U.S. patent application number 13/681934 was filed with the patent office on 2013-11-28 for use of cytokines and mitogens to inhibit pathological immune responses.
This patent application is currently assigned to University of Southern California. The applicant listed for this patent is University of Southern California. Invention is credited to David Horwitz.
Application Number | 20130315939 13/681934 |
Document ID | / |
Family ID | 46276782 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315939 |
Kind Code |
A1 |
Horwitz; David |
November 28, 2013 |
Use of Cytokines and Mitogens to Inhibit Pathological Immune
Responses
Abstract
The invention is generally related to methods of treating
autoimmune diseases, including both antibody-mediated and
cell-mediated disorders.
Inventors: |
Horwitz; David; (Santa
Monica, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Southern California |
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|
|
|
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Assignee: |
University of Southern
California
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Family ID: |
46276782 |
Appl. No.: |
13/681934 |
Filed: |
November 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12608527 |
Oct 29, 2009 |
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13681934 |
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11927575 |
Oct 29, 2007 |
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12608527 |
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11507908 |
Aug 21, 2006 |
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11927575 |
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10650157 |
Aug 27, 2003 |
7115259 |
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11507908 |
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10028944 |
Dec 21, 2001 |
6797267 |
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10650157 |
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09564436 |
May 4, 2000 |
6358506 |
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10028944 |
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09186771 |
Nov 5, 1998 |
6228359 |
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09564436 |
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60132616 |
May 5, 1999 |
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60064507 |
Nov 5, 1997 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
C12N 2501/23 20130101;
C12N 2501/15 20130101; C12N 2501/599 20130101; A61K 35/17 20130101;
C12N 5/0636 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 35/14 20060101
A61K035/14 |
Goverment Interests
[0002] This invention was made with government support under
Contract No. A1041768 awarded by the National Institutes of Health.
The Government has certain rights in the invention.
Claims
2. A method for treating an autoimmune disorder in a patient
comprising: a) removing peripheral blood mononuclear cells (PBMC)
from said patient; b) treating said cells with a regulatory
composition to generate regulatory T cells, said regulatory
composition comprising anti-CD2 and anti-CD3; and; c) reintroducing
said regulatory T cells to said patient to suppress an aberrant
immune response.
3. A method for treating an autoimmune disorder in a patient
comprising: a) removing peripheral blood mononuclear cells (PBMC)
from said patient; b) treating said cells with a regulatory
composition to induce said cells to produce immunosuppressive
levels of TGF-.beta.; said regulatory composition comprising
anti-CD2 and anti-CD3; and; c) reintroducing said cells to said
patient to suppress aberrant immune responses.
4. A method according to claim 1 or 2, said regulatory composition
further comprising TGF-.beta..
5. A method according to claim 1 or 2, said regulatory composition
further comprising Il-2.
6. A method according to claim 1 or 2, said regulatory composition
further comprising TGF-.beta. and IL-2.
7. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+.
8. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and said regulatory composition further comprises
TGF-.beta..
9. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and said regulatory composition further comprises IL-2.
10. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and said regulatory composition further comprises TGF-.beta.
and IL-2.
11. A method according to claim 1 or 2, wherein said PBMC comprise
CD4+.
12. A method according to claim 1 or 2, wherein said PBMC comprise
CD4+ and said regulatory composition further comprises
TGF-.beta..
13. A method according to claim 1 or 2, wherein said PBMC comprise
CD4+ and said regulatory composition further comprises IL-2.
14. A method according to claim 1 or 2, wherein said PBMC comprise
CD4+ and said regulatory composition further comprises TGF-.beta.
and IL-2.
15. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and CD4+.
16. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and CD4+ and said regulatory composition further comprises
TGF-.beta..
17. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and CD4+ and said regulatory composition further comprises
IL-2.
18. A method according to claim 1 or 2, wherein said PBMC comprise
CD8+ and CD4+ and said regulatory composition further comprises
TGF-.beta. and IL-2.
19. A method according to claim 1 or 2, wherein said PBMC comprise
NK T cells.
20. A method according to claim 1 or 2, wherein said PBMC comprise
NK T cells and said regulatory composition further comprises
TGF-.beta..
21. A method according to claim 1 or 2, wherein said PBMC comprise
NK T cells and said regulatory composition further comprises
IL-2.
22. A method according to claim 1 or 2, wherein said PBMC comprise
NK T cells and said regulatory composition further comprises
TGF-.beta. and IL-2.
23. A method according to claim 1 or 2, wherein said aberrant
immune response is a cell-mediated autoimmune disease selected from
the group consisting of Hashimoto's disease, polymyositis, disease
inflammatory bowel disease, multiple sclerosis, diabetes mellitus,
rheumatoid arthritis, and scleroderma.
24. A method according to claim 2 wherein said wherein said
aberrant immune response is an antibody mediated disease selected
from the group consisting of pemphigus vulgaris, myasthenia gravis,
hemolytic anemia, thrombocytopenia purpura, Grave's disease,
dermatomyositis and Sjogren's disease.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. Ser. No.
12/608,527, filed Oct. 29, 2009, which is a continuation of U.S.
Ser. No. 11/927,575, filed Oct. 29, 2007 (abandoned); which is a
continuation of Ser. No. 11/507,908, filed Aug. 21, 2006
(abandoned), which is a continuation of U.S. Ser. No. 10/650,157,
filed Aug. 27, 2003, now U.S. Pat. No. 7,115,259; which is a
continuation of U.S. Ser. No. 10/028,944, filed Dec. 12, 2001, now
U.S. Pat. No. 6,797,267, which is a continuation of Ser. No.
09/564,436, filed May 4, 2000, now U.S. Pat. No. 6,358,506, which
claims the benefit of the filing date of U.S. Ser. No. 60/132,616,
filed May 5, 1999, and is a continuation in part of U.S. Ser. No.
09/186,771, filed Nov. 5, 1998, now U.S. Pat. No. 6,228,359, which
claims the benefit of the filing date of U.S. Ser. No. 60/064,507,
filed Nov. 5, 1997.
