction of hematopoietic space.
[0318] As is discussed in PCT/US94/01616, hereby incorporated by
reference, and elsewhere herein, the engraftment of exogenously
supplied hematopoietic stem cells can be promoted by treating the
recipient of the cells so as to induce hematopoietic space in the
recipient. Hematopoietic space is commonly induced by radiation,
but other procedures can replace or reduce the need for WBI. For
example, space can be created by treating the recipient with a
monoclonal antibody against MHC class I antigens expressed by the
recipient (see e.g., Voralia, M. et al. (1987) Transplantation
44:487) or space can be created by treating the recipient with
myelosuppressive drugs (see e.g., Lapidot, T. et al. (1990) Proc.
Natl. Acad. Sci. USA 87:4595).
[0319] It has also been found that the direct introduction of donor
antigen, e.g., donor hematopoietic stem cells, into the thymus of a
recipient, can modify the immune response of the recipient. Thus,
embodiments of the invention include methods of promoting the
acceptance a graft (e.g., by prolonging the acceptance the graft)
by a recipient, by introducing into the recipient, donor antigen.
The graft can be an allograft, e.g., a graft from a primate e.g., a
human, which is introduced into a primate of the same species. The
graft can be a concordant or discordant xenograft. E.g., the graft
can be a miniature swine graft introduced into a second species,
e.g., a primate, e.g., a human.
Induction of Tolerance
[0320] The invention provides several methods of inducing tolerance
to foreign antigens, e.g., to antigens on allogeneic or xenogeneic
tissue or organ grafts. These methods can be used individually or
in combination. For example, it has been discovered that short-term
administration of a help reducing agent, e.g., a short high dose
course of cyclosporine A (CsA) significantly prolongs graft
acceptance. (Preferably the help reduction regimen of the invention
substantially eliminates the initial burst of IL-2 which
accompanies the first recognition of an antigen but does not
eliminate mature T cells. This is distinct from anti-T cell
antibody treatments which eliminate mature T cells.)
[0321] It has also been discovered that a short course of an
immunosuppressant, e.g., cyclosporine, can be used to inactivate T
cells which would otherwise promote the rejection of a graft.
[0322] Experiments which show the effect of cyclosporine-induced
tolerance on renal allografts in primates are described in section
I below. The help suppression methods of the invention can be
combined with other methods for prolonging graft acceptance.
Section II below discusses implantation of retrovirally transformed
bone marrow cells to induce tolerance to MHC disparity. This method
can be combined with help suppression methods of the invention,
e.g., a short course of high dose of cyclosporine to induce
tolerance to class I and other minor disparities.
[0323] Section III below discusses implantation of bone marrow
cells to induce tolerance to MHC disparity. This method can be
combined with a short course of high dose cyclosporine
administration to induce tolerance to class I and other minor
disparities. A short course of cyclosporine, to eliminate T cells,
can also be combined with bone marrow transplant.
I. A Short Post-Transplant Course of High Dose of Cyclosporine
(Administered in the Absence of Agents which Stimulate Cytokine
Release) Prolongs Acceptance of Partially Matched Allografts in
Primates.
[0324] Renal transplants were performed between cynomolgus monkeys
with or without a brief course of T cell-help-eliminating
immunosuppression in the form of a short course of high dose
cyclosporine. This regimen significantly prolonged acceptance of
the grafts. Monkeys which received a post-transplant course of high
dose cyclosporine (without Prednisone) were tolerant to kidney
grafts from MLC (mixed lymphocyte culture assay) nonreactive class
I and minor antigen mismatched donors for over 65 days, see below.
Monkeys which did not receive post-transplant cyclosporine rejected
grafts of the same disparity in less than 20 days. This work is
discussed in more detail below.
[0325] Animals. Cynomolgus monkeys were purchased from Charles
River Research Laboratories. The animals were quarantined, tested
for a full battery of pathogens, and then housed in environmentally
controlled rooms in strict conformance to the N.I.H. Guide for Care
and use of Laboratory Animals, in an AAALAC accredited
facility.
[0326] Typing. Animals were typed by a standard complement mediated
cytotoxicity assay for class I antigens and by MLC nonreactivity
for class II matching.
[0327] Immunosuppression. An intravenous preparation of CsA
(Sandimmune, i.v.) was obtained from Sandoz Pharmaceuticals
Corporation, Hanover, N.J. Monkeys received 12 doses of about 10
mg/kg with the first dose given 4 hours prior to graft
revascularization. The CsA was diluted in 250 ml of normal saline
and infused intravenously over 1 hour. The CsA was administered
without other immunosuppressants. The duration of therapy was 12
days. The suitable dosage in pigs is about 15 mg/kg delivered
intramuscularly. The dosage in either animal should be such that a
blood level of about 500-1,000 ng/ml is maintained.
[0328] Renal Function, Rejection, and Pathology. Renal function was
followed by creatinine and BUN levels in serum. Pathology was by
biopsy. Biopsies were performed at day 7, then weekly for 2 months,
then monthly.
[0329] Results. Control recipients (no cyclosporine) rejected
transplanted kidneys in less than 15 days. The results with 6
cyclosporine treated animals were as follows: animal 1, died on day
65 (i.e., 65 days after transplant), the transplanted was rejected,
(it should be noted that the blood cyclosporine level of this
animal was below 500 ng/ml during the first 7 days after
transplant); animal 2, died on day 65 from bleeding from a biopsy,
the transplanted kidney was normal at the time of death; animal 3
died on day 82, some rejection was apparent on day 55; animal 4 was
still normal at day 70 (at which time the experiment was still in
progress); animal 5 was still normal at day 40 (at which time the
experiment was still in progress); and animal 6 was still normal at
day 26 (at which time the experiment was still in progress).
[0330] II. A Short Course of High Dose Cyclosporine (Administered
in the Absence of Treatments which Stimulate the Release of
Cytokines, e.g. the Absence of Prednisone) to Induce Tolerance to
Class I and Other Minor Disparities Combined with Implantation of
Retrovirally Transformed Bone Marrow Cells to Induce Tolerance to
Class II Disparity.
Retroviral Transformation
[0331] Retroviral transformation allows the reconstitution of a
graft recipient's bone marrow with transgenic bone marrow cells,
preferably autologous bone marrow cells, expressing allogeneic or
xenogeneic MHC genes. Expression of the transgenic MHC genes
confers tolerance to grafts which exhibit the products of these or
closely related MHC genes. Thus, these methods provide for the
induction of specific transplantation tolerance by somatic transfer
of MHC genes. Retroviral introduction of MHC genes can be used
alone or combined with the T cell help reducing methods described
herein. This approach is discussed in detail below.
[0332] MHC Genes: MHC genes for a variety of species are well
studied. For example the HLA genes in man, see, e.g., Hansen et
al., 1989, The Major Histocompatibility Complex, In Fundamental
Immunology 2d ed., W. E. Paul, ed., Raven Press Ltd., New York,
hereby incorporated by reference, and the SLA genes in swine, see
e.g., Sachs et al., 1988, Immunogenetics 28:22-29, hereby
incorporated by reference, have been cloned and characterized.
[0333] A gene encoding a MHC antigen can be used in methods of the
invention to confer tolerance to a graft which displays that or a
closely related MHC antigen. Methods of the invention can be used
to confer tolerance to allogeneic grafts, e.g., wherein both the
graft donor and the recipient are humans, and to xenogeneic grafts,
e.g., wherein the graft donor is a nonhuman animal, e.g., a swine,
e.g., a miniature swine, and the graft recipient is a human.
[0334] The individual which supplies the MHC genes should be as
genetically similar as possible, particularly in terms of the MHC
genes, to the individual which supplies the graft. For example, in
allogeneic grafts wherein the implant donor is a human and the
implant recipient is a human it is preferable to use MHC genes from
the donor. In this embodiment, MHC probes, e.g., a probe from a
third person or a synthetic consensus probe, can be used to isolate
DNA encoding the MHC gene or genes of the implant donor individual.
This allows the closest match between the genes used to confer
tolerance and the genes which express MHC antigens on the
graft.
[0335] In xenogeneic grafts, the implant donor individual and the
individual which supplies the tolerance conferring DNA should be
the same individual or should be as closely related as possible.
For example, it is preferable to derive implant tissue from a
colony of donors which is highly inbred and, more preferably, which
is homozygous for the MHC genes. This allows the single cloned MHC
sequence to be used for many graft recipients.
[0336] Transformation of bone marrow cells: MHC genes can be
introduced into bone marrow cells by any methods which allows
expression of these genes at a level and for a period sufficient to
confer tolerance. These methods include e.g., transfection,
electroporation, particle gun bombardment, and transduction by
viral vectors, e.g., by retroviruses.
[0337] Recombinant retroviruses are a preferred delivery system.
They have been developed extensively over the past few years as
vehicles for gene transfer, see e.g., Eglitis et al., 1988, Adv.
Exp. Med. Biol. 241:19. The most straightforward retroviral vector
construct is one in which the structural genes of the virus are
replaced by a single gene which is then transcribed under the
control of regulatory elements contained in the viral long terminal
repeat (LTR). A variety of single-gene-vector backbones have been
used, including the Moloney murine leukemia virus (MoMuLV).
Retroviral vectors which permit multiple insertions of different
genes such as a gene for a selectable marker and a second gene of
interest, under the control of an internal promoter can be derived
from this type of backbone, see e.g., Gilboa, 1988, Adv. Exp. Med.
Biol. 241:29.
[0338] The elements of the construction of vectors for the
expression of a protein product, e.g., the choice of promoters is
known to those skilled in the art. The most efficient expression
from retroviral vectors is observed when "strong" promoters are
used to control transcription, such as the SV 40 promoter or LTR
promoters, reviewed in Chang et al., 1989, Int. J. Cell Cloning
7:264. These promoters are constitutive and do not generally permit
tissue-specific expression. However, in the case of class I genes,
which are normally expressed in all tissues, ubiquitous expression
is acceptable for functional purposes. Housekeeping gene promoters,
e.g., the thymidine kinase promoter, are appropriate promoters for
the expression of class II genes.
[0339] The use of efficient packaging cell lines can increase both
the efficiency and the spectrum of infectivity of the produced
recombinant virions, see Miller, 1990, Human Gene Therapy 1:5.
Murine retroviral vectors have been useful for transferring genes
efficiently into murine embryonic, see e.g., Wagner et al., 1985,
EMBO J. 4:663; Griedley et al., 1987 Trends Genet. 3:162, and
hematopoietic stem cells, see e.g., Lemischka et al., 1986, Cell
45:917-927; Dick et al., 1986, Trends in Genetics 2:165-170.
[0340] A recent improvement in retroviral technology which permits
attainment of much higher viral titers than were previously
possible involves amplification by consecutive transfer between
ecotropic and amphotropic packaging cell lines, the so-called
"ping-pong" method, see e.g., Kozak et al., 1990, J. Virol.
64:3500-3508; Bodine et al., 1989, Prog. Clin. Biol. Res. 319:
589-600.
[0341] Transduction efficiencies can be enhanced by pre-selection
of infected marrow prior to introduction into recipients, enriching
for those bone marrow cells expressing high levels of the
selectable gene, see e.g., Dick et al., 1985, Cell 42:71-79; Keller
et al., 1985, Nature 318:149-154. In addition, recent techniques
for increasing viral titers permit the use of virus-containing
supernatants rather than direct incubation with virus-producing
cell lines to attain efficient transduction, see e.g., Bodine et
al., 1989, Prog. Clin. Biol. Res. 319:589-600. Because replication
of cellular DNA is required for integration of retroviral vectors
into the host genome, it may be desirable to increase the frequency
at which target stem cells which are actively cycling e.g., by
inducing target cells to divide by treatment in vitro with growth
factors, see e.g., Lemischka et al., 1986, Cell 45:917-927, a
combination of IL-3 and IL-6 apparently being the most efficacious,
see e.g., Bodine et al., 1989, Proc. Natl. Acad. Sci. 86:8897-8901,
or to expose the recipient to 5-fluorouracil, see e.g., Mori et
al., 1984, Jpn. J. Clin. Oncol. 14 Suppl. 1:457-463, prior to
marrow harvest, see e.g., Lemischka et al., 1986, Cell 45:917-927;
Chang et al., 1989, Int. J. Cell Cloning 7:264-280.
[0342] N2A or other Moloney-based vectors are preferred retroviral
vectors for transducing human bone marrow cells.
[0343] Preparative Regimen For The Introduction of Transformed Bone
Marrow Cells To prepare for bone marrow cells the recipient must
undergo an ablation of the immune response which might otherwise
resist engraftment.
[0344] The preparative regimens necessary to permit engraftment of
modified autologous hematopoietic stem cells may be much less toxic
than those needed for allogeneic bone marrow
transplantation--preferably requiring only depletion of mature T
cells with monoclonal antibodies, as has been recently demonstrated
in a mouse model, see Sharabi et al., 1989, J. Exp. Med.
169:493-502. It is possible that transient expression may be
sufficient to induce tolerance, which may then be maintained by the
transplant even if expression on hematopoietic cells is lost, as
has been observed for heart transplants in a mixed xenogeneic bone
marrow transplant model, Ildstad et al., 1985, Transplant. Proc.
17: 535-538.
[0345] Graft and help reduction: The help reducing methods
described above can be administered in conjunction with
transplantation of the graft, as is described above.
Sustained Expression of a Swine Class II Gene in Murine Bone Marrow
Hematopoietic Cells by Retroviral-Mediated Gene Transfer
[0346] Overview: The efficacy of a gene transfer approach to the
induction of transplantation tolerance in miniature swine model was
shown by using double-copy retroviral vectors engineered to express
a drug-resistance marker (neomycin) and a swine class II DRB cDNA.
Infectious particles containing these vectors were produced at a
titer of >1.times.10.sup.6 G418-resistant colony-forming
units/ml using both ecotropic and amphotropic packaging cell lines.
Flow cytometric analysis of DRA-transfected murine fibroblasts
subsequently transduced with virus-containing supernatants
demonstrated that the transferred sequences were sufficient to
produce DR surface expression. Cocultivation of murine bone marrow
with high-titer producer lines leads to the transduction of 40% of
granulocyte/macrophage colony-forming units (CFU-GM) as determined
by the frequency of colony formation under G418 selection. After
nearly 5 weeks in long-term bone marrow culture, virus-exposed
marrow still contained G418-resistant CFU-GM at a frequency of 25%.
In addition, virtually all of the transduced and selected colonies
contained DRB-specific transcripts. These results show that a
significant proportion of very primitive myelopoietic precursor
cells can be transduced with the DRB recombinant vector and that
vector sequences are expressed in the differentiated progeny of
these cells. These experiments are described in detail below.
[0347] Construction and Screening of SLA-DRB Recombinant
Retroviruses As in man, Lee et al., 1982, Nature 299:750-752, Das
et al., 1983, Proc. Natl. Acad. Sci. USA 80:3543-3547, the sequence
of the swine DRA gene is minimally polymorphic. Therefore,
transduction of allogeneic DRB cDNAs into bone marrow cells should
be sufficient to allow expression of allogeneic class II DR
molecules on cells committed to express this antigen.
