U.S. patent application number 09/895713 was filed with the patent office on 2002-09-12 for specific tolerance in transplantation.
Invention is credited to Sachs, David H..
Application Number | 20020127713 09/895713 |
Document ID | / |
Family ID | 25171169 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127713 |
Kind Code |
A1 |
Sachs, David H. |
September 12, 2002 |
Specific tolerance in transplantation
Abstract
In general, the invention features, a method of inducing
tolerance in a recipient mammal, e.g., a human, of a first species
to a tissue obtained from a mammal, e.g., a swine, e.g., a
miniature swine, of a second species, which tissue expresses an MHC
antigen, including inserting DNA encoding an MHC antigen of the
second species into a bone marrow hematopoietic stem cell from the
recipient mammal, and allowing the MHC antigen encoding DNA to be
expressed in the recipient.
Inventors: |
Sachs, David H.; (Newton,
MA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Family ID: |
25171169 |
Appl. No.: |
09/895713 |
Filed: |
June 29, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09895713 |
Jun 29, 2001 |
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08910287 |
Aug 13, 1997 |
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6306651 |
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08910287 |
Aug 13, 1997 |
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08759404 |
Dec 4, 1996 |
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08759404 |
Dec 4, 1996 |
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08266427 |
Jun 27, 1994 |
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5614187 |
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08266427 |
Jun 27, 1994 |
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08126122 |
Sep 23, 1993 |
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08126122 |
Sep 23, 1993 |
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07797555 |
Nov 22, 1991 |
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Current U.S.
Class: |
435/325 ;
424/93.21; 435/456 |
Current CPC
Class: |
A61K 39/39541 20130101;
C07K 14/70539 20130101; A61K 39/001 20130101; A61K 39/39541
20130101; A61K 38/13 20130101; A61K 35/28 20130101; A61P 37/00
20180101; C07K 16/28 20130101; A61K 35/28 20130101; A61K 38/13
20130101; A61K 2039/5156 20130101; A61K 35/26 20130101; A61K 48/00
20130101; A61K 35/26 20130101; A61K 2300/00 20130101; A61K 2035/122
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
435/325 ;
424/93.21; 435/456 |
International
Class: |
A61K 048/00; C12N
015/867 |
Goverment Interests
[0001] This invention was made with U.S. Government support under
grants from the National Institutes of Health. The government has
certain rights in the invention.
Claims
1. A method of inducing tolerance in a recipient mammal of a first
species to a tissue obtained from a mammal of a second species,
which tissue expresses an MHC antigen, said method comprising
inserting DNA encoding an MHC antigen of said second species into a
bone marrow hematopoietic stem cell from said recipient mammal, and
allowing said MHC antigen encoding DNA to be expressed in the
recipient.
2. The method of claim 1, wherein said cell is removed from said
recipient mammal prior to said insertion and returned to said
recipient mammal after said insertion.
3. The method of claim 1, wherein said recipient is a human.
4. The method of claim 1, wherein said mammal is a swine.
5. The method of claim 4, wherein said swine is a miniature
swine.
6. The method of claim 1, wherein said DNA is obtained from the
individual mammal from which said tissue is obtained.
7. The method of claim 1, wherein said DNA is obtained from an
individual mammal which is syngeneic to the individual mammal from
which said tissue is obtained.
8. The method of claim 1, wherein said DNA is obtained from an
individual mammal which is MHC identical to the individual mammal
from which said tissue is obtained.
9. The method of claim 1, wherein said DNA comprises an MHC class I
gene.
10. The method of claim 1, wherein said DNA comprises an MHC class
II gene.
11. The method of claim 1, wherein said DNA is inserted into said
cell by transduction.
12. The method of claim 11, wherein said DNA is inserted into said
cell by a retrovirus.
13. The method of claim 12, wherein said DNA is recipient is a
human and said retrovirus is a Moloney-based retrovirus.
14. A method of inducing tolerance in a recipient mammal to a
tissue obtained from a donor mammal of the same species, which
tissue expresses an MHC antigen, said method comprising inserting
DNA encoding an MHC antigen of said donor into a bone marrow
hematopoietic stem cell from said recipient mammal, and allowing
said MHC antigen encoding DNA to be expressed in the recipient.
15. The method of claim 14, wherein said cell is removed from said
recipient prior to said insertion and returned to said recipient
after said insertion.
16. The method of claim 14, wherein said recipient is a human.
17. The method of claim 14, wherein said DNA comprises an MHC class
I gene.