FIELD OF THE INVENTION
[0003] The field of the invention is generally related to methods
of treating autoimmune diseases, including both antibody-mediated
and cell-mediated disorders.
BACKGROUND OF THE INVENTION
[0004] Autoimmune diseases are caused by the failure of the immune
system to distinguish self from non-self. In these diseases, the
immune system reacts against self tissues and this response
ultimately causes inflammation and tissue injury. Autoimmune
diseases can be classified into two basic categories:
antibody-mediated diseases such as systemic lupus erythematosus
(SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia,
thrombocytopenia purpura, Grave's disease, Sjogren's disease and
dermatomyositis; and cell-mediated diseases such as Hashimoto's
disease, polymyositis, disease inflammatory bowel disease, multiple
sclerosis, diabetes mellitus, rheumatoid arthritis, and
scleroderma.
[0005] In many autoimmune diseases, tissue injury is caused by the
production of antibodies to native tissue. These antibodies are
called autoantibodies, in that they are produced by a mammal and
have binding sites to the mammal's own tissue. Some of these
disorders have characteristic waxing and waning of the amount of
circulating autoantibodies causing varying symptoms over time.
[0006] Of the different types of antibody-mediated autoimmune
disorders, SLE is a disorder that has been well studied and
documented. SLE is a disorder of generalized autoimmunity
characterized by B cell hyperactivity with numerous autoantibodies
against nuclear, cytoplasmic and cell surface antigens. This
autoimmune disease has a multifactorial pathogenesis with genetic
and environmental precipitating factors (reviewed in Hahn, B. H.,
Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 69-76 (D. J.
Wallace et al. eds., Williams and Wilkins, Baltimore)). Among the
numerous lymphocyte defects described in SLE is a failure of
regulatory T cells to inhibit B cell function (Horwitz, D. A.,
Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 155-194 (D. J.
Wallace et al. eds., Williams and Wilkins, Baltimore)). Sustained
production of polyclonal IgG and autoantibodies in vitro requires T
cell help (Shivakumar, S. et al. (1989), J Immunol
143:103-112).
[0007] Regulatory T cells can down-regulate antibody synthesis by
lytic or cytokine-mediated mechanisms. The latter involve
transforming growth factor-beta (TGF-.beta.) and other inhibitory
cytokines (Wahl, S. M. (1994), J Exp Med 180:1587-190). Circulating
B lymphocytes spontaneously secreting antibodies are increased in
patients with active SLE (Klinman, D. M. et al. (1991), Arthritis
Rheum 34:1404-1410).
[0008] Clinical manifestations of SLE include a rash (especially on
the face in a "butterfly" distribution), glomerulonephritis,
pleurisy, pericarditis and central nervous system involvement. Most
patients are women, and are relatively young (average age at
diagnosis is 29).
[0009] The treatment of SLE depends on the clinical manifestations.
Some patients with mild clinical symptoms respond to simple
measures such as nonsteroidal anti-inflammatory agents. However,
more severe symptoms usually require steroids with potent
anti-inflammatory and immunosuppressive action such as prednisone.
Other strong immunosuppressive drugs which can be used are
azathioprine and cyclophosphamide. The steroids and other
immunosuppressive drugs have side effects due to the global
reduction of the mammal's immune system. There is presently no
ideal treatment for SLE and the disease cannot be cured.
[0010] Currently, considerable attention has been focused on the
identity of genes which enhance the susceptibility or resistance to
SLE, the identification of antigenic determinants that trigger the
disease, the molecular mechanisms of T cell activation which
results in survival or apoptosis, cytokines which determine T cell
function, and the properties of the autoantibody-forming B cells.
Many examples of T cell dysregulation in SLE have been described
(reviewed in Horwitz, D. A. et al., Dubois' Lupus Erythematosus,
5th Ed. (1997), pp. 83-96 (D. J. Wallace et al. eds., Williams and
Wilkins, Baltimore). Although it is well recognized that the
primary role of certain lymphocytes is to down-regulate immune
responses, progress in elucidating the identity and mechanisms
required for generation of these cells has been slow.
[0011] Interleukin-2 (IL-2) has previously been considered to have
an important role in the generation of antigen non-specific T
suppressor cells. Anti-IL-2 antibodies given to mice coincident
with the induction of graft-versus-host-disease resulted in several
features of SLE (Via, C. S. et al. (1993), International Immunol.
5:565-572). Whether IL-2 directly or indirectly is important in the
generation of suppression has been controversial (Fast, L. D.
(1992), J. Immunol. 149:1510-1515; Hirohata, S. et al. (1989), J.
Immunol. 142:3104-3112; Baylor, C. E. (1992), Advances Exp. Med.
Biol. 319:125-135). Recently, IL-2 has been shown to induce CD8+
cells to suppress HIV replication in CD4+ T cells by a non-lytic
mechanism. This effect is cytokine mediated, but the specific
cytokine has not been identified (Kinter, A. L. et al. Proc. Natl.
Acad. Sci. USA 92:10985-10989; Barker, T. D. et al. (1996), J.
Immunol. 156:4478-4483). T cell production of IL-2 is decreased in
SLE (Horwitz, D. A. et al. (1997), Dubois' Lupus Erythematosus, 5th
Ed. (1997), pp. 83-96, D. J. Wallace et al. eds., Williams and
Wilkins, Baltimore).
[0012] CD8+ T cells from subjects with SLE sustain rather than
suppress polyclonal IgG production (Linker-Israeli, M. et al.
(1990), Arthritis Rheum. 33:1216-1225). CD8+ T cells from healthy
donors can be stimulated to enhance antibody production (Takahashi,
T. et al. (1991), Clin. Immunol. Immunopath. 58:352-365). However,
neither IL-2 nor CD4+ T cells, by themselves, were found to induce
CD8+ T cells to develop strong suppressive activity. When NK cells
were included in the cultures, strong suppressive activity appeared
(Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942). It is
believed that the contribution of NK cells in the culture was to
produce transforming growth factor beta (TGF-.beta.) in its active
form. It was then discovered that non-immunosuppressive (2-10
pg/ml) concentrations of this cytokine served as a co-factor for
the generation of strong suppressive effects on IgG and IgM
production (Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942).