[0348] Details of retroviral constructs are given in FIG. 3. Two
types of retroviral constructs, GS4.4 and GS4.5, were prepared. The
diagram in FIG. 3 depicts the GS4.5 retroviral construct. The
arrows in FIG. 3 indicate the directions of transcription. In
GS4.5, the orientation of DRB cDNA transcription is the same as
viral transcription. In GS4.4 (not shown), the TK promoter and the
DRB cDNA were inserted into the 3' LTR of N2A in the reverse
orientation of transcription with respect to viral transcription
and the simian virus 40 3' RNA processing signal was added. pBSt
refers to Bluescript vector sequence (Stratagene). The thymidine
kinase (TK) promoter was contained within the 215-base-pair (bp)
Pvu II-Pst I fragment from the herpes simplex virus TK gene,
McKnight, 1980 Nucleic Acids Res. 8:5949-5964. The simian virus 40
3' RNA processing signal was contained within the 142-bp Hpa I-Sma
I fragment from the pBLCAT3 plasmid, Luckow et al., (1987) Nucleic
Acids Res. 15:5490-5497, (see FIG. 3). Sequence analysis of the
junctions of the promoter, the class II cDNA, and the vector
sequences confirmed that the elements of the constructs were
properly ligated.
[0349] These retroviral constructs were transfected into the
amphotropic packaging cell line PA317, and transfectants were
selected in G418-containing medium. A total of 24 and 36 clones,
transfected, respectively, with the GS4.4 and GS4.5 recombinant
plasmids, were tested by PEG precipitation of culture supernatants
and slot-blot analysis of viral RNA. Of these, 8 and 12 clones were
found, respectively, to be positive for DRB, although the DRB
signal was consistently weaker for the GS4.4-derived clones.
Analysis of genomic and spliced transcripts from GS4.5 cells by
dot-blot analysis of PEG-precipitated particles revealed
heterogeneity among viral transcripts in various clones transfected
by GS4.5. In one experiment, two clones contained
DRB.sup.+/Neo.sup.+ viral RNA, two contained DRB.sup.+/Neo.sup.-
RNA, two contained DRB.sup.-/Neo.sup.+ RNA, and one showed no class
II or Neo signal. G418-resistance (G418.sup.r) titer determination
of supernatants from DRB-positive clones confirmed that the average
titer produced by GS4.5-transfected clones (10.sup.3-10.sup.4
CFU/ml) was significantly higher than that of the GS4.4-transfected
clones (10.sup.2-10.sup.3 CFU/ml). Further transduction experiments
were, therefore, conducted with the best clone, named GS4.5 C4,
which produced an initial G418.sup.r titer of 3.times.10.sup.4
CFU/ml.
[0350] Plasmid preparation, cloning procedures, DNA sequencing, RNA
preparations, Northern blots, and RNA slot blots were performed by
standard methods, Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual 2nd Ed. (Cold Spring Harbor Lab., Cold Spring
Harbor). Final washes of blots were carried out in 0.1.times.SSPE
(1.times.SSPE =0.18 M NaCl/10 mM sodium phosphate, pH 7.4/1 mM
EDTA) at 60.degree. C. for 30 min.
[0351] The packaging cell lines PA317, Miller et al., 1986, Mol.
Cell. Biol. 6:2895-2902, GP+E-86, Markowitz et al., 1988, J. Virol
62:1120-1124, psiCRIP, Danos et al., 1988, Proc. Natl. Acad. Sci.
USA 85:6460-6464, and their derivatives were maintained at
37.degree. C. in Dulbecco's modified Eagle's medium (DMEM; GIBCO)
with 10% (vol/vol) fetal bovine serum (CELLect Silver; Flow
Laboratories) supplemented with 0.1 mM nonessential amino acids
(Whittaker Bioproducts), antibiotics penicillin (5 units/ml), and
streptomycin (5 .mu.g/ml).
[0352] Improvement of the Viral Titer of the C4 Clone
[0353] Since recent data indicated that supernatants containing
high retroviral titers were the best candidates for transducing
bone marrow cells, Bodine et al., 1990, Proc. Natl. Acad. Sci. USA
87:3738-3742, the titer of the C4 producer clone was increased by
"ping-pong" amplification, Bestwick et al., 1988, Proc. Natl. Acad.
Sci. USA 85:5404-5408. Supernatant from nearly confluent C4
cultures was used to transduce GP+E-86 ecotropic packaging cells
and G418 selection was applied. Forty-eight clones were isolated
and screened by PEG precipitation for production of viral
particles. Supernatants from 18 of these clones were DRB-positive
by dot-blot analysis of viral RNA and had G418.sup.r titers between
0.5 and 3.5.times.10.sup.4 CFU/ml). One positive clone was then
amplified by the ping-pong technique with the amphotropic
hygromycin-resistant packaging line psiCRIP. Supernatants from 48
hygromycin-resistant clones were examined for presence of
DRB-positive viral RNA by PEG precipitation and their G418.sup.r
titers were determined. All the clones were positive by dot-blot
analysis with the DRB probes and produced titers between
1.times.10.sup.5 and 1.times.10.sup.7 CFU/ml. Amphotropic clone
GS4.5 A4, which produced the highest titer, was tested for the
presence of helper virus by the S+L-assay. No replication-competent
helper virus was detected.
[0354] Amplification of virus titer was achieved by the ping-pong
technique. Since there is evidence that psiCRIP packaging cells are
less prone to produce helper virus than PA317 when using certain
types of vectors, Miller, 1990, Hum. Gene Therapy 1:5-14, DRB
recombinant virions were prepared using the psiCRIP/GP-E-86
producer combination. Titer values>1.times.10.sup.7 CFU/ml with
no detectable amphotropic helper viruses were obtained, confirming
that this strategy produced safe viral particles suitable for in
vivo experiments.
[0355] Northern blot analysis of GS4.5-producing clones C4, A9, and
A4, each derived from a different packaging cell line, showed a
conserved hybridization pattern. RNA species corresponding to the
full-length viral genome, the spliced Neo transcript, and the DRB
transcription unit were observed with additional RNA species. High
molecular size species observed in these experiments may constitute
a read-through transcript starting from the TK promoter and ending
in the other long terminal repeat (LTR). In contrast to many of the
virion-producer clones-obtained by transfection that presented
erratic DRB transcripts, those obtained by transduction showed
stable DRB hybridization patterns suggesting that no recombination
events occurred during the amplification procedure.
[0356] Retroviral titers were determined as follows.
Replication-defective retroviral particles were produced from
packaging cell lines initially transfected with recombinant
construct using the standard calcium phosphate precipitation
method, Wigler et al., 1978, Cell 14:725-733. Retrovirus production
was estimated by the drug-resistance titer (G418-resistant
colony-forming units/ml, CFU/ml) as described, Bodine et al., 1990,
Proc. Natl. Acad. Sci. USA 87:3738-3742. Except for the psiCRIP
line, G418 (GIBCO) selection was carried out in active component at
500 .mu.g/ml for 10-12 days. Hygromycin B selection was applied to
psiCRIP-derived packaging clones in medium containing active drug
at 50 .mu.g/ml for 10 days. Replication-competent helper virus
titer was assayed on PG4 feline cells by the S.sup.+L.sup.- method,
Bassen et al., 1971, Nature 229:564-566.
[0357] PEG precipitation of viral particles was performed as
follows. Virions contained in 1 ml of culture supernatant were
precipitated with 0.5 ml of 30% (wt/vol) polyethylene glycol (PEG)
for 30 min. at 4.degree. C. After centrifugation, the pellets were
treated with a mixture of RNase inhibitors (vanadyl ribonuclease
complex, BRL), phenol/chloroform-extracted, and
ethanol-precipitated. Pellets were then resuspended in 15.7%
(vol/vol) formaldehyde and serial dilutions were dotted onto
nitrocellulose membrane.
[0358] Analysis of DRB Transcription in Packaging Cell Clones To
test for accurate transcription of the introduced DRB cDNA within
the different producer clones, Northern blots containing RNAs
isolated from these clones were hybridized with the DRB and Neo
probes. FIG. 4 depicts the structure of the provirus genome and the
expected sizes of transcripts initiated from either the viral LTR
or the TK promoters. Each of the three GS4.5-containing clones,
which were derived from PA317 (clone C4), GP+E-86 (clone A9), and
psiCRIP (clone A4) cells, showed DRB-positive transcripts. As
reported, Hantzopoulos et al., 1989, Proc. Natl. Acad. Sci. USA
86:3519-3523, the unspliced genomic RNA (band a) and the spliced
Neo transcript (band b) were observed. In addition a transcript
uniquely hybridizable with the DRB probe was detected that
corresponds to the size predicted (1700 bases, band c) for the DRB
cDNA transcription unit.
[0359] Surface Expression of the SLA-DR Antigen on Transduced
Fibroblasts An in vitro assay was developed to examine surface
expression of the SLA-DR antigen on murine fibroblasts. Flow
cytometry (FCM) profiles shown in FIG. 5 demonstrate that
G418.sup.r titers of 3.times.10.sup.4 (clone C4) were sufficient to
promote expression of the DR antigen on the cell surface of
transduced DRA transfectants. In FIG. 5 solid lines indicate DR
cell surface expression (anti-DR antibody binding) (22% and 75% of
the bulk population of cells 3 days after transduction with GS4.5
C4, (B) and GS4.5 A4 (C), respectively); dashed lines indicate
anti-mouse class I antibody binding (positive control); dotted
lines indicate anti-pig CD8 antibody binding (negative control).
Twenty-two percent of the bulk population of transduced cells were
DR-positive and subclones maintained class II expression for more
than 5 months. The increase in titer (clone A4) correlated with an
increase in the number of cells transduced (75% of the transduced
population was DR-positive) and with the brightness of the DR
signal.
[0360] The class II transduction assay was performed as diagrammed
in FIG. 6. NIH 3T3 cells were transfected with the SLA-DRA.sup.d
cDNA inserted in a plasmid expression vector, Okayama et al., 1982,
Mol. Cell. Biol. 2:161-170. Approximately 3.times.10.sup.4 cells of
a stable DRA transfectant (clone 11/12.2F) that expressed a high
level of DRA mRNA were then transduced overnight with 1 ml of
DRB-containing retroviral supernatant. Cells were subsequently
cultivated in fresh DMEM supplemented with 10% fetal bovine serum
and antibiotics for 2 additional days and examined for cell surface
expression of the DR antigen by FCM analysis.
[0361] The class II transduction assay described here provides a
fast and simple method to test both the expression and functional
titer of retroviral constructs. By using cells transfected with
DRA, the need for lengthy double selection after transduction by
two separated vectors, Yang et al., 1987, Mol. Cell Biol.
1:3923-3928; Korman et al., 1987, Proc. Natl. Acad. Sci. USA
84:2150-2154, is obviated. Cell-surface expression of DR
heterodimers was demonstrated by FCM analysis 3 days after
transduction, providing direct evidence that the transferred
sequences were sufficient to produce significant level of DR .beta.
chain. More importantly, this test allows determination of
"functional" titers based on the expression of the gene of interest
rather than on that of the independently regulated drug-resistance
marker.
[0362] The SLA-DRB probe was an EcoRI cDNA fragment containing the
complete coding sequence of the DR .beta. chain, Gustafsson et al.,
1990, Proc. Natl. Acad Sci. USA 87:9798-9802. The neomycin
phosphotransferase gene (Neo) probe was the Bcl I-Xho I fragment of
the N2A retroviral plasmid, Hantzopoulos et al., 1989, Proc. Natl.
Acad Sci. USA 86:3519-3523.
[0363] Expression of Porcine DRB cDNA Transduced into Murine Bone
Marrow Progenitor Cells The efficiency with which myeloid
clonogenic precursors were transduced was determined by assaying
for CFU-GM with and without a selecting amount of G418 after
exposure of bone marrow cells to GS4.5-derived virions. Comparison
of the number of colonies that formed in the presence and absence
of the drug, for two experiments, indicated that .apprxeq.40% of
the initial population of myeloid progenitor cells were transduced.
The frequency of G418.sup.r CFU-GM was again determined after a
sample of the transduced marrow was expanded under long-term
culture conditions for 33 days. Twenty-five percent of the
progenitors present after 33 days in culture still gave rise to
colonies under G418 selection. Colonies of cells arisen from CFU-GM
were examined for the presence of DRB-specific transcripts by
converting RNA into cDNA and then performing PCR amplification as
described herein and in Shafer et al., 1991 Proc. Natl. Acad. Sci.
USA 88:9670. A 360-bp DRB-specific product was detected in five of
six G418-selected colonies from freshly transduced marrow, whereas
all six colonies similarly derived from transduced progenitors
present after 33 days in culture were positive. An additional band
of 100 bp observed in some of the samples probably reflects the
stochastic nature of nonspecific priming events. DRB-specific
transcripts were also detected in the bulk population of
drug-resistant colonies and in producer cells but were not detected
in controls such as a bulk population of untransduced colonies,
fibroblasts used to provide carrier RNA, and a bulk population of
transduced colonies processed as above but without reverse
transcriptase. These latter data demonstrate that the PCR signal
was dependent on the synthesis of cDNA, excluding the possibility
that provirus, rather than viral message, was responsible for the
amplified fragment.
[0364] Recent improvements including modifications of the virus
design, increase of viral titers, use of growth factors to
stimulate precursor cells, and selection of stem cells prior to
transduction have been shown to improve long-term expression of
transduced genes in the hematopoietic compartment, Bodine et al.,
1990, Proc. Natl. Acad. Sci. USA 87:3738-3742; Bodine et al., 1989,
Proc. Natl. Acad. Sci. USA 86:8897-8901; Wilson et al., 1990, Proc.
Natl. Acad. Sci. USA 87:439-443; Kang et al., 1990, Proc. Natl.
Acad. Sci. USA 87:9803-9807; Bender et al., 1989, Mol. Cell. Biol.
9:1426-1434. The experiments herein show the applicability of the
retroviral gene-transfer technique in achieving expression of major
histocompatibility complex class II genes transferred into
hematopoietic cells. To determine the efficiency with which
developmentally primitive hematopoietic cells were transduced, the
frequency of G418.sup.r CFU-GM was assessed after expanding
infected marrow cells kept for 33 days in long-term cultures.
Expression of the exogenous DRB cDNA was also monitored in cells
derived from transduced CFU-GM present either immediately after
infection or after an extended culture period. Virtually all of the
colonies individually tested were positive for DRB-specific
transcript, suggesting that the DRB recombinant vector is suitable
for expression in murine hematopoietic cells.
[0365] Bone marrow cells were obtained from the femora of 6- to
12-week-old female C57BL/10 mice and were prepared as described,
Ildstad et al., 1984, Nature 307:168-170. Methylcellulose colony
assays for granulocyte/macrophage colony-forming units (CFU-GM),
Eaves et al., 1978, Blood 52:1196-1210, were performed as described
using 5% (vol/vol) murine interleukin 3 culture supplement
(Collaborative Research). Long-term Dexter-type bone marrow
cultures were initiated in 60-mm culture dishes with
2.times.10.sup.7 nucleated cells, Eaves et al., 1987, CRC Crit.