18. The method of claim 14, wherein said DNA comprises an MHC class
II gene.
19. The method of claim 14, wherein said DNA is inserted into said
cell by transduction.
20. The method of claim 19, wherein said DNA is inserted into said
cell by a retrovirus.
21. The method of claim 20, wherein said retrovirus is a
Moloney-based retrovirus.
Description
BACKGROUND OF THE INVENTION
[0002] The invention relates to conferring tolerance to foreign
tissue.
[0003] Tolerance to self major histocompatibility (MHC) antigens
occurs during T cell maturation in the thymus (McDuffie et al.,
1988, J. Immunol. 141:1840). It has been known since the landmark
experiments of Billingham, Brent and Medawar (Billingham et al.,
1953, Nat. 172:603) that exposure of the immune system to
allogeneic MHC antigens during ontogeny can cause the immune system
to lose reactivity to those antigens, thus leaving the animal
specifically tolerant into adult life. Ever since that time,
transplantation immunologists have sought means of inducing
tolerance in adult animals by production of lymphohema topoietic
chimeras. The induction of tolerance across MHC barriers in adult
mice by whole body irradiation (WBI) and bone marrow
transplantation (BMT) has been studied extensively in murine models
(Rayfield et al., 1983, Transplan. 36:183; Mayumi et al., 1989, J.
Exp. Med. 169:213; Sykes et al., 1988, Immunol. Today 9:23). This
approach has also recently been extended to the miniature swine
large animal model, in which it was demonstrated that bone marrow
transplants across MHC barriers led to the induction of long-term,
specific transplantation tolerance to kidney grafts from donors MHC
matched to the bone marrow donors (Guzzetta et al., 1991,
Transplan. 51:862).
[0004] The use of MHC mismatched BMT as a means of inducing
tolerance to organ allografts is usually accompanied by several
major disadvantages: the preparative regimen required for
allogeneic BMT involves lethal irradiation, with its inherent risks
and toxicities; clinical applicability is limited by the fact that
most potential recipients do not have an appropriate MHC-matched
donor, and BMT across MHC barriers causes severe graft-vs-host
disease (GVHD); and removing the T lymphocytes in allogeneic bone
marrow inocula (Rodt et al., 1971, Eur. J. Immunol. 4:25), to
prevent GVHD is associated with increased rates of engraftment
failure (Martin et al., 1988, Bone Marrow Transplant 3:445;
O'Reilly et al., 1985, Transplant. Proc 17:455; Soderling et al.
1985, J. Immunol. 135:941). While these drawbacks are generally
considered acceptable for the treatment of otherwise lethal
malignant diseases (e.g., leukemia), they would severely limit the
application of this methodology as a preparative regimen for organ
transplantation, in which non-specific immunosuppressive agents,
while not without major complications, are nevertheless
effective.
[0005] There have been previous reports of the use of cell lines
transfected with allogeneic class I and class II genes to
selectively modify the immune response to subsequent tissue grafts
bearing the foreign gene (Madsen et al., 1989, Transplant. Proc.
21:477). Graft prolongation by this technique was not permanent,
and the mechanism is unclear. Several laboratories have
demonstrated the utility of retroviral mediated gene transfer for
the introduction of new genetic material into totipotent
hematopoietic stem cells of mice. In general, these protocols
involve the transduction of bone marrow by recombinant retroviral
vectors ex vivo, with the subsequent reintroduction of the treated
cells into myeloablated recipients (for review, see Dick et al.,
1986, Trends in Genetics 2:165)
SUMMARY OF THE INVENTION
[0006] In general, the invention features a method of inducing
tolerance in a recipient mammal, e.g., a human, of a first species
to a tissue obtained from a mammal, e.g., a swine, e.g., a
miniature swine, of a second species, which tissue expresses an MHC
antigen, including inserting DNA encoding an MHC antigen of the
second species into a bone marrow hematopoietic stem cell from the
recipient mammal, and allowing the MHC antigen encoding DNA to be
expressed in the recipient.
[0007] 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 tissue is
obtained; the DNA is obtained from an individual mammal which is
syngeneic with the individual mammal from which the tissue is
obtained; the DNA is obtained from an individual mammal which is
MHC matched, and preferably identical, with the individual mammal
from which the tissue 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 14, preferably
30, more preferably 60, and most preferably 120 days, after the DNA
is introduced into the recipient.