In addition, it is believed that NK cells are the principal source
of TGF-.beta. in unstimulated lymphocytes (Gray, J. D. et al.
(1998), J. Immunol. 160:2248-2254).
[0013] TGF-.beta.s are a multifunctional family of cytokines
important in tissue repair, inflammation and immunoregulation
(Massague, J. (1980), Ann. Rev. Cell Biol. 6:597). TGF-.beta. is
unlike most other cytokines in that the protein released is
biologically inactive and unable to bind to specific receptors
(Sporn, M. B. et al. (1987) J. Cell Biol. 105:1039-1045). The
latent complex is cleaved extracelluarly to release active cytokine
as discussed below. The response to TGF-.beta. requires the
interaction of two surface receptors (TGF-.beta.-R1) and
TGF-.beta.-R2) which are ubiquitously found on mononuclear cells
(Massague, J. (1992), Cell 69:1067-1070). Thus, the conversion of
latent to active TGF-.beta. is the critical step which determines
the biological effects of this cytokine.
[0014] It was found that SLE patients have decreased production of
TGF-.beta.1 by NK cells. Defects in constitutive TGF-.beta.
produced by NK cells, as well induced TGF-.beta. were documented in
a study of 38 SLE patients (Ohtsuka, K. et al. (1998), J. Immunol.
160:2539-2545). Neither addition of recombinant IL-2 or TNF-alpha,
or antagonism of IL-10 normalized the TGF-.beta. defect in SLE.
Decreased production of TGF-.beta. in SLE did not correlate with
activity of disease and, therefore, may be a primary defect.
[0015] Systemic administration of TGF-.beta., IL-2, or a
combination of both can lead to serious side effects. These
cytokines have numerous effects on different body tissues and are
not very safe to deliver to a patient systemically. It is,
therefore, an object of the invention to provide methods and kits
for treating mammalian cells that are responsible for controlling
the regulation of autoantibodies to increase the population of
cells that down regulate auto-antibody production.
SUMMARY OF THE INVENTION
[0016] In accordance with the objects outlined herein, the present
invention provides methods for inhibiting immune responses in a
sample of ex vivo peripheral blood mononuclear cells (PBMCs)
comprising adding an regulatory composition to the cell
population.
[0017] In an additional aspect, the present invention provides
methods for treating an autoimmune disorder in a patient. The
methods comprise removing peripheral blood mononuclear cells (PBMC)
from the patient and treating the cells with an regulatory
composition for a time sufficient to suppress inflamation and
tissue injury. In particular, the methods of the present invention
suppress antibody production or induce cells to down regulate
antibody production and enhance cell mediated immune responses in
patients with antibody mediated autoimmune diseases. The treated
cells are then reintroduced to the patient, with a resulting
amelioration of the autoimmune symptoms. The regulatory composition
preferably comprises TGF-.beta. and agents which enable T cells to
respond to TGF-.beta..
[0018] In an additional aspect, the present invention provides
methods for treating cell-mediated autoimmune diseases. The methods
comprise removing peripheral blood mononuclear cells (PBMC) from
the patient and treating the cells with an regulatory composition
for a time sufficient to suppress tissue injury by immune cells.
The treated cells are then reintroduced to the patient, with a
resulting amelioration of the autoimmune symptoms. The regulatory
composition preferably comprises TGF-.beta. and agents which enable
T cells to respond to TGF-.beta..
[0019] In an additional aspect, the invention provides kits for the
treatment of an autoimmune disorder in a patient. The kits comprise
a cell treatment container adapted to receive cells from a patient
with an antibody-mediated autoimmune disorder or a cell-mediated
disorder and at least one dose of an regulatory composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows that incubation of SLE patients PBMC with IL-2
and TGF-.beta. decreases spontaneous immunoglobulin production.
PBMC (2.times.10.sup.5/well) were cultured in AIM-V serum free
medium with or without IL-2 (10 U/ml) and TGF-.beta. (10 pg/ml).
After 3 days, the wells were washed three times and fresh AIM-V
medium added. Supernatants were collected from the wells after a
further 7 days and IgG content determined by an ELISA.
[0021] FIG. 2 shows that both IL-2 and TGF-.beta. significantly
decrease spontaneous IgG production. The values represent the
mean.+-.SEM of IgG (.mu.g/ml) produced by the 12 SLE patients PBMC
cultured as described in legend to FIG. 1 except some cells were
also incubated with IL-2 (10 U/ml) or TGF-.beta. (10 pg/ml)
only.
[0022] FIGS. 3A and 3B show that anti-TGF-.beta. can reverse the
effects of IL-2. SLE patients PBMC was cultured for three days in
the presence (solid bars) or absence (spotted bars) of IL-2 (10
U/ml). Included in these cultures was medium, anti-TGF-.beta. (10
m/ml) or control mouse IgG1 (10 m/ml). After 3 days the wells were
washed and fresh AIM-V medium added. Supernatants were collected
after a further seven days and assayed for IgG (FIG. 3A) or
anti-nucleoprotein (NP) (FIG. 3B) content by an ELISA.
[0023] FIGS. 4A, 4B and 4C depict regulatory effects of CD8+ T
cells on antibody production. (A) Synergism between NK cells and
CD8+ cells in the suppression of IgG production in a healthy
subject. CD4+ cells and B cells were stimulated with anti-CD2 and
the effects of CD8+ cells and NK cells were examined. The
combination of NK and CD8+ cells markedly inhibited anti-CD2
induced IgG production we previously reported (Gray, J. D. et al.