Rev. Oncol. Hematol. 7:125-138.
[0366] Bone marrow cells were transduced essentially as described,
Bodine et al., 1989, Proc. Natl. Acad. Sci. USA 86:8897-8901.
Briefly, bone marrow was harvested for 6-12-week-old female
C57BL/10 donors that had been treated 2 days with 5-fluorouracil
(150 mg/kg). Prestimulation was performed by incubating
1.times.10.sup.6 cells per ml for 2 days in long-term Dexter-type
bone marrow culture medium to which was added 7.5% interleukin 3
culture supplement and recombinant human interleukin 6 (200
units/ml; gift from J. Jule, National Institutes of Health,
Bethesda, Md.). Marrow cells were transduced for 48 hr by adding
5.times.10.sup.6 cells per 10-cm plate containing nearly confluent
virus-producers, Polybrene (8 mg/ml), and the cytokines described
above.
[0367] Detection of DRB-Specific Transcripts in CFU-Derived
Colonies was performed as follows. Cells corresponding to
individual CFU colonies and to colonies present on an entire plate
(bulk) were first extracted from methylcellulose cultures by
dilution in phosphate-buffered saline and centrifugation. These
cells were then combined with 1.times.10.sup.6 NIH 3T3 cells (to
provide carrier RNA), and total RNA was prepared using the
guanidine isothiocyanate/CsCl method. First-strand cDNA was
prepared from 20 .mu.g of total RNA using the Invitrogen Red Module
kit. cDNA was then subjected to 50 cycles of PCR amplification in
the presence of the SLA DRB-specific oligonucleotides 04
(5'-CCACAGGCCTGATCCCTAATGG) (Seq. I.D. No. 1) and 17
(5'-AGCATAGCAGGAGCCTTCTCATG) (Seq. I.D. No. 2) using the Cetus
GeneAmp kit as recommended (Perkin-Elmer/Cetus). Reaction products
were visualized after electrophoresis on a 3% NuSieve agarose gel
(FMC) by staining with ethidium bromide.
[0368] FCM analysis was performed with a FAC-Scan II
fluorescence-activated cell sorter (Becton Dickenson) on cells
stained with the anti-DR monoclonal antibody 40D, Pierres et al.,
1980, Eur. J. Immunol. 10:950-957, an anti-H-2.sup.d allo
antiserum, or the anti-porcine CD8 monoclonal antibody 76-2-11,
Pescovitz et al., 1984, J. Exp. Med. 160:1495-1505, followed by
fluorescein isothiocyanate-labeled goat anti-mouse antibodies
(Boehringer Mannheim).
Expression of Allogeneic Class II cDNA in Swine Bone Barrow Cells
Transduced with a Recombinant Retrovirus
[0369] A MHC gene (DRB) was transferred into clonogenic progenitor
cells from swine using a recombinant retroviral vector (GS4.5) and
a transduction protocol designed to be applicable in vivo. Both the
selectable drug resistance gene and the allogeneic class II cDNA
transferred by this vector were expressed in the progeny of these
transduced progenitors. Expression of the Neo gene was monitored
functionally by colony formation under G418 selection, while the
presence of class II transcripts was detected by PCR analysis. With
this latter method, the transcriptional expression of both
endogenous and virally derived DRB genes in transduced and selected
colonies were demonstrated.
[0370] Primary porcine fibroblasts were cultured with high titer
viral supernatants, and then analyzed by northern blotting using
probes specific for DRB and Neo. A specific transcript was observed
which was uniquely hybridizable with the DRB probe and migrated at
the position predicted (1700 bases) for the DRB cDNA transcription
unit arising from the TK promoter and terminating at the LTR 3' RNA
processing site.
[0371] To determine whether GS4.5 containing virions could
transduce swine myelopoietic progenitor cells a colony assay
adapted for swine CFU-GM was used. Transductions were carried out
by incubating bone marrow from a donor of the SLAC haplotype in
high titer viral supernatant. Comparisons of the number of colonies
which formed in the presence and absence of G418 for a total of 5
independent experiments indicated that 5% to 14% of CFU-GM were
transduced.
[0372] Colonies of cells originating from transduced CFU-GM were
examined for the presence of DRB-specific transcripts by converting
RNA into cDNA, and then performing PCR amplification. Utilizing a
polymorphic Sau3AI restriction site absent from the endogenous
DRB.sup.c gene, the presence of DRB.sup.d-specific transcripts was
unambiguously demonstrated. Gel electrophoresis of the PCR product
demonstrated that a 183/177 bp doublet indicative of the
vector-derived DRB.sup.d transcript was amplified in samples
derived not only from pools of transduced and selected CFU-GM
progeny, but also from at least 4 out of 6 individual colonies
tested. A 360 bp PCR fragment, indicative of endogenous DRB.sup.c
transcripts, was also amplified not only as expected from PBL
isolated from an SLAC donor, but also from both of the pooled
colony samples and a number of the individual colony samples.
[0373] Construction of the retrovirus GS4.5, and production of high
titer viral supernatants was as described above. Detection of
DRB-specific transcripts in CFU-derived colonies by PCR of cDNA
were described above and as follows. Bone marrow from an SLA.sup.c
donor was exposed to GS4.5-containing virions, and G418 selected
colonies were tested for the presence of DRB.sup.c (endogenous) and
DRB.sup.d (vector derived) specific transcripts by PCR of cDNA
followed by digestion with Sau3AI and agarose gel electrophoresis.
Controls were as follows: template synthesized either in the
presence or absence of reverse transcriptase; template derived from
cells producing GS4.5-containing virions, from PBL isolated from
SLA.sup.c or SLA.sup.d donors, and from untransduced producer cells
used as carrier RNA.
[0374] Transduction of bone marrow was performed as follows. Swine
bone marrow was harvested as previously described (Pennington et
al., 1988, Transplantation 45:21-26) and transductions were carried
out by incubating marrow cells in high titer viral supernatants at
an m.o.i. of 3-5 in the presence of 8 .mu.g of polybrene per ml at
37.degree. C. for 5 hr. Myeloid progenitors were assayed by colony
formation in methylcellulose cultures using PHA-stimulated swine
lymphocyte conditioned medium as a source of growth factors.
Selective medium contained 1.2 mg/ml active G418.
[0375] Transduced bone marrow was administered to a lethally
irradiated miniature swine. At 5 weeks peripheral blood lymphocytes
were analyzed by Southern, northern, and cell-surface FACS
analyses. By all of these test there was evidence of presence of
the transduced allogeneic class II gene in these cells and for
expression of the product of this gene. In particular, northern
analysis showed bands characteristic of the transcribed cDNA, and
FACS analysis with a combination of alloantisera and monoclonal
antibodies to DR showed presence of the transduced allele of DR
beta on the surface of peripheral lymphocytes.
Allogeneic Tolerance
[0376] Development of the B10.MBR-B10.AKM Strain Combination In an
attempt to maintain strains which are truly congenic for the MHC, a
program of continuous backcrossing of each congenic line to a
common background partner strain was instituted more than 15 years
ago. Backcross animals were intercrossed and appropriate progeny
selected by serologic typing in order to reestablish each congenic
line. Thus, C57BL/10 was used as one reference background strain
and all other congenic lines on the B10 background were backcrossed
once every six to ten generations to this C57BL/10 line.
[0377] During the backcrossing of each congenic line to its
pedigreed reference line, there is of course the chance for an
intra-MHC recombination event to occur. Typing of the intercross
(F2) generation serologically reveals such recombinant events, and
when the recombinant provides a new haplotype of potential interest
for genetic studies, it is outcrossed and then intercrossed to
produce a homozygous new recombinant H-2 haplotype. One of the most
valuable of such recombinants originating in this colony is the
B10.MBR line, Sachs et al., 1979, J. Immunol. 123:1965-1969, which
was derived from a recombination event during the backcrossing of
B10.AKM to C57BL/10. Because this strain was the first to separate
K.sup.b from I.sup.k it has been used extensively in studies of R-2
immunogenetics. In addition, in combination with the parental
B10.AKM strain, the B10.MBR offers the possibility of examining an
isolated K gene as the only MHC difference between these two
strains. Thus, as illustrated in FIG. 7, introduction of the
K.sup.b gene into B10.AKM bone marrow stem cells, could
theoretically lead to expression of all cell surface MHC antigens
of the B10.MBR. Expression on bone marrow derived cell populations
produces transplantation tolerance to the product of the transduced
gene, and this tolerance can be tested by a tissue graft from the
B10.MBR strain.
[0378] Reconstitution of Myeloablated Mice with Transduced Bone
Marrow Eighty prospective donor B10.AKM mice were treated with 150
mg/kg 5 FU on day -7. Bone marrow was harvested from these mice on
day -5, treated with anti-CD4 and anti-CD8 monoclonal antibodies
(mAbs) plus complement to remove mature T cells, and cultured for
five days with N2-B19-H2b virus-containing supernatant (H2) from
the psi-Crip packaging cell line. As a control, one-half of the
marrow was cultivated with supernatant from control packaging cells
not containing N2-B19-H2b (A2). On day zero, 45 B10.AKM recipients
received 10 Gy total body irradiation (TBI), followed by
administration of various concentrations of cultured bone marrow
cells (A2 or H2). K.sup.b expression On day 13 several animals
receiving the lowest doses of cultured bone marrow were sacrificed
and individual spleen colonies were harvested and analyzed by PCR
for the presence of N2-B19-H2b DNA. In addition, spleen cell
suspensions were prepared and analyzed for cell surface expression
of K.sup.b by flow microfluorometry on a fluorescence-activated
cell sorter (FACS). FACS analyses indicated that all animals
receiving the H2-treated marrow showed some Level of K.sup.b
expression above control staining with the non-reactive antibody.
The results are shown in FIG. 8 which is a FACS profile of spleen
cells from a recipient of transduced bone marrow: A=Anti K.sup.b
antibody; B=control antibody. Spleen cells from recipients of
non-transduced marrow were also negative. In addition, the PCR
analysis showed every colony examined to contain the transduced
DNA. Animals were thereafter followed by FACS and PCR on peripheral
blood lymphocytes (PBL). On day 28 and again on day 40, PCR
analyses were positive. However, FACS analysis for cell-surface
expression was variable, with PBL from most H2 animals showing only
a slight shift of the entire peak for staining with anti-K.sup.b,
as compared to PBL from A2 animals stained with the same antibody,
or as compared to PBL from H2 animals stained with the non-reactive
HOPC antibody.
[0379] Allogeneic grafts On day 40 skin from B10.MBR (K.sup.b
specific) and B10.BR (control, third party class I disparate)
donors was grafted onto all animals. Graft survivals were scored
daily by a blinded observer (i.e., readings were made without
knowledge of which graft was from which donor strain) until
rejection was complete. The survival times are shown in FIG. 9, and
indicate marked specific prolongation of survival of the B10.MBR
skin grafts on the recipients of K.sup.b-transduced BMC (FIG. 9B),
but not on recipients of control marrow (FIG. 9A). One of the
animals with a long-standing intact B10.MBR skin graft was
sacrificed at day 114 and cell suspensions of its lymphoid tissues
were examined by FACS and compared to similar suspensions of cells
from an animal which had rejected its B10.MBR skin graft. A
striking difference was noted in staining of thymus cells with an
anti-K.sup.b mAb. Cell suspensions were prepared and stained either
with the anti-K.sup.b mAB 28-8-6 or the control antibody HOPC1. A
subpopulation of thymus cells from the tolerant animal showed a
marked shift toward increased staining with 28-8-6 compared to
HOPC1, while there was essentially no change in the staining
pattern of thymocytes from the animal which had lost its graft.
FIG. 10 shows FACS analysis on thymocytes from skin graft rejecter
(FIGS. 10A, B) and skin graft acceptor (FIGS. 10C, D). Staining
with control HOPC1 antibody (FIGS. 10A, C) and with specific
anti-K.sup.b antibody (FIGS. 10B, D). A similar comparison of
staining patterns on bone marrow cells showed the presence of low
level K.sup.b expression on a cell population in the marrow of the
tolerant mouse, but not of the mouse which had rejected its skin
graft. These results indicate that a pluripotent stem cell or early
progenitor cell population expressed K.sup.b in the tolerant mouse
but not in the rejecter mouse, and that this BMC stem cell provided
a continuous source of K.sup.b antigen in the thymus on cells which
are critical for the inactivation of developing thymocytes with
K.sup.b-reactive TCR. It is of interest to note that K.sup.b
expression was not detected on splenocytes of the tolerant mouse,
and that, in general, splenocyte expression did not correlate with
skin graft tolerance. Since the spleen contains T cells which
mature in the thymus, these results suggest that either thymocytes
lose expression of K.sup.b as they mature, or that the
K.sup.b-bearing thymocytes of this animal were cells of a
non-lymphoid lineage, such as macrophages.
[0380] Long-term expression As discussed above, the B10.AKM and
B10.MBR congenic mouse strains are identical except in the MHC
class I region. A recombinant retrovirus containing the class I
gene from the B10.MBR stain (H-2K.sup.b) linked to a B19 parvovirus
promoter (B19-H2K.sup.b) and a neomycin resistance (neo.sup.r) gene
was introduced into B10.AKM (H-2K.sup.k) marrow cells. As a
control, a recombinant retrovirus containing only the neo.sup.r
gene was introduced into B10.AKM marrow cells. The transduced
marrow was injected into lethally irradiated AKM recipients
pre-treated with an anti-CD8 monoclonal antibody. Twelve weeks post
BMT, quantitative PCR was used to show that the B19-H-2K.sup.b
proviral sequences were present in 5%-30% of peripheral blood cells
in all recipient animals. Reverse transcriptase PCR was used to
demonstrate the B19-H-2K.sup.b mRNA in RNA isolated from bone
marrow and spleen of a subset of recipient animals.
[0381] Construction of the K.sup.b Retroviral Vector The retroviral
vectors used the Maloney murine leukemia virus based vector N2,
Armentano et al., 1987, J. Virol. 61:1647-1650. The coding regions
within this virus were deleted during its construction, and
replaced with the selectable marker gene, neomycin
phosphotransferase (Neo), which is transcribed from the viral LTR
promoter, and provides drug resistance to G418. This conventional
N2 virus was then further modified by insertion of a
parvovirus-derived promoter, B19, Liu et al., 1991, J. Virol. (In
Press), downstream from Neo, followed by 1.6 kb of cDNA coding for
the class I antigen H-2K.sup.b to form the new recombinant virus
N2-B19-H26. FIG. 11 depicts the N2-B19-H26 retroviral vector:
P=PstI; X=XhoI; H=HinDIII; E=EcoRI; B=BamHI. This latter cDNA was
derived by Waneck et al. during the construction of an H-2.sup.b
cDNA library for their purposes, Waneck et al., 1987, J. Exp. Med.