[0008] In another aspect, the invention features a method of
inducing tolerance in a recipient mammal, e.g., a human, to a
tissue obtained from a donor mammal of the same species, which
tissue expresses an MHC antigen, including: inserting DNA encoding
an MHC antigen of the donor into a bone marrow hematopoietic stem
cell of the recipient mammal; and allowing the MHC antigen encoding
DNA to be expressed in the recipient.
[0009] 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
14, preferably 30, more preferably 60, and most preferably 120
days, after the DNA is introduced into the recipient.
[0010] Tolerance, as used herein, refers to the inhibition of a
graft recipient's ability to mount an immune response which would
otherwise occur, e.g., in response to the introduction of a nonself
MHC antigen into the recipient. Tolerance can involve humoral,
cellular, or both humoral and cellular responses.
[0011] Bone marrow hematopoietic stem cell, as used herein, refers
to a bone marrow cell which is capable of developing into mature
myeloid and/or lymphoid cells.
[0012] 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., of the DRB gene.
[0013] Miniature swine, as used herein, refers to partially inbred
miniature swine.
[0014] Graft, as used herein, refers to a body part, organ, tissue,
or cells.
[0015] The invention allows 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, methods
of the invention provide for the induction of specific
transplantation tolerance by somatic transfer of MHC genes.
[0016] Methods of the invention avoid the undesirable side effects
of broad spectrum immune suppressants which are often used in
transplantation. Drugs such as Prednisone, Imuran, CyA, and most
recently FK506, have all had 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 must stay on chronic immunosuppressive
therapy for the remainder of their lives, facing the major
complications of too much or too little immunosuppression,
infection and rejection, respectively.
[0017] Tolerance to transplantation antigens can be achieved
through induction of lymphohematopoietic chimerism by bone marrow
transplantation (BMT). However, successful BMT across MHC barriers
has 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. Methods of the invention, which induce specific
tolerance by reconstitution of the recipients 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.
[0018] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
DETAILED DESCRIPTION
[0019] The drawings are first briefly described.
[0020] Drawings
[0021] FIG. 1 is a diagram of the GS4.5 retroviral construct.
[0022] FIG. 2 is a diagram of the GS4.5 proviral genome and the
expected transcripts.
[0023] FIG. 3 is a representation of flow cytrometry profile of
tranduced cells.
[0024] FIG. 4 is a diagram of the transduction assay.
[0025] FIG. 5 is a diagram of genetic maps of the C57BL/10,
B10.AKM, and B10.MBR strains.
[0026] FIG. 6 is a diagram of the FACS profile of spleen cells from
a recipient of transduced bone marrow.
[0027] FIG. 7 is a graph of survival versus time in skin graft
experiments.
[0028] FIG. 8 is a diagram of FACS analysis of thymocytes from
graft rejectors, receptors, and controls.
[0029] FIG. 9 is a diagram of the N2-B19-H2b vector.
[0030] MHC Genes
[0031] 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 Histocompatability 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, Immunogentics 28:22-29, hereby incorporated by reference,
have been cloned and characterized.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] Transformation of bone marrow cells
[0036] 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, electoporation, particle gun bombardment, and
transduction by viral vectors, e.g., by retroviruses.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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, F. Virol.
64:3500-3508; Bodine et al., 1989, Prog. Clin. Biol. Res.
319:589-600.
[0041] Transduction efficiencies can be enhanced by preselection 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.
[0042] N2A or other Moloney-based vectors are preferred retroviral
vectors for transducing human bone marrow cells.
[0043] Preparative Regimen For The Introduction of Transformed Bone
Marrow Cells
[0044] To prepare for bone marrow cells the recipient must undergo
an ablation of the immune response which might otherwise resist
engraftment.
[0045] 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.
EXAMPLE 1
Sustained Expression of a Swine Class II Gene in Murine Bone Marrow
Hematopoietic Cells by Retroviral-mediated Gene Transfer
[0046] 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.
[0047] 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.
[0048] Details of retroviral constructs are given in FIG. 1. Two
types of retroviral constructs, GS4.4 and GS4.5, were prepared. The
diagram in FIG. 1 depicts the GS4.5 retroviral construct. The
arrows in FIG. 1 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. 1). 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.
[0049] 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 (G.sub.418.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.
[0050] 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.
[0051] 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).
[0052] Improvement of the Viral Titer of the C4 Clone 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 of 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.
[0053] 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.
[0054] Northern blot analysis of GS4.5-producing clones C4, A9, and
A4, each derived form 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.
[0055] 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.