(1998), J Immunol 160:2248-2254; Gray, J. D. et al. (1994), J Exp
Med 180:1937-1942). (B) NK cells and CD8+ cells enhance IgG
synthesis in SLE. CD4+ cells from a patient with active SLE and
resting B cells from a healthy subject were stimulated with
anti-CD2. Enhancement of IgG production by SLE CD8+ cells was
markedly increased by the addition of NK cells. (C) Cytokine
normalization of CD8+ T cell function in SLE. In parallel with the
study shown in FIG. 4B, CD4+ T cells from this patient were
stimulated with anti-CD2 in the presence or absence of CD8+ T
cells. IL-2 (10 U/ml) and/or TGF-.beta. (2 pg/ml) was added where
indicated. These cytokines abolished the helper effects of these
CD8+ cells and enabled them to inhibit IgG production by 75%.
[0024] FIGS. 5A and 5B depict the lymphocyte production of
TGF-.beta.1 by unstimulated and anti-CD2 stimulated cells. PBL from
healthy donors and patients of SLE and RA were added to microtiter
plates at 1.times.10.sup.5/well. Some wells received the anti-CD2
mAbs GT2 (1:40) and T11 (1:80). After 2 days at 37 DC, supernatants
were harvested and assayed for active and total TGF-131.
Significant p values are indicated.
[0025] FIGS. 6A and 6B depict the effects of TGF-.beta. on T cell
production of TNF-.alpha. and IL-10. Purified T cells
(1.times.10.sup.5 cells/well) in serum free AIM V medium were added
to flat bottomed microwells and stimulated with a low dose (0.5
.mu.g/ml) or high dose (5 .mu.g/ml) of Con A with or without IL-2
(10 U/ml) in the presence or absence of TGF-.beta. (1 ng/ml).
Supernatants were collected at 2 days and 5 days and tested for
TNF-.alpha. and IL-10 by ELISA. Maximal production of TGF-.beta.
was found at 2 days and for IL-10 at 5 days. TGF-.beta. abolished
IL-10 production and up-regulated TNF-.alpha. production.
[0026] FIGS. 7A and 7B show that TNF-.alpha. is an essential
intermediate for the generation of regulatory T cells by
TGF-.beta.. Purified CD8+ cells were incubated overnight with Con A
(2.5 .mu.g/ml), 11-2 (10U) and TGF-.beta. (10 pg/ml). After washing
these cells were added to CD4+ B cells and stimulated with
anti-CD2. To some wells anti-TNF-.alpha. antibody (10 .mu.g/ml) or
isotype control antibody (10 .mu.g/ml) was included. After 7 days,
supernatants were evaluated for IgG content by an ELISA. The
regulatory activity of conditioned CD8+ cells was reversed by
anti-TNF-.alpha..
[0027] FIGS. 8A-8C depict that enhanced production of Th1 cytokines
by TGF-.beta. primed T cells is dependent upon TNF-.alpha..
Purified naive T cells were cultured with Con A (5 .mu.g/ml) an
IL-2 (10 U/ml) in the presence of TGF-.beta. (1 ng/ml). Some wells
also received neutralizing anti-TNF-.alpha. antibody (10 .mu.g/ml)
or isotype control antibody (10 .mu.g/ml). After 5 days of culture,
the cells were washed and replated at 1.times.10.sup.5 cells/well
in fresh medium. The next day they were restimulated with Con A and
IL-2 for 6 hours and, in the presence of brefeldin A (10 .mu.g/ml),
the cells were stained for CD8 and the cytokines indicated. The
percentage of CD8+ and CD8- cells expressing TNF-.alpha., IL-2 and
IFN-.gamma. is shown. Note that neutralization of TNF-.alpha. in
primary cultures abolished the enhancing effects of TGF-.beta. on
production of Th1 cytokines.
[0028] FIGS. 9A-9C depict the effect of TGF-.beta. in generating
suppressors of cytotoxic T cell activity. T cells from donor A
prepared by E rosetting were divided into two portions. One portion
was used as responders for an allogeneic mixed lymphocyte reaction
(allo-MLR). The other portion was used to prepare the T cell
subsets indicated by negative selection after staining the cells
with appropriate monoclonal antibodies and removing the stained
cells using immunomagnetic beads. The responder T cells were mixed
with stimulator cells from donor B (irradiated T cell depleted
peripheral blood mononuclear cells) and cultured for 5 days to
generate killer cells. Controls consisted of the T cell subsets
cultured for 5 days with or without stimulator cells. Afterwards,
the cells were washed, counted and used to assess allo-cytotoxic T
cell activity. The responder cells from donor A were mixed with
chromium labeled lymphoblasts from donor B in the effector to
target cell ratios shown and chromium release was measured in a
standard 4 hour assay (open squares). T cells subsets cultured with
stimulators were added in a ratio of 1 regulatory cell per 4
responder cells (open circles). T cell subsets cultured with
stimulators with TGF-beta are shown as closed circles. In all
experiments, the maximal effects of TGF-beta were on naive CD4
CD45RA+CD45RO-- cells.
[0029] FIGS. 10A and 10B depict the effect of CD4 cells primed with
TGF-beta on allo-cytotoxic T lymphocyte (CTL) activity. The
addition of CD4 CD45RA cells that had been cultured for 5 days
without stimulators had no effect on CTL activity (result not
shown). Culturing these T cells with stimulator cells resulted in
modest to moderate suppressive activity. In all experiments,
culture of these T cells with TGF-beta 1 ng/ml markedly suppressed,
or abolished allo-CTL activity.
[0030] FIGS. 11A and 1 lB demonstrate that regulatory T cells
require cell contact to inhibit CTL activity. Regulatory CD4 cells
were prepared from CD4 CD45RA cells cultured with TGF-beta as
described above. Some of these cells were mixed with responder and
chromium-labeled target cells, while others were separated from the
killer cells by a membrane. Inhibition of cytotoxic T lymphocyte
activity (CTL) was only observed when the regulatory T cells were
in direct contact with the killer cells.