165:1358-1370.
[0382] Viral producer cell lines were developed using the packaging
cell lines for amphotropic (psi-Crip), Danos et al., 1988, Proc.
Natl. Acad. Sci 85:6460-6464, and ecotropic (psi-2), Sambrook et
al., 1989, Molecular cloning: A laboratory manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, viral production.
These cell lines have been specially designed to produce structural
viral proteins for the recombinant defective virus to be produced.
Viral production was achieved by transfecting psi-Crip with
N2-B19-H2b. Both amphotropic and ecotropic producer cell lines were
then co-cultivated allowing multiple integration events and high
expression [i.e. the "ping-pong" technique see Bestwick et al.,
1988, Proc. Natl. Acad. Sci 85:5404-5408]. In this technique,
co-cultivation overcomes viral antigen receptor blockage by
endogenously secreted proteins since amphotropic and ecotropic
viruses recognize different receptors. Ecotropic psi-2 viral
producer clones were then selected which produced titers of G418
resistance on 3T3 cells of greater than 10.sup.7 cfu/ml.
[0383] In order to ensure that K.sup.b was being expressed from the
recombinant virus, transduced 3T3 cells were stained with a
monoclonal antibody specific to this antigen and analyzed by flow
microfluorometry. These experiments clearly demonstrated high level
expression of virally derived K.sup.b.
[0384] Animals and husbandry were as follows. The B10.BMR strain,
[Sachs et al., 1979, J. Immunol. 123:1965-1969, was provided to the
Jackson Laboratory, Bar Harbor, Me. about 6 years ago, and specific
pathogen-free stock animals of this strain are now available from
that source. Upon arrival in animals should be transferred to
autoclaved microisolator cages containing autoclaved feed and
autoclaved acidified drinking water. Sterile animal handling
procedures which are effective in maintaining animals free of
pathogens so that interpretable survival studies can be performed
should be used.
[0385] Bone marrow transplantation was performed as follows.
Techniques for bone marrow transplantation in mice are known to
those skilled in the art, see e.g., Sykes et al., 1988, J. Immunol.
140:2903-2911. Briefly, recipient B10.AKM mice aged 12 to 16 weeks
are lethally irradiated (1025R, 137Cs source, 110R/min) and
reconstituted within 8 hours with 2.5.times.10.sup.6 bone marrow
cells, obtained from the tibiae and femora of sex-matched donors
aged 6-14 weeks. Animals are housed in sterilized microisolator
cages, and receive autoclaved food and autoclaved acidified
drinking water. For these studies some modifications of this
general technique are required, since the syngeneic bone marrow
will have been transduced with an allogeneic gene, and since the
bone marrow will come from 5FU-treated mice, which should have
lower total cell counts but higher stem cell content than normal
mice. The protocol is therefore as follows:
[0386] 1. Donors will be treated with 5-Fluorouracil, 150 mg/kg
i.v. on day -7 in order to induce pluripotent stem cells to
cycle.
[0387] 2. Marrow will be harvested from donors on day -5, and T
cell depleted with mAbs and complement.
[0388] 3. Marrow will than be cultured for 5 days in supernatant
from an ecotropic packaging cell line (B17H2Kb-18) which produces a
high titer of non-infectious retroviral particles containing the
K.sup.b gene (see below). IL-3 and IL-6 will be added to the
cultures.
[0389] 4. On day 0, recipient B10.AKM mice will be lethally
irradiated (10.25 Gy), and will be reconstituted with
2.5.times.10.sup.6 BMC transduced with the K.sup.b gene. Control
animals will be similarly treated, except that they will receive
marrow exposed to supernatant from a similar ecotropic packaging
line not exposed to a K.sup.b-containing vector. The recipient may
also be pre-treated with anti-CD8 monoclonal antibody.
[0390] Cellular and serological assays are performed as
Follows.
[0391] Anti-class I Cell-Mediated Lympholysis (CM) Assay: Spleens
are removed from BMT recipients and normal mice, red cells are
lysed using ACK buffer, and a single cell suspension is prepared.
Cells are filtered through 100-mesh nylon, washed, and resuspended
at 4.times.10.sup.6/ml in complete medium consisting of RPMI 1640
with 10% fetal calf serum, 0.025 mM 2-mercapteothanol, 0.01M Hepes
buffer, 0.09 mM nonessential amino acids, 1 mM sodium pyruvate, 2
mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin. 90
.mu.l of responder cells are added to Costar 96-well round-bottomed
plates along with irradiated (30 Gy) stimulator splenocytes.
Cultures are set up in two rows of 3 replicates each, and after 5
days of incubation in 6% CO.sub.2 at 37.degree. C., twofold serial
dilutions are prepared from the second row oftriplicates, so that
cytolytic capacity can be examined at a total of 5 different
responder:target ratios. 51Cr-labelled 2-day concanavalin A-induced
lymphoblasts are then added at 10.sup.4 blasts per well and
incubated for 4 hr at 37.degree. C., 6% CO.sub.2. Plates are
harvested using the Titertek supernatant collection system
(Skatron, Inc., Sterling, Va.) and .sup.51Cr release is determined
using an automated gamma counter. Cytolytic capacity is measured
directly in the original cell culture plated, so that the
measurement is based on the number of responders plated, rather
than on the number of live cells present at the end of the 5-day
incubation period. This methodology has been developed and used
successfully in this laboratory for several years for analysis of
spleen cell responses from individual animals [Sykes, M., et al.,
1988 J. Immunol. 140:2903-2911]. Percent specific lysis is
calculated using the formula: % .times. .times. Specific .times.
.times. Lysis = Experimental .times. .times. release - Spontaneous
.times. .times. release .times. 100 .times. .times. % Maximum
.times. .times. release - Spontaneous .times. .times. release
.times. 100 .times. .times. % ##EQU1## Limiting dilution analyses:
Responder and stimulator (6.times.10.sup.5, 30 Gy irradiated) cells
are co-cultured for 7 days in complete medium containing 13% TCGF
[lectin-inactivated con A supernatant obtained from BALB/c con
A-Activated splenocytes) in 96-well plates. Wells containing
10.sup.5 (24 wells), 3.times.10.sup.4 (24 wells), 10.sup.4 (30
wells), 3000 (30 wells), 1000 (30 wells), 300 (30 wells), and 100
(30 wells) responder cells are prepared. Three thousand
.sup.51Cr-labeled con A blasts are added to each well on day 7, and
4 hour .sup.51Cr release is measured. Wells are considered positive
if .sup.51Cr release is 3 standard deviations greater than the mean
.sup.51Cr release in 24 wells containing stimulator cells only plus
similar numbers of target cells. The Poisson distribution is used
to determine the frequency of precursor CTL's which recognize each
target, and statistical analysis is performed by the Chi square
method of Taswell, Taswell, 1981, J. Immunol. 126:1614.
[0392] Flow microfluorometry: One-color and two-color flow
cytometry will be performed, and percentages of cells expressing a
particular phenotype will be determined from 2-color data, as
previously described in detail Sykes, 1990, J. Immunol.
145:3209-3215. The Lysis II software program (Becton Dickinson)
will be used for distinguishing granulocytes from lymphocytes by
gating on the basis of forward angle and 90.degree. light scatter.
Cell sorting will be performed on a Coulter Epics Elite cell
sorter. Cell suspensions for flow cytometry: PBL, BMC, thymocyte,
splenocyte, and lymph node suspensions will be prepared as
previously described, Sykes, M. et al., 1988, J. Immunol.
140:2903-2911; Sykes, M. 1990, J. Immunol. 145:3209-3215; Sharabi,
Y. et al., 1990, J. Exp. Med. 172:195-202. Whole peripheral white
blood cell suspensions (including granulocytes) will be prepared by
centrifugation of heparinized blood for 2 minutes at 14,000 RPM in
an Eppendorf centrifuge, followed by aspiration of the buffy coat
layer. These cells will be transferred to a 15 ml. conical tube and
washed. Red blood cells (RBC) contaminating the remaining pellet
will be lysed by exposure for 5 seconds to 4.5 ml of distilled
H.sub.2O followed by rescue with 0.5 ml of 10.times.PBS.
[0393] Cell staining: One-color and two-color staining will be
performed as we have previously described, Sykes, M., 1990, J.
Immunol. 145:3209-3215; Sykes et al., 1988, J. Immunol. 141:
2282-2288. Culture supernatant of rat anti-mouse Rc.tau.R mAb
2.4G2, Unkeless, J. C., 1979, J. Exp. Med. 150:580-596, will be
used for blocking of non-specific staining due to Fc.tau.R binding,
whenever only direct staining is used. The following mAbs are used:
biotinylated murine K.sup.b-specific IgG.sub.2a mAb 28-8-6, Ozato
et al., 1981, J. Immunol. 126:317-321, and control murine
IgG.sub.2a, mAb HOPC1 (with no known specific binding to murine
antigens) are prepared by purification on a protein A-Sepharose
column, and are biotinylated by standard procedures used in our
laboratory; rat anti-MAC1 mAb M1/70, Springer et al, 1979, Eur. J.
Immunol. 9:301, is used as culture supernatant, and will be stained
by mouse anti-rat IgG-specific mAb MAR18.5; FITC-labeled
rat-anti-mouse granulocyte antibody Gr1 is purchased from Zymed;
FITC-labeled rat-anti-mouse Thy1.2 mAb will be purchased from
Becton-Dickinson; FITC-labeled mouse-anti-human CD3 mAb Leu4
(Becton Dickenson) is used as a directly FITC labeled negative
control antibody.
[0394] Thymic tissue immunofluorescence: The tissue is incubated in
L15 medium for 24 hours to reduce background staining, and is then
cut and embedded in O.C.T. compound for freezing in Isopentane.
Frozen sections are prepared (thickness 4 .mu.m) on a cryostat,
dried, fixed in acetone, then washed in PBS. The first antibody
incubation (with 28-8-6) is performed in the presence of 2% normal
mouse serum, in order to saturate Fc receptors. After 45 minutes,
the slides are washed 4 times, and FITC-conjugated secondary
reagent (monoclonal rat-anti-mouse IgG2a-FITC, purchased from
Pandex) is added. After 45 minutes' incubation with the secondary
reagent, four washes are performed and the tissue is mounted.
Sections are examined under a fluorescence microscope by an
observer who is unaware of the group of animals from which the
tissue was obtained.
[0395] Bone Marrow Manipulations and Assays were Performed as
Follows:
[0396] Transduction of murine bone marrow stem cells: The
methodology used for transduction of bone marrow cells has been
described previously, Karlsson et al., 1988, Proc. Natl. Acad. Sci.
85:6062-6066. Bone marrow is harvested from 6-12 week old female
B10.AKM donors treated 2 days previously with 150 mg/kg 5-FU.
Following T cell depletion (see above), the marrow is divided and
10.sup.7 cells per 10 cm plate are cultured for 5 days in the
presence of 8 .mu.g of Polybrene per ml, 10% FCS, 0.6%
IL-3-containing supernatant, 0.6% IL-6-containing supernatant, and
fresh supernatants from B19H2K.sup.b or N2 cells. IL-3- and
IL-6-containing supernatant is 48 hour supernatant of COS 7 cells
transfected with the murine rIL-3 gene-containing plasmid pCD-IL-3
or with the murine rIL-6 gene-containing plasmid pCD-IL-6,
respectively (both plasmids provided by Dr. Frank Lee, DNAX Corp.).
IL-3-containing supernatants are tittered by testing proliferation
of the IL-3-dependent cell line 32D in the presence of dilutions of
these supernatants, and IL-6 is tittered in a similar manner using
the IL-6-dependent line T1165 as the indicator cell line. We will
also test the effect of murine SCF on bone marrow transduction, as
recently described, Zsebo et al., 1990, Cell 63:125-201.
[0397] The virus-containing supernatants are refreshed on a daily
basis by harvesting the non-adherent layer of each plate, pelleting
the cells, and resuspending in freshly harvested filtered
virus-containing B19H2K.sup.b or N2 supernatant with additives.
After 5 days, the non-adherent and adherent BMC are harvested,
washed, and resuspended at 2.5.times.10.sup.6/ml in Medium 199 with
Hepes buffer and Gentamycin plus Heparin 10 U/Ml. One ml. of this
suspension is injected i.v. to irradiated mice.
[0398] Murine CFU-GM assay: To test for the bone marrow progenitor
cells known as CFU-GM (colony forming unit-granulocyte/macrophage),
bone marrow cells are suspended in plating medium consisting of
IMDM medium containing 30% defined fetal bovine serum (FBS)
(HyClone, Logan, Utah), 10.sup.-4 M .beta.-mercaptoethanol,
antibiotics, 5% v/v murine IL-3 culture supplement (Collaborative
Research Inc., Bedford, Mass.) and 0.8% methylcellulose (achieved
by adding 36% v/v of a commercially prepared solution purchased
from the Terry Fox Laboratory, Vancouver). 1.1 ml of this
suspension is then dispensed into 35 mm tissue culture plates (in
duplicate), and placed in a 37.degree. C. incubator. The resulting
CFU-GM derived colonies are enumerated microscopically after 5-7
days. Transduced CFU-GM are selected by including 0.9 .mu.g/ml
active G418 in the culture medium. The transduction frequency is
then determined by the ratio of CFU-GM which form colonies in the
presence and in the absence of the drug.
[0399] Molecular methods were as Follows:
[0400] Construction of N2-B19-H2b vector: This vector was
constructed staring from the original retroviral vector N2, Eglitis
et al., 1985, Science 230:1395-1398, as modified by Shimada to
include an additional BamHI site immediately 3' of the XhoI site.
It includes the K.sup.b cDNA previously cloned in the vector
pBG367, as described by G. Waneck, Waneck et al., 1987, J. Exp.
Med. 165:1358-1370. This gene has been placed under control of the
B19 promoter, a highly efficient parvo virus derived promoter, Liu
et al., 1991, J. Virol. In Press:] to produce the N2-B19-H2b
construct.
[0401] Southern blot analysis can be performed on DNA extracted
from PBL, thymocyte, BMC, splenocyte or lymph node cell suspensions
using standard methods, Ausubel et al., 1989, Current protocols in
molecular biology. John Wiley & Sons, New York, and probing
will be performed with the fragment of K.sup.b cDNA released from
pBG367 by EcoRI. The genomic DNA will be cut with enzymes capable
of distinguishing the transduced K.sup.b from other class I genes
of the B10.AKM strain. From known sequences it would appear that
EcoRI may be satisfactory for this purpose, since it should
liberate a 1.6 kb band from the transduced K.sup.b cDNA, which is
distinct from both the expected endogenous K.sup.k and D.sup.q
class I bands of B10.AXM, Arnold et al., 1984, Nucl. Acids Res.
12:9473-9485; Lee et al., J. Exp. Med 168:1719-1739. However, to
assure that there is no confusion with bands liberated from other
class I and class I-like genes we will test several enzymes first
on DNA from B10.AKM and choose appropriate restriction enzyme
combinations.