[0056] 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-extract- ed, and
ethanol-precipitated. Pellets were then resuspended in 15.7%
(vol/vol) formaldehyde and serial dilutions were dotted onto
nitrocellulose membrane.
[0057] 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. 2 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.
[0058] 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. 3 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. 3 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.
[0059] The class II transduction assay was performed as diagramed
in FIG. 4. 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.
[0060] 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.
7:3923-3928; 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.
[0061] The SLA-DRB probe was an EcoRI cDNA fragment containing the
complete coding sequence of the DR p 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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).
EXAMPLE 2
Expression of Allogeneic Class II cDNA in Swine Bone Barrow Cells
Transduced With A Recombinant Retrovirus
[0068] 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.
[0069] 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.
[0070] 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 SLA.sup.c 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.
[0071] 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 SLA.sup.c donor, but also from both of the pooled
colony samples and a number of the individual colony samples.
[0072] 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 narrow 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.
[0073] 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 ug of polybrene per ml at
37.degree. C. for 5 hr. Myeloid progenitors were assayed by colony
formation in methylcellulose cultures using PRA-stimulated swine
lymphocyte conditioned medium as a source of growth factors.
Selective medium contained 1.2 mg/ml active G418.
[0074] 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.
EXAMPLE 3
Allogenic Tolerance
[0075] 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.
[0076] 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
(F.sub.2) 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 H-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.
5, 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.
[0077] Reconstitution of Myeloablated Mice with Transduced Bone
Marrow Eighty prospective donor B10.AKM mice were treated with 150
mg/kg 5FU 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).
[0078] 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. 6 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.
[0079] 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. 7, and
indicate marked, specific prolongation of survival of the B10.MBR
skin grafts on the recipients of K.sup.b-transduced BMC (FIG. 7B),
but not on recipients of control marrow (FIG. 7A). 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 animals 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. 8 shows FACS analysis on thymocytes from skin graft rejector
(FIGS. 8A, B) and skin graft acceptor (FIGS. 8C, D). Staining with
control HOPC1 antibody (FIGS. 8A, C) and with specific anti-K.sup.b
antibody (FIGS. 8B, 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 rejector 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.
[0080] 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.
[0081] 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-H2b. FIG. 9 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-.sub.2.sup.b cDNA library for other purposes, Waneck et al.,
1987, J. Exp. Med. 165:1358-1370.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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:
[0086] 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.
[0087] 2. Marrow will be harvested from donors on day -5, and T
cell depleted with mAbs and complement.
[0088] 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.
[0089] 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.
[0090] Cellular and serological assays are performed as
follows.
[0091] Anti-class I Cell-Mediated Lympholysis (CML) 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 of triplicates, so that
cytolytic capacity can be examined at a total of 5 different
responder:target ratios. .sup.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 a 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:
% Specific Lysis=
Experimental release-Spontaneous release.times.100%
Maximum release-Spontaneous release
[0092] Limiting dilution analyses: Responder and stimulator
(6.times.10.sup.5, 30 Gy irradiated) cells are concultured 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-labelled 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.
[0093] 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 ob 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.
[0094] 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 sued:
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-labelled
rat-anti-mouse granulocyte antibody Gr1 is purchased from
Pharmingen; FITC-labelled rat-anti-mouse IgM mAb is purchased from
Zymed; FITC-labelled rat-anti-mouse Thy1.2 mAb will be purchased
from Becton-Dickinson; FITC-labelled mouse-anti-human CD3 mAb Leu4
(Becton Dickenson) is used as a directly FITC labelled negative
control antibody.
[0095] 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.
[0096] Bone Marrow Manipulations and Assays were performed as
follows:
[0097] 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.
[0098] 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.
[0099] 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 ug/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.
[0100] Molecular methods were as follows:
[0101] 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.
[0102] 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.AKM, 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.
[0103] PCR analysis of DNA can be performed using primers
previously shown to be effective in our preliminary studies (see
FIG. 4):
[0104] 5' primer: 5'-GGCCCACACTCGCTGAGGTATTTCGTC-3' (covers 5' end
of .alpha.1 exon) (Seq. ID No. 3)
[0105] 3' primer: 5'-GCCAGAGATCACCTGAATAGTGTGA-3' (covers 5' end of
.alpha.2 exon) (Seq. ID No. 4)
[0106] 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.
[0107] 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 a
miniaturized RNA preparation procedure.
[0108] 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).
[0109] 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 K.sup.b
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.
[0110] 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 a 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.).
Sequence CWU 1
1
* * * * *