[0031] FIG. 12 depicts suppression of lymphocyte proliferation by
regulatory CD4+ T cells induced with TGF-.beta.. Naive CD4+ T cells
from donor A were mixed with stimulator cells as described above
and added to fresh responder and stimulator cells at the indicated
ratios. The bars show the uptake of tritiated thymidine.+-.SEM
after 7 days of culture. The lightly shaded bar (Nil) indicates the
proliferative response of the responder T cells without added CD4+
cells. The darkly shaded bar indicates the effect of control CD4+
cells which had been cultured with stimulator cells without
TGF-.beta.. The black bar indicates the effect of CD4+ cells that
had been mixed with stimulator cells in the presence of TGF-.beta.
(1 ng/ml). The effect of these CD4+ cells on the proliferative
response of fresh responder cells added to irradiated stimulator
cells after 7 days of culture is shown. The bars indicate the mean
uptake of tritiated thymidine.
[0032] FIG. 13 depicts the regulatory activity of CD25+ CD4 T
cells. CD4+ cells were stimulated with irradiated allogeneic non-T
cells+TGF-.beta. (1 ng/ml) for 5 days. After washing, the CD4+
cells were stained with DII and fresh responder T cells were
stained with carboxyfluorescein (CFSE). control or TGF-.beta.
primed CD4+ cells were added to the responder T cells and
allo-stimulator cells in a 1:4 ratio. After 5 days, the cells were
harvested and analyzed by flow cytometry. The intensity of CFSE in
CD8+ cells was determined by gating on DII negative cells. Note
that the addition of TGF-.beta. primed CD4+ cells to responder T
cells markedly decreased cell division by CD8+ cells.
[0033] FIGS. 14A and 14B depict that regulatory CD4+ cells express
CD25+ (IL-2) receptors on their surface. Control and TGF-.beta.
induced CD4+ regulatory T cells were prepared as described above.
After conditioning with allo-stimulator cells and TGF-.beta., the
CD4+ cells were divided into CD25+ and CD25- subsets by cell
sorting and added to fresh responder T cells and irradiated
stimulator cells. The capacity of these responder cells to kill
stimulator T lymphoblasts is shown in a standard 4 hour chromium
release assay.
[0034] In FIG. 14A, the open boxes show CTL activity without
additional CD4+ cells. Control or TGF-.beta. induced regulatory T
cells were added in a 1:4 ratio with responder cells. The open
circles show that the control CD4+ cells did not alter CTL
activity. The solid circles show that TGF-.beta. induced CD4+ cells
almost completely suppressed CTL activity. The solid diamonds show
that the suppressive activity was contained exclusively in the
CD25+ subset. The CD25- subset (solid squares) did not have
suppressive activity.
[0035] FIG. 14B shows the effect of decreasing the numbers of CD4+
regulatory cells added to the MLR. Decreasing the number to only 3%
had a minimal effect in decreasing the suppressive effects.
[0036] FIGS. 15A and 15B depict that repeated stimulation of T
cells with a low dose of staphylococcal enterotoxin B (SEB) induces
T cells to produce immunosuppressive levels of TGF-.beta.. CD4+ T
cells were stimulated with SEB (0.01 ng/ml) and irradiated B cells
as superantigen presenting cells with our without TGF-.beta. at the
times indicated by the arrows. Active TGF-.beta. was measured 2 or
5 days later.
[0037] FIG. 16 depicts that repeated stimulation of CD4+ T cells
with a low dose of SEB enables these cells to produce
immunosuppressive levels of TGF-.beta.. CD4+ T cells were
stimulated with SEB (0.01 ng/ml) and irradiated B cells as
superantigen presenting cells with or without TGF-.beta. at the
times indicated by the arrows. Active TGF-.beta. was measured 2 or
5 days later.
[0038] FIGS. 17A-17D show the effects of SEB on naive
(CD45RA+CD45RO--) CD4+ and CD8+ T cells. The cells were stimulated
with SEB every 5th day for a total of three stimulations. The
percentages of each T cell subset and the cells expressing the CD25
IL-2 receptor activation marker were determined after each
stimulation. Figures A and C show that if TGF-.beta. 1 ng/ml was
included in the initial stimulation, CD4+ T cells became the
predominant subset in the cultures after repeated stimulation.
Figures B and D show that CD25 expression by SEB stimulated cells
decreases by the third stimulation in control cultures. However,
CD25 expression remains high if the T cells have been primed with
TGF-.beta..
DETAILED DESCRIPTION
[0039] The present invention is directed to methods of treating
autoimmune disorders, including both cell-mediated and
antibody-mediated disorders such as systemic lupus erythematosus
(SLE). The methods involve removing cells from a patient and
treating them with a composition that can act in one of two ways.
In one embodiment, symptoms of antibody-mediated autoimmune
disorders are ameliorated using the compositions of the invention.
The compositions down-regulates B cell hyperactivity thereby
inhibiting the production of antibodies, including autoantibodies.
In addition, the compositions enhance cell mediated immune
responses that are frequently defective in patients with SLE and
certain other antibody-mediated autoimmune disorders; that is,
patients with antibody-mediated autoimmune disorders can be treated
to ameliorate their defective cell-mediated symptoms.
[0040] Alternatively, the compositions are used to treat
cell-mediated autoimmune disease. In this embodiment, the
compositions induce immune cells to generate suppressor T cells.
These suppressor T cells prevent other T cells from becoming
cytotoxic and attacking the cells and tissue of an affected
individual. Thus, the composition decrease cytotoxicity and thereby
ameliorate the symptoms of cell-mediated autoimmune disorders.
[0041] This strategy is unlike almost all other treatment
modalities currently in use which are either anti-inflammatory or
immunosuppressive. Commonly used corticosteroids suppress cytokine
production and block the terminal events which cause tissue injury,
but generally do not alter the underlying autoimmune response.