[0402] PCR analysis of DNA can be performed using primers
previously shown to be effective in our preliminary studies (see
FIG. 6): TABLE-US-00005 (Seq. ID No. 3) 5' primer:
5'-GGCCCACACTCGCTGAGGTATTTCGTC-3' (covers 5' end of .alpha.1 exon)
(Seq. ID No. 4) 3' primer: 5'-GCCAGAGATCACCTGAATAGTGTGA-3' (covers
5' end of .alpha.2 exon)
[0403] DNA is subjected to 25 cycles of PCR amplification using
these specific oligonucleotides and the Cetus GeneAmp kit (Perkin
Elmer Cetus, Norwalk, Conn.) according to the manufacturer's
directions. In addition, [.sup.32]PdCTP will be included in the PCR
reaction in order to visualize products by autoradiography
following electrophoresis.
[0404] RNA can be isolated from 5.times.10.sup.6 to
5.times.10.sup.7 cells using the guanidine isothiocyanate and CsCl
methods, Chirgwin et al., 1979, Biochem. 18:5294-5308, and will be
used for northern analyses, RNase protection analyses, and for PCR
analyses of products formed by reverse transcriptase. For
situations in which less then 5.times.10.sup.6 cells are available,
for example following tail bleedings of individual mice, we will
utilize the QuickPrep mRNA Purification Kit (Amgen) as miniaturized
RNA preparation procedure.
[0405] Northern analyses can be carried out using standard methods,
Ausubel et al., 1989, Current protocols in molecular biology John
Wiley & Sons, New York, and the same K.sup.b cDNA-derived
probe. Vector-derived K.sup.b mRNA is larger than endogenous class
I transcripts (2.5 kb vs. 1.6 kb) due to the inclusion of vector
sequences between the 3' end of the cDNA and the poly-adenylation
site in the viral 3' LTR. It should therefore be easy to
distinguish the vector-derive K.sup.b mRNA from endogenous
transcripts that might cross-hybridize with a K.sup.b cDNA probe.
We will also utilize probes derived from unique non-K.sup.b
sequences of the transcript (e.g., from B19 or N2 derived vector
sequences).
[0406] RNAse protection analyses-are more sensitive than standard
northern blots, yet still quantitative. Procedures based on
published methods, Sambrook et al., 1989, Molecular cloning: A
laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, will be used to derive riboprobes. Briefly, the Kb cDNA
will be cloned into a plasmid vector containing the T3 and T7 RNA
polymerase promoter sequences (bluescript or Bluescribe plasmids
from Stratagene). Using appropriate polymerase and
.sup.32P-nucleotides, transcription of the insert will be initiated
and the radioactive K.sup.b RNA will be purified. This probe will
then be incubated with various RNA preparations followed by
treatment with ribonuclease. Presence of RNA will be assessed by
electrophoresis on a sequencing gel.
[0407] PCR following reverse transcriptase treatment of RNA will be
used as a highly sensitive procedure for detecting the K.sup.b
transcript. Appropriate primers will be designed in order to
specifically amplify retroviral derived transcripts (one primer
covering the 5'UT region of the construct and second derived from
the cDNA sequence). Briefly, RNA will be prepared by the GuSCN/CsCl
method and first strand cDNA will be prepared from 5 ug of total
RNA using the SuperScript preamplification system (BRL/Life
Technologies, Inc., Gaithersburg, Md.). PCR amplifications will be
conducted for 50 cycles, Hansen et al., J. Immunol. 118:1403-1408,
using the Cetus GeneAmp kit (Perkin Elmer Cetus, Norwalk, Conn.).
Reaction products will be visualized following electrophoresis on a
3% NuSieve agarose gel (FMC BioProducts, Rockland, Me.).
Allogeneic MHC Gene Transfer Plus Cyclosporine
[0408] It has been shown previously in partially inbred miniature
swine that differences in class II MHC loci are of critical
importance in determining the fate of primarily vascularized
allografts. Cyclosporine given early in the post-transplant period
uniformly leads to tolerance of class II matched class I mismatched
kidney allografts. However, cyclosporine alone does not produce
tolerance across a full-MHC barrier. Consistent with the importance
of class II allogeneic bone marrow transplantation across class II
barriers induces tolerance to kidney transplants matched to the
class II of the bone marrow donors, but completely disparate to the
recipients. In the following experiment specific transplantation
tolerance to complete SLA-disparate kidney transplant was induced
with autologous bone marrow transplantation in which the
recipient's marrow was genetically modified prior to transplant by
transduction with a retroviral expression vector carrying an
allogeneic SLA class II gene. The retroviral expression vectors
contained cDNA for either SLA-DRB.sup.a or -DRB.sup.c and a drug
selection marker (Neo), and high tittered viral supernatants were
prepared using amphotropic packaging cell lines. Bone marrow from
five animals included in this study was harvested on day 2, and
then cultured with virus-containing supernatant for either an
allogeneic (n=4) or syngeneic (n-1) MHC gene for a approximately 48
hours. After lethal irradiation (10 Gy in two fractions 24 hours
apart) in days -1 and 0, animals were transplanted with 0.4 to
1.3.times.10.sup.8 cells/kg on day 0. The effectiveness of the gene
transfer was tested using a colony forming unit assay for
granulocyte/macrophage progenitors (CFU-GM) in the presence of G418
to select for neomycin resistance. The frequency of G418 resistant
CFU-GM varied significantly between animals (6.5% to 25.9%)
immediately after transduction and dimnished slowly with time. All
animals regained their responsiveness to allogeneic stimuli by the
third month post-bone marrow transplantation, as tested by MLR.
mAbs specific for DQ and DR molecules of the class II MHC were used
for "blocking" MLR studies, to separate the effects of recognition
of DQ and DR. In assays using cells from recipients of bone marrow
transplantation transduced with the allogeneic DRB gene, the DR
portion of the response to the gene-donor type cells was strongly
diminished, demonstrating the effectiveness of the transduced gene
at inducing DR-specific unresponsiveness. This effect was observed
in all experimental animals, although more pronounced in the
DRB.sup.d to cc combination than in DRB.sup.a to gg direction. In
MRL using cells from a control animal transduced with a syngeneic
gene, blocking of DR or DQ in MLR showed a pattern of reactivity
identical to that observed in naive animals of the same haplotype.
Five months after BMT, animals were challenged with kidney
transplants matched for class II of the gene-donor type, and fully
mismatched to the original recipient haplotype. Cyclosporine 10 to
15 mg/kg/day iv was given for 12 days, to tolerize for the class I
MHC and minor antigen disparity. Three animals rejected their
kidney transplants at days 8, 22 and 40. The accelerated manner of
rejection at day 8 suggested sensitization as an undesirable effect
of the expression of the allogeneic gene product. In none of these
recipients could the presence of anti-donor type antibodies be
detected by flow cytometry. One animal became tolerant and
exhibited normal creatinine levels at 101 days post-transplant. The
animal which received the bone marrow transduced with a syngeneic
gene underwent severe rejection with high creatinine levels and
vascular changes in pathology. The recipient of the longest
surviving kidney transplant also received the most efficiently
transduced autologous bone marrow, as judged on the initial
frequency of G418r CFU-GM. In this one case, recombinant cytokines
(Pixy 321 (Pixy is a human-GM-CSF/IL3 fusion protein) 100 Units/ml;
mouse stem cell growth factor 20 Units/ml; although these cytokines
were used, cytokines from the same species as the cell being
transformed can also be used) were included in the culture medium
during transduction with the allogeneic DRB retroviral expression
vector. The cytokines may have led to the transduction of
multilineage pluripotent hematopoietic stem cells, including the
precursors of dendritic cells which ultimately induced DRB-specific
hyporesponsiveness. These experiments demonstrate that somatic
class II MHC DRB gene transfer into bone marrow cells has profound
functional consequences upon the immune responses of the recipient.
In vitro, and more importantly in vivo, immune function was
significantly modulated, with the induction of donor specific
prolongation of fully mismatched kidney transplant survival. The
transduction of allogeneic bone marrow stem cells with MHC genes
provides a method of inducing tolerance across MHC barriers by a
mechanism comparable to lymphohematopoietic chimerism. The lethal
irradiation used in the experiments described herein can be
replaced with a non-myeloablative conditioning regimen that would
permit bone marrow engraftment in a more clinically acceptable
fashion.
III. The Induction of Tolerance with Bone Marrow
Transplantation
[0409] A Short Course of High Dose of Cyclosporine (Administered in
Absence of Treatments. e.g. Treatment with Prednisone which
Stimulate Cytokine Release) to Induce Tolerance to Class I and
Other Minor Disparities Combined with Implantation of Bone Marrow
Cells Induce Tolerance to Class II Disparity.
[0410] Xenografts: The following procedure was designed to lengthen
the time an implanted organ (a xenograft) survives in a xenogeneic
host prior to rejection. The organ can be any organ, e.g., a liver,
e.g., a kidney, e.g., a heart. The main strategies are elimination
of natural antibodies by organ perfusion, transplantation of
tolerance-inducing bone marrow, optionally, the implantation of
donor stromal tissue, and, optionally, the administration of a
short course of a help reducing agent at about the time of
introduction of the graft, as described above preparation of the
recipient for transplantation includes any or all of these steps.
Preferably they are carried out in the following sequence.
[0411] First, a preparation of horse anti-human thymocyte globulin
(ATG) is intravenously injected into the recipient. The antibody
preparation eliminates mature T cells and natural killer cells. If
not eliminated, mature T cells would promote rejection of both the
bone marrow transplant and, after sensitization, the xenograft
itself. Of equal importance, the ATG preparation also eliminates
natural killer (NK) cells. NK cells probably have no effect on the
implanted organ, but would act immediately to reject the newly
introduced bone marrow. Anti-human ATG obtained from any mammalian
host can also be used, e.g., ATG produced in pigs, although thus
far preparations of pig ATG have been of lower titer than
horse-derived ATG. ATG is superior to anti-NK monoclonal
Antibodies, as the latter are generally not lytic to all host NK
cells, while the polyclonal mixture in ATG is capable of lysing all
host NK cells. Anti-NK monoclonal antibodies can, however, be
used.
[0412] The presence of donor antigen in the host thymus during the
time when host T cells are regenerating post-transplant is critical
for tolerizing host T cells. If donor hematopoietic stem cells are
not able to become established in the host thymus and induce
tolerance before host T cells regenerate repeated doses of
anti-recipient T cell antibodies may be necessary throughout the
non-myeloablative regimen. Continuous depletion of host T cells may
be required for several weeks. Alternatively, e.g. if this approach
is not successful, and tolerance (as measured by donor skin graft
acceptance, specific cellular hyporesponsiveness in vitro, and
humoral tolerance) is not induced in these animals, the approach
can be modified to include host thymectomy. In thymectomized
recipients, host T cells do not have an opportunity to
differentiate in a host thymus, but must differentiate in the donor
thymus. If this is not possible, then the animal has to rely on
donor T cells developing in the donor thymus for immunocompetence.
Immunocompetence can be measured by the ability to reject a
non-donor type allogeneic donor skin graft, and to survive in a
pathogen-containing environment.
[0413] It may also be necessary or desirable to splenectomize the
recipient in order to avoid anemia.
[0414] Second, the recipient is administered low dose radiation in
order to make room for newly injected bone marrow cells. A
sublethal dose of between 100 rads and 400 rads whole body
radiation, plus 700 rads of local thymic radiation, has been found
effective for this purpose.
[0415] Third, natural antibodies are adsorbed from the recipient's
blood by hemoperfusion of a liver of the donor species. Pre-formed
natural antibodies (nAB) are the primary agents of graft rejection.
Natural antibodies bind to xenogeneic endothelial cells and are
primarily of the IgM class. These antibodies are independent of any
known previous exposure to antigens of the xenogeneic donor. B
cells that produce these natural antibodies tend to be T
cell-independent, and are normally tolerized to self antigen by
exposure to these antigens during development. The mechanism by
which newly developing B cells are tolerized is unknown. The liver
is a more effective adsorber of natural antibodies than the
kidney.
[0416] The fourth step in the non-myeloablative procedure is to
implant donor stromal tissue, preferably obtained from fetal liver,
thymus, and/or fetal spleen, into the recipient, preferably in the
kidney capsule. Stem cell engraftment and hematopoiesis across
disparate species barriers is enhanced by providing a hematopoietic
stromal environment from the donor species. The stromal matrix
supplies species-specific factors that are required for
interactions between hematopoietic cells and their stromal
environment, such as hematopoietic growth factors, adhesion
molecules, and their ligands.
[0417] As liver is the major site of hematopoiesis in the fetus,
fetal liver can also serve as an alternative to bone marrow as a
source of hematopoietic stem cells. The thymus is the major site of
T cell maturation. Each organ includes an organ specific stromal
matrix that can support differentiation of the respective
undifferentiated stem cells implanted into the host. Although adult
thymus may be used, fetal tissue obtained sufficiently early in
gestation is preferred because it is free from mature T lymphocytes
which can cause GVHD. Fetal tissues also tend to survive better
than adult tissues when transplanted. As an added precaution
against GVHD, thymic stromal tissue can be irradiated prior to
transplantation, e.g., irradiated at 1000 rads. As an alternative
or an adjunct to implantation, fetal liver cells can be
administered in fluid suspension.
[0418] Fifth, bone marrow cells (BMC), or another source of
hematopoietic stem cells, e.g., a fetal liver suspension, of the
donor are injected into the recipient. Donor BMC home to
appropriate sites of the recipient and grow contiguously with
remaining host cells and proliferate, forming a chimeric
lymphohematopoietic population. By this process, newly forming B
cells (and the antibodies they produce) are exposed to donor
antigens, so that the transplant will be recognized as self.
Tolerance to the donor is also observed at the T cell level in
animals in which hematopoietic stem cell, e.g., BMC, engraftment
has been achieved. When an organ graft is placed in such a
recipient several months after bone marrow chimerism has been
induced, natural antibody against the donor will have disappeared,
and the graft should be accepted by both the humoral and the
cellular arms of the immune system. This approach has the added
advantage of permitting organ transplantation to be performed
sufficiently long following transplant of hematopoietic cells,
e.g., BMT, e.g., a fetal liver suspension, that normal health and
immunocompetence will have been restored at the time of organ
transplantation. The use of xenogeneic donors allows the
possibility of using bone marrow cells and organs from the same
animal, or from genetically matched animals.
[0419] Finally, a short course of a help reducing agent, e.g., a
short course of high dose CsA is administered to the recipient. As
is described above, the course is begun at about the time of
implantation, or a little before, and is continued for a time about
equal to the time it takes for a mature T cell to be stimulated and
initiate rejection. While any of these procedures may aid the
survival of an implanted organ, best results are achieved when all
steps are used in combination. Methods of the invention can be used
to confer tolerance to allogeneic grafts, e.g., wherein both the
graft donor and the recipient are humans, and to xenogeneic grafts,
e.g., wherein the graft donor is a nonhuman animal, e.g., a swine,
e.g., a miniature swine, and the graft recipient is a primate,
e.g., a human.
[0420] While any of these procedures may aid the survival of an
implanted organ, best results are achieved when all steps are used
in combination. Methods of the invention can be used to confer
tolerance to allogeneic grafts, e.g., wherein both the graft donor
and the recipient are humans, and to xenogeneic grafts, e.g.,
wherein the graft donor is a nonhuman animal, e.g., a swine, e.g.,
a miniature swine, and the graft recipient is a primate, e.g., a
human.
[0421] In the case of xenogeneic grafts, the donor of the implant
and the individual that supplies either the tolerance-inducing
hematopoietic cells or the liver to be perfused should be the same
individual or should be as closely related as possible. For
example, it is preferable to derive implant tissue from a colony of
donors that is highly inbred.
Detailed Protocol
[0422] In the following protocol for preparing a cynomolgus monkey
for receipt of a kidney from a miniature swine donor, zero time is
defined as the moment that the arterial and venous cannulas of the
recipient are connected to the liver to be perfused.
[0423] On day -1 a commercial preparation (Upjohn) of horse
anti-human anti-thymocyte globulin (ATG) is injected into the
recipient. ATG eliminates mature T cells and natural killer cells
that would otherwise cause rejection of the bone marrow cells used
to induce tolerance. The recipient is anesthetized, an IV catheter
is inserted into the recipient, and 6 ml of heparinized whole blood
are removed before infection. The ATG preparation is then injected
(50 mg/kg) intravenously. Six ml samples of heparinized whole blood
are drawn for testing at time points of 30 min., 24 hours and 48
hours. Blood samples are analyzed for the effect of antibody
treatment on natural killer cell activity (testing on K562 targets)
and by FACS analysis for lymphocyte subpopulations, including CD4,
CD8, CD3, CDllb, and CD16. Preliminary data from both assays
indicate that both groups of cells are eliminated by the
administration of ATG. If mature T cells and NK cells are not
eliminated, ATG can be re-administered at later times in the
procedure, both before and after organ transplantation.
[0424] Sublethal irradiation is administered to the recipient
between days -1 and -8. Irradiation is necessary to eliminate
enough of the recipient's endogenous BMC to stimulate hematopoiesis
of the newly introduced foreign BMC. Sublethal total body
irradiation is sufficient to permit engraftment with minimal toxic
effects to the recipient. Whole body radiation (150 Rads) was
administered to cynomolgus monkey recipients from a bilateral
(TRBC) cobalt teletherapy unit at 10 Rads/min. Local irradiation of
the thymus (700 Rads) was also employed in order to facilitate
engraftment.
[0425] Natural antibodies are a primary cause of organ rejection.
To remove natural antibodies from the recipient's circulation prior
to transplantation, on day 0 an operative adsorption of natural
antibodies (nAB) is performed, using a miniature swine liver, as
follows. At -90 minutes the swine donor is anesthetized, And the
liver prepared for removal by standard operative procedures. At -60
minutes the recipient monkey is anesthetized. A peripheral IV
catheter is inserted, and a 6 ml sample of whole blood is drawn.
Through mid-line incision, the abdominal aorta and the vena cava
are isolated. Silastic cannulas containing side ports for blood
sampling are inserted into the blood vessels.
[0426] At -30 minutes the liver is perfused in situ until it turns
pale, and then removed from the swine donor and placed into cold
Ringers Lactate. The liver is kept cold until just prior to
reperfusion in the monkey. A liver biopsy is taken. At -10 minutes
the liver is perfused with warm albumin solution until the liver is
warm (37 degrees).
[0427] At 0 time the arterial and venous cannulas of the recipient
are connected to the portal vein and vena cava of the donor liver
and perfusion is begun. Liver biopsies are taken at 30 minutes and
60 minutes, respectively. Samples of recipient blood are also drawn
for serum at 30 minutes and 60 minutes respectively. At 60 minutes
the liver is disconnected from the cannulas and the recipient's
large blood vessels are repaired. The liver, having served its
function of adsorbing harmful natural antibodies from the recipient
monkey, is discarded. Additional blood samples for serum are drawn
from the recipient at 2, 24, and 48 hours. When this procedure was
performed on two sequential perfusions of swine livers, the second
liver showed no evidence of mild ischemic changes during perfusion.
At the end of a 30 minute perfusion the second liver looked grossly
normal and appeared to be functioning, as evidenced by darkening of
the venous outflow blood compared to the arterial inflow blood in
the two adjacent cannulas. Tissue sections from the livers were
normal, but immunofluorescent stains showed IgM on endothelial
cells. Serum samples showed a decrease in natural antibodies.
[0428] To promote long-term survival of the implanted organ through
T-cell and B-cell mediated tolerance, donor bone marrow cells are
administered to the recipient to form chimeric bone marrow. The
presence of donor antigens in the bone marrow allows newly
developing B cells, and newly sensitized T cells, to recognize
antigens of the donor as self, and thereby induces tolerance for
the implanted organ from the donor. To stabilize the donor BMC,
donor stromal tissue, in the form of tissue slices of fetal liver,
thymus, and/or fetal spleen are transplanted under the kidney
capsule of the recipient. Stromal tissue is preferably implanted
simultaneously with, or prior to, administration of hematopoietic
stem cells, e.g., BMC, or a fetal liver cell suspension.
[0429] To follow chimerism, two color flow cytometry can be used.
This assay uses monoclonal antibodies to distinguish between donor
class I major histocompatibility antigens and leukocyte common
antigens versus recipient class I major histocompatibility
antigens. BMC can in turn be injected either simultaneously with,
or preceding, organ transplant. Bone marrow is harvested and
injected intravenously (7.5.times.10.sup.8/kg) as previously
described (Pennington et al., 1988, Transplantation 45:21-26).
Should natural antibodies be found to recur before tolerance is
induced, and should these antibodies cause damage to the graft, the
protocol can be modified to permit sufficient time following BMT
for humoral tolerance to be established prior to organ
grafting.
[0430] The approaches described above are designed to
synergistically prevent the problem of transplant rejection. When a
kidney is implanted into a cynomolgus monkey following liver
adsorption of natural antibodies, without use of bone marrow
transplantation to induce tolerance, renal functions continued for
1-2 days before rejection of the kidney. When four steps of the
procedure were performed (adsorption of natural antibodies by liver
perfusion, administration of ATG, sublethal irradiation and bone
marrow infusion, followed by implant of a porcine kidney into
primate recipient), the kidney survived 7 days before rejection.
Despite rejection of the transplanted organ, the recipient remained
healthy.
[0431] When swine fetal liver and thymic stromal tissue were
implanted under the kidney capsule of two sublethally irradiated
SCID mice, 25-50% of peripheral blood leukocytes were of donor
lineage two weeks post-transplantation. A significant degree of
chimerism was not detected in a third animal receiving fetal liver
without thymus.
[0432] The methods of the invention may be employed in combination,
as described, or in part.
[0433] The method of introducing bone marrow cells may be altered,
particularly by (1) increasing the time interval between injecting
hematopoietic stem cells and implanting the graft; (2) increasing
or decreasing the amount of hematopoietic stem cells injected; (3)
varying the number of hematopoietic stem cell injections; (4)
varying the method of delivery of hematopoietic stem cells; (5)
varying the tissue source of hematopoietic stem cells, e.g., a
fetal liver cell suspension may be used; or (6) varying the donor
source of hematopoietic stem, cells. Although hematopoietic stem
cells derived from the graft donor are preferable, hematopoietic
stem cells may be obtained from other individuals or species, or
from genetically-engineered inbred donor strains, or from in vitro
cell culture.
[0434] Methods of preparing the recipient for transplant of
hematopoietic stem cells may be varied. For instance, recipient may
undergo a splenectomy or a thymectomy. The latter would preferably
be administered prior to the non-myeloablative regimen, e.g., at
day -14.
[0435] Hemoperfusion of natural antibodies may: (1) make use of
other vascular organs, e.g., liver, kidney, intestines; (2) make
use of multiple sequential organs; (3) vary the length of time each
organ is perfused; (4) vary the donor of the perfused organ.
Irradiation of the recipient may make use of: (1) varying the
absorbed dose of whole body radiation below the sublethal range;
(2) targeting different body parts (e.g., thymus, spleen); (3)
varying the rate of irradiation (e.g., 10 rads/min, 15 rads/min);
or (4) varying the time interval between irradiation and transplant
of hematopoietic stem cells; any time interval between 1 and 14
days can be used, and certain advantages may flow from use of a
time interval of 4-7 days. Antibodies introduced prior to
hematopoietic cell transplant may be varied by: (1) using
monoclonal antibodies to T cell subsets or NK cells (e.g.,
anti-NKH1.sub.A, as described by U.S. Pat. No. 4,772,552 to
Hercend, et al., hereby incorporated by reference); (2) preparing
anti-human ATG in other mammalian hosts (e.g., monkey, pig, rabbit,
dog); or (3) using anti-monkey ATG prepared in any of the above
mentioned hosts.
[0436] The methods of the invention may be employed with other
mammalian recipients (e.g., rhesus monkeys) and may use other
mammalian donors (e.g., primates, sheep, or dogs). As an
alternative or adjunct to hemoperfusion, host antibodies can be
depleted by administration of an excess of hematopoietic cells.
[0437] Stromal tissue introduced prior to hematopoietic cell
transplant, e.g., BMT, may be varied by: (1) administering the
fetal liver and thymus tissue as a fluid cell suspension; (2)
administering fetal liver or thymus stromal tissue but not both;
(3) placing a stromal implant into other encapsulated,
well-vascularized sites, or (4) using adult thymus or fetal spleen
as a source of stromal tissue.
[0438] Tolerance to fully MHC mismatched renal allografts in
chimeric swine
[0439] Overwhelming importance of major histocompatibility complex
(MHC) class II matching for achieving tolerance of kidney
transplants (KTx) in miniature swine has been demonstrated
previously. When class II antigens are matched, long-term specific
tolerance across MHC class I and minor antigens (MA) barrier, can
uniformly be induced by a short course of cyclosporine. However,
cyclosporine does not produce this effect across a full MHC
barrier. Bone marrow transplantation (BMT) across single-haplotype
class II MHC+MA barriers creates fully chimeric animals, as
confirmed by FCM. These chimeras recover normal cellular immune
function 2-3 months after BMT, as tested by MLR and CML. Four such
chimeric animals (see Table V, numbers 1-4) received kidney
transplants from donors class II matched to BMT donors and fully
mismatched to the recipients. A 12-day course of cyclosporine (10
mg/kg/day) was the only immunosuppression following kidney
transplantation. All 4 pigs have maintained normal creatinine (Cr)
values (<2 mg %) for longer than 300 days, and one recipient is
alive over 3 years with good kidney function (Cr<2 mg %) and
graft histology showing minimal borderline rejection. These results
demonstrate that induction of tolerance to class II antigens by BMT
allows a short course of cyclosporine to induce specific tolerance
(as tested by skin grafts) to fully allogeneic kidney transplants.
Subsequently, we have examined the specificity of this phenomenon
by determining if single-haplotype class II+MA mismatched BMT will
facilitate cyclosporine induced long-term acceptance of kidney
transplants completely mismatched to both the recipient and BMT
donor (Table V, numbers 5-10). A 12-day course of cyclosporine
allowed long-term survival of such kidney transplants in chimeric
recipients. Animal #5 was still alive and clinically well, with
normal Cr levels; histology however reveals borderline rejection.
Animal #6 was sacrificed 18 months after kidney transplant, with
deteriorating kidney function (Cr>11 mg %). Animal #7 was
sacrificed at 6 months after kidney transplant due to sepsis,
kidney transplants showed moderate tubulointestinal infiltrate
without signs of vascular injury. Both long-term survivors (pigs #3
& 5) were recently tested for anti-donor reactivity. CML and
MLR revealed specific unresponsiveness to the kidney transplant
donor type cells. Pigs #8-10 received kidney transplant from
outbred Yorkshire donors. These animals developed irreversible
renal failure, starting shortly after cessation of the cyclosporine
therapy. TABLE-US-00006 TABLE V # Recipient BMT Donor KTx Donor
Outcome (funct./pathol.) 1 aa (I.sup.aaII.sup.aa) aj
(I.sup.aaII.sup.ac) cc (I.sup.ccI.sup.cc) sac 1 y (good/normal) 2
ac (I.sup.acII.sup.ac) ag (I.sup.acII.sup.ad) dd
(I.sup.ddII.sup.dd) died >2.5 y (good/ chronic rej) 3 ac
(I.sup.acII.sup.ac) ag (I.sup.acII.sup.ad) dd (I.sup.ddII.sup.dd)
alive >3 y (good/border rej) 4 ac (I.sup.acII.sup.ac) ag
(I.sup.acII.sup.ad) dd (I.sup.ddII.sup.dd) sac 1 y (good/normal) 5
aa (I.sup.aaII.sup.aa) ah (I.sup.aaII.sup.ad) cc
(I.sup.ccII.sup.cc) alive >2.5 y (good/border rej) 6 aa
(I.sup.aaII.sup.aa) ah (I.sup.aaII.sup.ad) cc (I.sup.ccII.sup.cc)
sac >1.5 y (poor/chronic rej) 7 aa (I.sup.aaII.sup.aa) aj
(I.sup.aaII.sup.ac) dd (I.sup.ddII.sup.dd) sac 0.5 y
(good/infiltrate) 8 aa (I.sup.aaII.sup.aa) aj (I.sup.aaII.sup.ac)
YORK (I.sup.?II.sup.?) sac 30 d (poor/acute rej) 9 ac
(I.sup.acII.sup.ac) ch (I.sup.acII.sup.ad) YORK (I.sup.?II.sup.?)
sac 70 d (poor/acute rej) 10 ac (I.sup.acII.sup.ac) ch
(I.sup.acII.sup.ad) YORK (I.sup.?II.sup.?) sac 38 d (poor/acute
rej) sac = sacrificed; rej = rejection
[0440] Thus, a short postoperative course of cyclosporine in MHC
class II mismatched BMT recipients allows tolerance to be induced
to kidney transplants that are class II matched to the BMT donor.
Long-term unresponsiveness to kidney transplants that are fully
mismatched to both the recipient and BMT donor can be achieved in
some cases, apparently dependent on the degree of disparity at
multiple loci (compare with the difference between inbred and
outbred donors).
A Short Course of Cyclosporine to Suppress T Cell Function in
Primate Allogeneic Kidney Transplantation.
[0441] The following experiment shows that mixed chimerism,
obtained during a non-myeloablative protocol to achieve
engraftment, is capable of producing multilineage
lymphohematopoietic chimerism and long-term tolerance to renal
allografts between fully MHC mismatched cynomolgus monkeys.
Complete ablation of host lymphohematopoietic elements is neither
necessary nor desirable when bone marrow transplantation is
utilized as a tolerance-inducing regimen. Instead, it is
advantageous to achieve a state of mixed chimerism, in which the
presence of certain donor-derived elements induce specific
tolerance, while host-type antigen presenting cells maintain normal
immunocompetence.