Cytotoxic drugs or experimental genetically engineered biologicals
such as monoclonal antibodies may also deplete specific lymphocyte
populations or interfere with their function. These drugs are
generally only moderately successful and have severe adverse side
effects. Certain cytokines have been given systemically to
patients, but these agents also have broad actions with associated
serious adverse side effects.
[0042] By contrast, the strategy of the present invention is to
produce remission by restoring normal regulatory cell function and,
thus, "resetting" the immune system. Another significant potential
advantage of this strategy is a low probability of serious adverse
side effects. Since only trace amounts of regulatory compositions
such as cytokines will be returned to the patient, there should be
minimal toxicity.
[0043] Circulating B lymphocytes spontaneously secreting IgG are
increased in patients with active SLE (Blaese, R. M., et al.
(1980), Am J. Med 69:345-350; Klinman, D. M. et al. (1991)
Arthritis Rheum 34: 1404-1410). Sustained production of polyclonal
IgG and autoantibodies in vitro requires T cell help (Shivakumar,
S. et al. (1989), J Immunol 143:103-112). Previous studies of T
cell regulation of spontaneous IgG production shows that while CD8+
T cells inhibit antibody production in healthy individuals, in SLE
these cells support B cell function instead (Linker-Israeli, M. et
al. (1990), Arthritis Rheum 33:1216-1225). In other autoimmune
diseases such as rheumatoid arthritis and mutliple sclerosis, T
cells rather than antibody are responsible for tissue injury and
the resulting inflammation (Panayi G S, et al. Arthritis Rheum
(1992) 35:725-773), Allegretta M et al. Science (1990)
247:718-722.
[0044] Accordingly, in a preferred embodiment, the present
invention is drawn to methods of treating antibody- and T
cell-mediated autoimmune diseases that comprise removing peripheral
blood mononuclear cells (PBMCs) from the patient with the
autoimmune disease and treating certain of these cells with an
regulatory composition.
[0045] Without being bound by theory, it appears there are several
ways the methods of the invention may work. First of all, the
treatment of the cells by an regulatory composition leads to the
direct suppression of antibody production in the treated cells,
which can lead to amelioration of antibody-mediated autoimmune
symptoms. Alternatively or additionally, the treatment of the cells
induces regulatory cells to down regulate antibody production in
other cells. Antibody in this context includes all forms of
antibody, including IgA, IgM, IgG, IgE, etc. The net result is a
decrease in the amount of antibody in the system.
[0046] Additionally, the treatment of the cells enhances
cell-mediated immune responses in patients with antibody-mediated
autoimmune symptoms. Without being bound by theory, it appears that
the treatment of the cells restores the balance between IL-10 and
TNF-.alpha. leading to an enhanced production of Th1 cytokines and
normalization of cell mediated immunity.
[0047] Furthermore, stimulation of immune cells with regulatory
compositions including TGF-.beta. can suppress cell-mediated immune
responses. Without being bound by theory, it appears that CD4+ T
cells can be stimulated to produce immunosuppressive levels of
active TGF-.beta., that then suppresses cell-mediated immune
responses. Alternatively, CD4+ T cells can be stimulated to
suppress the activation and/or effector functions of other T cells
by a contact-dependent mechanism of action. These effects require
CD4+ cells to be activated in the presence of TGF-.beta..
[0048] Thus, the present invention inhibits aberrant immune
responses. In patients with antibody-mediated autoimmune disorders,
the present invention restores the capacity of peripheral blood T
cells to down regulate antibody production and restores cell
mediated immune responses by treating them with an regulatory
composition ex vivo. In patients with cell-mediated disorders, the
present invention generates regulatory T cells which suppress
cytotoxic T cell activity in other T cells.
[0049] By "immune response" herein is meant host responses to
foreign or self antigens. By "aberrant immune responses" herein is
meant the failure of the immune system to distinguish self from
non-self or the failure to respond to foreign antigens. In other
words, aberrant immune responses are inappropriately regulated
immune responses that lead to patient symptoms. By "inappropriately
regulated" herein is meant inappropriately induced, inappropriately
suppressed and/or non-responsiveness. Aberrant immune responses
include, but are not limited to, tissue injury and inflammation
caused by the production of antibodies to an organism's own tissue,
impaired production of IL-2, TNF-.alpha. and IFN-.gamma. and tissue
damage caused by cytotoxic or non-cytotoxic mechanisms of
action.
[0050] Accordingly, in a preferred embodiment, the present
invention provides methods of treating antibody-mediated autoimmune
disorders in a patient. By "antibody-mediated autoimmune diseases"
herein is meant a disease in which individuals develop antibodies
to constituents of their own cells or tissues. Antibody-mediated
autoimmune diseases include, but are not limited to, systemic lupus
erythematosus (SLE), pemphigus vulgaris, myasthenia gravis,
hemolytic anemia, thrombocytopenia purpura, Grave's disease,
dermatomyositis and Sjogren's disease. The preferred autoimmune
disease for treatment using the methods of the invention is
SLE.
[0051] In addition, patients with antibody-mediated disorders
frequently have defects in cell-mediated immune responses. By
"defects in cell mediated immune response" herein is meant impaired
host defense against infection. Impaired host defense against
infection includes, but is not limited to, impaired delayed
hypersensitivity, impaired T cell cytotoxicity and impaired
production of TGF-.beta.. Other defects, include, but are not
limited to, increased production of IL-10 and decreased production
of IL-2, TNF-.alpha. and IFN-.gamma.. Using the methods of the
present invention, purified T cells are stimulated to increase
production of IL-2, TNF-.alpha. and IFN-.gamma. and decrease
production of IL-10. T cells which can be stimulated using the
current methods include, but are not limited to, CD4+ and CD8+.
[0052] In one embodiment, antibody-mediated disorders are not
treated.