[0442] It has been demonstrated in murine studies that removal of
mature host T cells is important in order to achieve mixed
chimerism. In initial studies using fully MHC mismatched cynomolgus
monkeys, a variety of monoclonal antibodies were tested to mature T
cell subsets (anti-CD4 and anti-CD8) as well as several sources of
anti-thymocyte globulin (ATG) as T cell depleting reagents.
Although these antibody treatments led to marked depletion of T
cells in the peripheral blood, biopsies of lymph nodes demonstrated
that residual T cells remained, often coated with antibody. In
order to further suppress T cell function, a one-month course of
treatment with an i.m. preparation of cyclosporine(CyA) in oil was
added to the preparative regimen. This treatment led to therapeutic
levels of cyclosporine during drug administration and to tapering
levels over a period of 3 weeks after the drug was discontinued.
The basic protocol for nonlethal preparative regimen was as
follows: Cynomolgus monkeys weighing 6 to 10 kg. (Charles River
Primates, Wilmington, Mass.) were treated with 300 Rads of WBI
either as a single dose (#M393) on day -6 or as two fractions of
150 Rads each on days -6 and -5 (#M3093 and #M3293). 700 Rads of
thymic irradiation was administered on day -1. Horse anti-human
thymocyte globulin (ATG) (Upjohn) was administered at 50 mg/kg i.m.
on days -2, -1 and 0. Orthotopic kidney transplantation was
performed on day 0 through a midline incision using end to side
anastamoses of the donor renal artery and renal vein into the
recipient aorta and vena cava, respectively, and using a
ureteroureteral anastomosis for urinary drainage. Bone marrow was
harvested from two donor ribs, prepared as a single cell
suspension, and infused i.v. into the recipient at the end of the
renal transplant. Treatment with cyclosporine (Sandimmune.RTM., 15
mg/kg/day, suspended in olive oil) i.m. was begun on day 0 and
continued for 27 days.
[0443] Monkey #393 became pancytopenic on day 8, and required three
blood transfusions with blood group matched, irradiated whole blood
over the next two weeks. However, peripheral blood components
recovered gradually thereafter, and were normal by day 30. Renal
function has remained normal for over 250 days, and a biopsy on day
215 showed a normal kidney.
[0444] Sequential flow cytometric (FCM) analyses were performed on
this animal utilizing a monoclonal anti-class I antibody previously
determined to distinguish donor from host, and analyzing lymphoid,
monocytic and neutrophil populations as determined by scatter
profiles. Clear evidence for chimerism in all three subpopulations
was detected first on day 10, and persisted at similarly high
levels until cyclosporine treatment was discontinued on day 27.
Thereafter, the levels of chimerism detected in each subpopulation
decreased, but chimerism was still detectable by FCM among
lymphocytes (1.5%) and monocytes (29%) as late as day 203, the last
day tested. In addition, a bone marrow aspirate on day 203 showed
11.2% donor cells by FCM.
[0445] Mixed lymphocyte reactions performed pre-transplant and on
day 159 post-transplant revealed a specific loss of anti-donor
reactivity (Table VI). TABLE-US-00007 TABLE VI 3.sup.rd Time Medium
Autologous Donor Party #1 3.sup.rd Party #2 Pre- 888 2434 5946 5571
6986 Transplant (CPM) Pre- -- 1.0 3.3 3.0 3.9 Transplant (Stim.
Index) Day 159 703 3410 2324 11298 9127 (CPM) Day 159 -- 1.0 0.6
4.2 3.1 (Stim. Index)
[0446] This result, combined with normal renal function and normal
kidney histology without any additional exogenous immunosuppression
since day 27, lead us to conclude that specific transplantation
tolerance has been induced in this animal through the establishment
of mixed chimerism. Two additional animals were treated by the same
protocol, but with an intravenous preparation of cyclosporine which
led to an abrupt fall of cyclosporine levels in the blood after
discontinuation rather than gradual tapering of levels over a
three-week period. One of these animals died of sepsis on day 12
during the period of aplasia, and the other lost evidence for
chimerism after discontinuation of cyclosporine and although still
alive on day 100, has shown a course consistent with chronic
rejection both by clinical and pathological criteria.
[0447] In order to reduce the toxicity of the preparative regimen,
we have subsequently modified the irradiation protocol. In one
animal (#3893) the WBI was decreased to 1.5 Gy. This animal failed
to develop mixed chimerism and rejected the kidney transplant
(Creatinine=12.1 on day 47). In two additional animals (#3093 and
#3292) the WBI was maintained at 3.0 Gy, but was fractionated to
1.5 Gy on two successive days (-6 and -5) rather than administered
as a single dose. Both of these animals developed mixed
multilineage chimerism, first detectable on day 11 and day 20
respectively. They showed much less toxicity from the preparative
regimen than did the animals receiving unfractionated irradiation,
and both remain chimeric with normal renal function at the time of
this writing (day 40 and day 25, respectively).
Pig to Monkey Kidney Xenotransplantation by a Mixed Chimerism
Approach
[0448] The following experiment shows the induction of tolerance in
monkeys to pig organs by means of a xenogeneic lymphohematopoietic
chimerism approach which has previously been shown effective in
concordant rodent systems. To date 16 Cynomolgus monkeys have
received pig kidney transplants along with xenogeneic bone marrow
from the same donor. The preparative regimen for these xenografts
included: 1) conditioning with non-myeloablative whole body
irradiation (WBI) and thymic irradiation; 2) removal of preformed
mAbs by perfusion of monkey blood through a pig liver; 3)
splenectomy; 4) T cell depletion with ATG and/or mAbs; and 5)
postoperative immunosuppression with cyclosporine and in some
animals anti-IgM mAbs. Ten animals have survived more than 4 days,
with the longest surviving 13 days, with normal renal function to
day 11. In this animal pig cells were detected in the peripheral
blood only at day 10 post-transplant, suggesting transient
xenogeneic chimerism. Two monkeys received only splenectomy and pig
liver perfusion prior to the kidney xenograft. In one of these
animals, in which no further immunosuppression was administered
post-transplant, the kidney functioned for 3 days, then rapidly
lost function, with complete rejection by day 5. Analysis of this
monkey's sera by flow cytometry indicated return of high titers of
IgM, which correlated with rejection. In the second animal
cyclosporine 15 mg/kg/day iv and 15 deoxyspergualin (DSG) 6
mg/kg/day iv were administered post-transplant. The kidney
functioned until day 7, then failed and was removed on day 8.
Pathologic examination showed a focal inflammatory infiltrate in
addition to patchy interstitial hemorrhage. The infiltrate
contained approximately 20% T cells, as determined by staining with
mAbs to CD3, CD4 and CD8. IgM natural antibodies were effectively
removed during liver perfusion in this animal, and strikingly, they
did not appear in the serum thereafter, IgG levels started to rise
on day 7, correlating with the beginning of renal dysfunction.
These results show 1) that natural antibody (IgM) responses can be
effectively eliminated by components of the preparative regimen
involving pig liver adsorption and post-operative suppression with
DSG; and 2) that T cell suppressive components of the preparative
regimen (i.e., irradiation, cyclosporine and ATG) are required to
prevent cellular and secondary (IgG) responses in these
experiments.
Other Embodiments
[0449] Stromal tissue introduced prior to hematopoietic cell
transplant, e.g., BMT, may be varied by: (1) administering the
fetal liver and thymus tissue as a fluid cell suspension; (2)
administering fetal liver or thymus stromal tissue but not both;
(3) placing a stromal implant into other encapsulated,
well-vascularized sites, or (4) using adult thymus or fetal spleen
as a source of stromal tissue.
[0450] The methods described herein for inducing tolerance to, or
promoting the acceptance of, an allogeneic antigen or allogeneic
graft can be used where, as between the donor and recipient, there
is any degree of mismatch at MHC loci or other loci which influence
graft rejection. Preferably, there is a mismatch at least one MHC
locus or at least one other locus that mediates recognition and
rejection, e.g., a minor antigen locus. With respect to class I and
class II MHC loci, the donor and recipient can be: matched at class
I and mismatched at class II; mismatched at class I and matched at
class II; mismatched at class I and mismatched at class II; matched
at class I, matched at class II. In any of these combinations other
loci which control recognition and rejection, e.g., minor antigen
loci, can be matched or mismatched. As stated above, it is
preferable that there is mismatch at least one locus. Mismatched at
MHC class I means mismatched for one or more MHC class I loci,
e.g., in the case of humans, mismatched at one or more of HLA-A,
HLA-B, or HLA-C, or in the case of swine, mismatch at one or more
SLA class I loci, e.g., the swine A or B loci. Mismatched at MHC
class II means mismatched at one or more MHC class II loci, e.g.,
in the case of humans, mismatched at one or more of a DP .alpha., a
DP.beta., a DQ .alpha., a DQ .beta., a DR .alpha., or a DR .beta.,
or in the case of swine, mismatch at one or SLA class II loci,
e.g., mismatch at DQ .alpha. or .beta., or DR .alpha. or
.beta..
[0451] The methods described herein for inducing tolerance to an
allogeneic antigen or allogeneic graft can be used where, as
between the donor and recipient, there is any degree of reactivity
in a mixed lymphocyte assay, e.g., wherein there is no, low,
intermediate, or high mixed lymphocyte reactivity between the donor
and the recipient. In preferred embodiments mixed lymphocyte
reactivity is used to define mismatch for class II, and the
invention includes methods for performing allogeneic grafts between
individuals with any degree of mismatch at class II as defined by a
mixed lymphocyte assay. Serological tests can be used to determine
mismatch at class I or II loci and the invention includes methods
for performing allogeneic grafts between individuals with any
degree of mismatch at class I and or II as measured with
serological methods. In a preferred embodiment, the invention
features methods for performing allogeneic grafts between
individuals which, as determined by serological and or mixed
lymphocyte reactivity assay, are mismatched at both class I and
class II.
[0452] The methods of the invention are particularly useful for
replacing a tissue or organ afflicted with a neoplastic disorder,
particularly a disorder which is resistant to normal modes of
therapy, e.g., chemotherapy or radiation therapy. Methods of the
invention can be used for inducing tolerance to a graft, e.g., an
allograft, e.g., an allograft from a donor which is mismatched at
one or more class I loci at one or more class II loci or at one or
more loci at each of class I and class II. In preferred
embodiments: the graft includes tissue from the digestive tract or
gut, e.g., tissue from the stomach, or bowel tissue, e.g., small
intestine, large intestine, or colon; the graft replaces a portion
of the recipient's digestive system e.g., all or part of any of the
digestive tract or gut, e.g., the stomach, bowel, e.g., small
intestine, large intestine, or colon.
[0453] Tolerance, as used herein, refers not only to complete
immunologic tolerance to an antigen, but to partial immunologic
tolerance, i.e., a degree of tolerance to an antigen which is
greater than what would be seen if a method of the invention were
not employed.
[0454] As is discussed herein, it is often desirable to expose a
graft recipient to irradiation in order to promote the development
of mixed chimerism. The inventor has discovered that it is possible
to induce mixed chimerism with less radiation toxicity by
fractionating the radiation dose, i.e., by delivering the radiation
in two or more exposures or sessions. Accordingly, in any method of
the invention calling for the irradiation of a recipient, e.g., a
primate, e.g., a human, recipient, of a xenograft or allograft, the
radiation can either be delivered in a single exposure, or more
preferably, can be fractionated into two or more exposures or
sessions. The sum of the fractionated dosages is preferably equal,
e.g., in rads or Gy, to the radiation dosage which can result in
mixed chimerism when given in a single exposure. The fractions are
preferably approximately equal in dosage. For example, a single
dose of 700 rads can be replaced with, e.g., two fractions of 350
rads, or seven fractions of 100 rads. Hyperfractionation of the
radiation dose can also be used in methods of the invention. The
fractions can be delivered on the same day, or can be separated by
intervals of one, two, three, four, five, or more days. Whole body
irradiation, thymic irradiation, or both, can be fractionated.
[0455] The inventor has also discovered that much or all of the
preparative regimen can be delivered or administered to a
recipient, e.g., an allograft or xenograft recipient, within a few
days, preferably within 72, 48, or 24 hours, of transplantation of
tolerizing stem cells and/or the graft. This is particularly useful
in the case of humans receiving grafts from cadavers. Accordingly,
in any of the methods of the invention calling for the
administration of treatments prior to the transplant of stem cells
and/or a graft, e.g., treatments to inactivate or deplete host
antibodies, treatments to inactivate host T cells or NK cells, or
irradiation, the treatment(s) can be administered, within a few
days, preferably within 72, 48, or 24 hours, of transplantation of
the stem cells and/or the graft. In particular, primate, e.g.,
human, recipients of allografts can be given any or all of
treatments to inactivate or deplete host antibodies, treatments to
inactivate host T cells or NK cells, or irradiation, within a few
days, preferably within 72, 48, or 24 hours, of transplantation of
stem cells and/or the graft. For example, treatment to deplete
recipient T cells and/or NK cells, e.g., administration of ATG, can
be given on day -2, -1, and 0, and WBI, thymic irradiation, and
stem cell, e.g., bone marrow stem cells, administered on day 0.
(The graft, e.g., a renal allograft, is transplanted on day 0).
[0456] Methods of the invention can include recipient
splenectomy.
[0457] As is discussed herein, hemoperfusion, e.g., hemoperfusion
with a donor organ, can be used to deplete the host of natural
antibodies. Other methods for depleting or otherwise inactivating
natural antibodies can be used with any of the methods described
herein. For example, drugs which deplete or inactivate natural
antibodies, e.g., deoxyspergualin (DSG) (Bristol), or anti-IgM
antibodies, can be administered to the recipient of an allograft or
a xenograft. One or more of, DSG (or similar drugs), anti-IgM
antibodies, and hemoperfusion, can be used to deplete or otherwise
inactivate recipient natural antibodies in-methods of the
invention. DSG at a concentration of 6 mg/kg/day, i.v., has been
found useful in suppressing natural antibody function in pig to
cynomolgus kidney transplants.
[0458] Some of the methods described herein use lethal irradiation
to create hematopoietic space, and thereby prepare a recipient for
the administration of allogeneic, xenogeneic, syngeneic, or
genetically engineered autologous, stem cells. In any of the
methods described herein, particularly primate or clinical methods,
it is preferable to create hematopoietic space for the
administration of such cells by non-lethal means, e.g., by
administering sub-lethal doses of irradiation, bone marrow
depleting drugs, or antibodies. The use of sublethal levels of bone
marrow depletion allows the generation of mixed chimerism in the
recipient. Mixed chimerism is generally preferable to total or
lethal ablation of the recipient bone marrow followed by complete
reconstitution of the recipient with administered stem cells.