[0053] In a preferred embodiment, the present invention provides
methods of treating cell-mediated autoimmune disorders in a
patient. By "cell-mediated autoimmune diseases" herein is meant a
disease in which the cells of an individual are activated or
stimulated to become cytotoxic and attack their own cells or
tissues. Alternatively, the autoimmune cells of the individual may
stimulate other cells to cause tissue damage by cytotoxic or
non-cytotoxic mechanisms of action. Cell-mediated autoimmune
diseases include, but are not limited to, Hashimoto's disease,
polymyositis, disease inflammatory bowel disease, multiple
sclerosis, diabetes mellitus, rheumatoid arthritis, and
scleroderma.
[0054] By "treating" an autoimmune disorder herein is meant that at
least one symptom of the autoimmune disorder is ameliorated by the
methods outlined herein. This may be evaluated in a number of ways,
including both objective and subjective factors on the part of the
patient. For example, immunological manifestations of disease can
be evaluated; for example, the level of spontaneous antibody and
autoantibody production, particularly IgG production in the case of
SLE, is reduced. Total antibody levels may be measured, or
autoantibodies, including, but not limited to, anti-double-stranded
DNA (ds DNA) antibodies, anti-nucleoprotein antibodies, anti-Sm,
anti-Rho, and anti-La. Cytotoxic activity can be evaluated as
outlined herein. Physical symptoms may be altered, such as the
disappearance or reduction in a rash in SLE. Renal function tests
may be performed to determine alterations; laboratory evidence of
tissue damage relating to inflammation may be evaluated. Decreased
levels of circulating immune complexes and levels of serum
complement are further evidence of improvement. In the case of SLE,
a lessening of anemia may be seen. The ability to decrease a
patient's otherwise required drugs such as immunosuppressives can
also be an indication of successful treatment. Other evaluations of
successful treatment will be apparent to those of skill in the art
of the particular autoimmune disease.
[0055] By "patient" herein is meant a mammalian subject to be
treated, with human patients being preferred. In some cases, the
methods of the invention find use in experimental animals, in
veterinary application, and in the development of animal models for
disease, including, but not limited to, rodents including mice,
rats, and hamsters; and primates.
[0056] The methods provide for the removal of blood cells from a
patient. In general, peripheral blood mononuclear cells (PBMCs) are
taken from a patient using standard techniques. By "peripheral
blood mononuclear cells" or "PBMCs" herein is meant lymphocytes
(including T-cells, B-cells, NK cells, etc.) and monocytes. As
outlined more fully below, it appears that in one embodiment, the
main effect of the regulatory composition is to enable CD8+ or CD4+
T lymphocytes to suppress harmful autoimmune responses.
Accordingly, the PBMC population should comprise CD8+ T cells.
Preferably, only PBMCs are taken, either leaving or returning
substantially all of the red blood cells and polymorphonuclear
leukocytes to the patient. This is done as is known in the art, for
example using leukophoresis techniques. In general, a 5 to 7 liter
leukophoresis step is done, which essentially removes PBMCs from a
patient, returning the remaining blood components. Collection of
the cell sample is preferably done in the presence of an
anticoagulant such as heparin, as is known in the art.
[0057] In some embodiments, a leukophoresis step is not
required.
[0058] In general, the sample comprising the PBMCs can be
pretreated in a wide variety of ways. Generally, once collected,
the cells can be additionally concentrated, if this was not done
simultaneously with collection or to further purify and/or
concentrate the cells. The cells may be washed, counted, and
resuspended in buffer.
[0059] The PBMCs are generally concentrated for treatment, using
standard techniques in the art. In a preferred embodiment, the
leukophoresis collection step results a concentrated sample of
PBMCs, in a sterile leukopak, that may contain reagents and/or
doses of the regulatory composition, as is more fully outlined
below. Generally, an additional concentration/purification step is
done, such as Ficoll-Hypaque density gradient centrifugation as is
known in the art.
[0060] In a preferred embodiment, the PBMCs are then washed to
remove serum proteins and soluble blood components, such as
autoantibodies, inhibitors, etc., using techniques well known in
the art. Generally, this involves addition of physiological media
or buffer, followed by centrifugation. This may be repeated as
necessary. They can be resuspended in physiological media,
preferably AIM-V serum free medium (Life Technologies) (since serum
contains significant amounts of inhibitors) although buffers such
as Hanks balanced salt solution (HBBS) or physiological buffered
saline (PBS) can also be used.
[0061] Generally, the cells are then counted; in general from
1.times.10.sup.9 to 2.times.10.sup.9 white blood cells are
collected from a 5-7 liter leukophoresis step. These cells are
brought up roughly 200 mls of buffer or media.
[0062] In a preferred embodiment, the PBMCs may be enriched for one
or more cell types. For example, the PBMCs may be enriched for CD8+
T cells or CD4+ T cells. This is done as is known in the art, as
described in Gray et al. (1998), J. Immunol. 160:2248, hereby
incorporated by reference. Generally, this is done using
commercially available immunoabsorbent columns, or using research
procedures (the PBMCs are added to a nylon wool column and the
eluted, nonadherent cells are treated with antibodies to CD4, CD16,
CD11b and CD74, followed by treatment with immunomagnetic beads,
leaving a population enriched for CD8+ T cells).
[0063] In a preferred embodiment, the PBMCs are separated in a
automated, closed system such as the Nexell Isolex 300i Magnetic
Cell Selection System. Generally, this is done to maintain
sterility and to insure standardization of the methodology used for
cell separation, activation and development of suppressor cell
function.
[0064] Once the cells have undergone any necessary pretreatment,
the cells are treated with an regulatory composition. By "treated"
herein is meant that the cells are incubated with the regulatory
composition for a time period sufficient to develop the capacity to
inhibit immune responses, including antibody and autoantibody
production, particularly when transferred back to the patient. The
incubation will generally be under physiological temperature. As
noted above, this may happen as a result of direct suppression of
Antibody production by the treated cells, or by inducing regulatory
cells to down regulate the production of antibody in the patient's
lymphoid organs.