[0459] Alternative methods for the inactivation of thymic T cells
are also included in embodiments of the invention. Some of the
methods described herein include the administration of thymic
irradiation to inactivate host thymic-T cells or to otherwise
diminish the host's thymic-T cell mediated responses to donor
antigens. It has been discovered that the thymic irradiation called
for in allogeneic or xenogeneic methods of the invention can be
supplemented with, or replaced by, other treatments which diminish
(e.g., by depleting thymic-T cells and/or down modulating one or
more of the T cell receptor (TCR), CD4 co-receptor, or CD8
co-receptor) the host's thymic-T cell mediated response. For
example, thymic irradiation can be supplemented with, or replaced
by, anti-T cell antibodies (e.g., anti-CD4 and/or anti-CD8
monoclonal antibodies) administered a sufficient number of times,
in sufficient dosage, for a sufficient period of time, to diminish
the host's thymic-T cell mediated response.
[0460] For best results, anti-T cell antibodies should be
administered repeatedly. E.g., anti-T cell antibodies can be
administered one, two, three, or more times prior to donor bone
marrow transplantation. Typically, a pre-bone marrow
transplantation dose of antibodies will be given to the patient
about 5 days prior to bone marrow transplantation. Additional,
earlier doses 6, 7, or 8 days prior to bone marrow transplantation
can also be given. It may be desirable to administer a first
treatment then to repeat pre-bone marrow administrations every 1-5
days until the patient shows excess antibodies in the serum and
about 99% depletion of peripheral T cells and then to perform the
bone marrow transplantation. Anti-T cell antibodies can also be
administered one, two, three, or more times after donor bone marrow
transplantation. Typically, a post-bone marrow transplant treatment
will be given about 2-14 days after bone marrow transplantation.
The post bone marrow administration can be repeated as many times
as needed. If more than one administration is given the
administrations can be spaced about 1 week apart. Additional doses
can be given if the patient appears to undergo early or unwanted T
cell recovery. Preferably, anti-T cell antibodies are administered
at least once (and preferably two, three, or more times) prior to
donor bone marrow transplantation and at least once (and preferably
two, three, or more times) after donor bone marrow
transplantation.
[0461] The following experiments show that additional T
cell-depleting antibodies can replace thymic irradiation in a
non-myeloablative conditioning regimen and allow allogeneic bone
marrow engraftment and donor-specific tolerance induction.
[0462] A low toxicity, non-myeloablative conditioning regimen that
allows allogeneic bone marrow engraftment and donor-specific
tolerance induction in mice has been previously described. A
regimen which includes pre-treatment with depleting doses of
anti-CD4 and anti-CD8 monoclonal antibodies on day -5,
administration of 3 Gy whole body irradiation and 7 Gy of thymic
irradiation on day 0 followed by administration of fully
MHC-mismatched donor bone marrow cells, allows the induction of
permanent mixed chimerism and skin graft tolerance. The thymic
irradiation step in this protocol was replaced with additional
anti-CD4 and anti-CD8 monoclonal antibody treatment. Multilineage
chimerism was compared in B10 (H-2.sup.b) mice receiving allogeneic
(B10.A, H-2.sup.a) bone marrow transplantation on day 0 following 3
Gy whole body irradiation with or without thymic irradiation, and
treatment with monoclonal antibodies by a variety of schedules pre
and post-bone marrow transplantation. Most (50 of 52) animals that
either received thymic irradiation or that received at least two
pre-bone marrow transplantation monoclonal antibody treatments
demonstrated long-term multilineage peripheral blood mixed
allogeneic chimerism (as demonstrated by flow cytometric analysis).
In contrast, only 1 of 8 animals receiving only one pre-bone marrow
transplantation monoclonal antibody treatment without thymic
irradiation developed lasting (more than 20 weeks) mixed chimerism.
AU chimeric animals accepted donor skin grafts for more than 100
days and rejected third party BALB/c grafts within 14 days.
Therefore, mixed chimerism and donor-specific skin graft acceptance
could be induced without the use of thymic irradiation if at least
2 pre-bone marrow transplantation monoclonal antibody treatments
were given. (The monoclonal antibody treatments were spaced about 5
days apart with the final treatment 1 day prior to bone marrow
transplantation.) However, levels of donor T cell reconstitution
were highest in animals receiving thymic irradiation or receiving
additional anti-T cell monoclonal antibody treatments following
bone marrow transplantation. Eleven of 20 mice receiving two
pre-bone marrow transplantation monoclonal antibody treatments (the
monoclonal antibody treatments were spaced about 5 days apart with
the final treatment 1 day prior to bone marrow transplantation) and
no thymic irradiation showed relatively low levels of donor T cell
reconstitution (less than 20% donor, more than 80% host) at 6
weeks, and 9 of these showed a marked loss of donor cells in all
lineages by 20 weeks. In contrast, 12 of 12 similarly-treated mice
receiving 1 or 2 additional post-bone marrow transplantation
monoclonal antibody treatments (the monoclonal antibody treatments
were spaced about 7 days apart with the first treatment 7 day after
bone marrow transplantation) showed high levels of donor T cell
reconstitution at 6 weeks (mean 86.+-.12% donor), and high levels
of donor reconstitution persisted in all lineages at 20 weeks.
Thus, a second dose of pre-bone marrow transplantation T
cell-depleting monoclonal antibodies can replace thymic irradiation
and allow tolerance induction in our regimen, but additional
monoclonal antibodies administered at one and two weeks post-bone
marrow transplantation may increase the ability to reliably induce
durable mixed chimerism and tolerance. The capacity of repeated
anti-T cell monoclonal antibody treatments to replace thymic
irradiation in this regimen most likely reflects their ability to
deplete host thymocytes that escape depletion by the initial
monoclonal antibody treatment. These monoclonal antibodies deplete
most host T cells and induce down-modulation of both TCR and CD4
and CD8 co-receptors on the few remaining cells. In these animals,
early migration of donor bone marrow-derived cells to the host
thymus is associated with complete clonal deletion of mature
host-type thymocytes with TCR that recognize donor antigens.
Although a small population of host T cells with such TCR persists
in the spleens of chimeras, these cells are anergic to stimulation
through their TCR. These cells may have escaped depletion by
down-modulating CD4 or CD8 after monoclonal antibody treatment.
Thus, this relatively non-toxic regimen achieves pluripotent
hematopoietic stem cell engraftment and specific tolerance by
ablating most of the existing T cell repertoire and allowing new T
cell development in the presence of intrathymic donor antigen, and
by inducing anergy among the few remaining host T cells in the
periphery.
[0463] Some of the methods herein include the administration of
hematopoietic stem cells to a recipient. In many of those methods,
hematopoietic stem cells are administered prior to or at the time
of the implantation of a graft (an allograft or a xenograft), the
primary purpose of the administration of hematopoietic stem cells
being the induction of tolerance to the graft. The inventors have
found that one or more subsequent administrations (e.g., a second,
third, fourth, fifth, or further subsequent administration) of
hematopoietic stem cells can be desirable in the creation and/or
maintenance of tolerance. Thus, the invention also includes methods
in which hematopoietic stem cells are administered to a recipient,
e.g., a primate, e.g., a human, which has previously been
administered hematopoietic stem cells as part of any of the methods
referred to herein.
[0464] While not wishing to be bound by theory the inventor
believes that repeated stem cell administration may promote
chimerism and possibly long-term deletional tolerance in graft
recipients. Accordingly, any method referred to herein which
includes the administration of hematopoietic stem cells can further
include multiple administrations of stem cells. In preferred
embodiments: a first and a second administration of stem cells are
provided prior to the implantation of a graft; a first
administration of stem cells is provided prior to the implantation
of a graft and a second administration of stem cells is provided at
the time of implantation of the graft. In other preferred
embodiments: a first administration of stem cells is provided prior
to or at the time of implantation of a graft and a second
administration of stem cells is provided subsequent to the
implantation of a graft. The period between administrations of
hematopoietic stem cells can be varied. In preferred embodiments a
subsequent administration of hematopoietic stem cell is provided:
at least two days, one week, one month, or six months after the
previous administration of stem cells; at least two days, one week,
one month, or six months after the implantation of the graft.
[0465] The method can further include the step of administering a
second or subsequent dose of hematopoietic stem cells: when the
recipient begins to show signs of rejection, e.g., as evidenced by
a decline in function of the grafted organ, by a change in the host
donor specific antibody response, or by a change in the host
lymphocyte response to donor antigen; when the level of chimerism
decreases; when the level of chimerism falls below a predetermined
value; when the level of chimerism reaches or falls below a level
where staining with a monoclonal antibody specific for a donor PBMC
antigen is equal to or falls below staining with an isotype control
which does not bind to PBMC's, e.g. when the donor specific
monoclonal stains less than 1-2% of the cells; or generally, as is
needed to maintain tolerance or otherwise prolong the acceptance of
a graft. Thus, method of the invention can be modified to include a
further step of determining if a subject which has received a one
or more administrations of hematopoietic stem cells is in need of a
subsequent administration of hematopoietic stem cells, and if so,
administering a subsequent dose of hematopoietic stem cells to the
recipient.
[0466] Any of the methods referred to herein can include the
administration of agents, e.g., 15-deoxyspergualin, mycophenolate
mofetil, brequinar sodium, or similar agents, which inhibit the
production, levels, or activity of antibodies in the recipient. One
or more of these agents can be administered: prior to the
implantation of donor tissue, e.g., one, two, or three days, or
one, two, or three weeks before implantation of donor tissue; at
the time of implantation of donor tissue; or after implantation of
donor tissue, e.g., one, two, or three days, or one, two or three
weeks after, implantation of a graft.
[0467] The administration of the agent can be initiated: when the
recipient begins to show signs of rejection, e.g., as evidenced by
a decline in function of the grafted organ, by a change in the host
donor specific antibody response, or by a change in the host
lymphocyte response to donor antigen; when the level of chimerism
decreases; when the level of chimerism falls below a predetermined
value; when the level of chimerism reaches or falls below a level
where staining with a monoclonal antibody specific for a donor PBMC
antigen is equal to or falls below staining with an isotype control
which does not bind to PBMC's, e.g. when the donor specific
monoclonal stains less than 1-2% of the cells; or generally, as is
needed to maintain tolerance or otherwise prolong the acceptance of
a graft.
[0468] The period over which the agent is administered (or the
period over which clinically effective levels are maintained in the
subject) can be long term, e.g., for six months or more or a year
or more, or short term, e.g., for less than a year, more preferably
six months or less, more preferably one month or less, and more
preferably two weeks or less. The period will generally be at least
about one week and preferably at least about two weeks in duration.
In preferred embodiments the period is two or three weeks long.
[0469] Preferred embodiments include administration of
15-deoxyspergualin (6 mg/kg/day) for about two weeks beginning on
the day of graft implantation.
[0470] Some of the methods referred to herein include steps in
which antibodies, e.g., preformed natural antibodies, are removed
from the blood of a recipient. For example, in some methods
antibodies are removed by hemoperfusion of an organ from the donor
species. The inventor has discovered that an .alpha.1-3 galactose
linkage epitope-affinity matrix, e.g., in the form of an affinity
column, is useful for removing antibodies from the recipient's
blood. Accordingly, the use of an .alpha. 1-3 galactose linkage
epitope-affinity matrix, e.g., matrix bound linear B type VI
carbohydrate, can be added to any method referred to herein and can
be used in addition to or in place of any antibody perfusion or
removal technique, e.g., organ perfusion, in any method referred to
herein.
[0471] Some of the methods referred to herein include the
administration of hematopoietic stem cells to a recipient. In many
of those methods hematopoietic stem cells are administered prior to
or at the time of the administration of a graft (an allograft or a
xenograft), the primary purpose of the administration of
hematopoietic stem cells being the induction of tolerance to the
graft. The inventors have found that administration of one or more
cytokines, preferably a cytokine from the species from which the
stem cells are derived, can promote tolerance or otherwise prolong
acceptance of a graft. Thus, the invention also includes methods in
a subject which has previously been administered donor
hematopoietic stem cells, is administered one or more cytokine,
e.g., a donor-species cytokine.
[0472] Although not wishing to be bound by theory, the inventor
believes that the cytokines, particularly donor species cytokines,
promote the engraftment and/or function of donor stem cells or
their progeny cells. Accordingly, any method referred to herein
which includes the administration of hematopoietic stem cells can
further include the-administration of a cytokine, e.g., SCF, IL-3,
or GM-CSF. In preferred embodiments the cytokine one which is
species specific in its interaction with target cells.
[0473] Administration of a cytokine can begin prior to, at, or
after the implantation of a graft or the implantation of stem
cells.
[0474] The method can further include the step of administering a
first or subsequent dose of a cytokine to the recipient: when the
recipient begins to show signs of rejection, e.g., as evidenced by
a decline in function of the grafted organ, by a change in the host
donor specific antibody response, or by a change in the host
lymphocyte response to donor antigen; when the level of chimerism
decreases; when the level of chimerism falls below a predetermined
value; when the level of chimerism reaches or falls below a level
where staining with a monoclonal antibody specific for a donor PBMC
antigen is equal to or falls below staining with an isotype control
which does not bind to PBMC's, e.g. when the donor specific
monoclonal stains less than 1-2% of the cells; or generally, as is
needed to maintain tolerance or otherwise prolong the acceptance of
a graft. Thus, method of the invention can be modified to include a
further step of determining if a subject is in need of cytokine
therapy and if so, administering a cytokine.
[0475] The period over which the cytokine(s) is administered (or
the period over which clinically effective levels are maintained in
the subject) can be long term, e.g., for six months of more or a
year or more, or short term, e.g., for a year or less, more
preferably six months or less, more preferably one month or less,
and more preferably two weeks or less. The period will generally be
at least about one week and preferably at least about two weeks in
duration.
[0476] In preferred embodiments the recipient is a primate, e.g., a
human, and the donor is from a different species, e.g., the donor
is a pig and: pig SCF is administered; pig IL-3 is administered; a
combination of pig SCF and pig IL-3 is administered; a pig specific
hematopoiesis enhancing factor, e.g., pig GM-SCF, is administered,
e.g., after the implantation of stem cells, e.g., about a month
after the implantation of stem cells.
[0477] A particularly preferred embodiment combines a short course,
e.g., about a month, of cyclosporine or a similar agent, a short
course, e.g., about two weeks, of 15-deoxyspergualin or a similar
agent, and a short course, e.g., about two weeks, of donor specific
cytokines, e.g., SCF and IL-3. In Cynomolgus monkeys receiving pig
grafts and pig stem cells, treatment which included the combination
of cyclosporine (15 mg/kg/day for 28 days), 15-deoxyspergualin (6
mg/kg/day for two weeks), and recombinant pig cytokines (SCF and
IL-3, each at 10 .mu.g/kg/day, i.v., for two weeks) was found to be
useful. Administration began at the time of graft implant. (The
monkeys were also given a preparative regimen consisting of
3.times.100 cGy total body irradiation on day -6, and -5 and
hemoperfusion with a pig liver just prior to stem cell
administration.)
[0478] An anti-CD2 antibody, preferably a monoclonal, e.g.,
BTI-322, or a monoclonal directed at a similar or overlapping
epitope, can be used in addition to or in place of any anti-T cell
antibodies (e.g., ATG) in any method referred to herein.
Sequence CWU 1
1
* * * * *