[0065] By "regulatory composition" or "antibody production
inhibitor composition" or "humoral inhibitor composition" or
"non-specific immune cell inhibitor" or specific T cell inhibitor"
or "inhibitory composition" or "suppressive composition" herein is
meant a composition that can cause suppression of immune responses,
including inhibition of T cell activation, inhibition of
spontaneous antibody and autoantibody production, or cytotoxicity,
or both. Generally, these compositions are cytokines. Suitable
regulatory compositions include, but are not limited to, T cell
activators such as anti-CD2, including anti-CD2 antibodies and the
CD2 ligand, LFA-3, and mixtures or combinations of T cell
activators such as Concanavalin A (Con A), staphylococcus
enterotoxin B (SEB), anti-CD3, anti-CD28 and cytokines such as
IL-2, IL-4, TGF-.beta. and TNF-.alpha.. A preferred regulatory
composition for antibody suppression is a mixture containing a T
cell activator, IL-2 and TGF-.beta.. The preferred regulatory
composition for suppression of cytotoxicity is TGF-.beta..
[0066] The concentration of the regulatory composition will vary on
the identity of the composition. In a preferred embodiment,
TFG-.beta. is a component the regulatory composition. By
"transforming growth factor-.beta." or "TGF-.beta." herein is meant
any one of the family of the TGF-.beta.s, including the three
isoforms TGF-.beta.1, TGF-.beta.2, and TGF-.beta.3; see Massague,
J. (1980), J. Ann. Rev. Cell Biol 6:597. Lymphocytes and monocytes
produce the .beta.1 isoform of this cytokine (Kehrl, J. H. et al.
(1991), Int J Cell Cloning 9: 438-450). The TFG-.beta. can be any
form of TFG-.beta. that is active on the mammalian cells being
treated. In humans, recombinant TFG-.beta. is currently preferred.
A preferred human TGF-.beta. can be purchased from Genzyme
Pharmaceuticals, Farmington, Mass. In general, the concentration of
TGF-.beta. used ranges from about 2 picograms/ml of cell suspension
to about 5 nanograms, with from about 10 pg to about 4 ng being
preferred, and from about 100 pg to about 2 ng being especially
preferred, and 1 ng/ml being ideal.
[0067] In a preferred embodiment, IL-2 is used in the regulatory
composition. The IL-2 can be any form of IL-2 that is active on the
mammalian cells being treated. In humans, recombinant IL-2 is
currently preferred. Recombinant human IL-2 can be purchased from
Cetus, Emeryville, Calif. In general, the concentration of IL-2
used ranges from about 1 Unit/ml of cell suspension to about 100
U/ml, with from about 5 U/ml to about 25 U/ml being preferred, and
with 10 U/ml being especially preferred. In a preferred embodiment,
IL-2 is not used alone.
[0068] In a preferred embodiment, CD2 activators, such as a
combination of mitogenic anti CD2 antibodies, which may include the
CD2 ligand LFA-3, are used as the regulatory composition. CD2 is a
cell surface glycoprotein expressed by T lymphocytes. By "CD2
activator" herein is meant compound that will initiate the CD2
signaling pathway. A preferred CD2 activator comprises anti CD2
antibodies (OKT11, American Type Culture Collection, Rockville Md.
and GT2, Huets, et al., (1986) J. Immunol. 137:1420). In general,
the concentration of CD2 activator used will be sufficient to
induce the production of TGF-.beta.. The concentration of anti CD2
antibodies used ranges from about 1 ng/ml to about 10 .mu.g/ml,
with from about 10 ng/ml to about 100 ng/ml being especially
preferred.
[0069] In some embodiments it is desirable to use a mitogen to
activate the cells; that is, many resting phase cells do not
contain large amounts of cytokine receptors. The use of a mitogen
such as Concanavalin A or staphylococcus enterotoxin B (SEB) can
allow the stimulation of the cells to produce cytokine receptors,
which in turn makes the methods of the invention more effective.
When a mitogen is used, it is generally used as is known in the
art, at concentrations ranging from 1 .mu.g/ml to about 10 .mu.g/ml
is used. In addition, it may be desirable to wash the cells with
components to remove the mitogen, such as .alpha.-methyl mannoside,
as is known in the art.
[0070] In a preferred embodiment, T cells are strongly stimulated
with mitogens, such as anti-CD2, anti-CD3, anti-CD28 or
combinations of monoclonal antibodies, or a specific autoantigen,
if known, and anti-CD28 or IL-2 as a co-stimulator. ConA is also
used to stimulate T cells. The presence of TGF-.beta. in the
suppressive composition induces T cells to develop potent
suppressive activity. Repeated stimulation of the T cells with our
without TGF-.beta. in secondary cultures may be necessary to
develop maximal suppressive activity.
[0071] In a preferred embodiment, the invention provides methods
comprising conditioning T cells, including, but not limited to CD8+
T or CD4+ T cells, and other minor T cell subsets such as
CD8.sup.-CD4.sup.-, NK T cells, etc., with TGF-.beta.. These T
cells prevent other T cells from becoming cytotoxic effector
cells.
[0072] In a preferred embodiment, the invention provides methods
comprising conditioning CD4+ or CD8+ T cells with TGF-.beta. to
produce immunosuppresive levels of TGF-.beta..
[0073] In a preferred embodiment, the invention provides methods
comprising conditioning CD4+ or CD8+ T cells with TGF-.beta. to
produce T cells that suppress by a contact-dependent mechanism.
[0074] In a preferred embodiment, the invention provides methods
comprising treating naive CD4+ T cells with a stimulant such that
said CD4+ cells produce immunosuppressive levels of active
TGF-.beta.. By "stimulant" is generally meant a generalized
stimulant that triggers all T cells, such as anti-CD2 or
anti-CD3.
[0075] In a preferred embodiment, the invention provides methods
comprising stimulating naive CD4+ T cells in the presence of
TGF-.beta. to expand the CD4+ cell population.
[0076] In a preferred embodiment, the invention provides methods
which decrease production of IL-10 and correspondingly increase
TNF-.alpha. production.
[0077] The regulatory composition is incubated with the cells for a
period of time sufficient to cause an effect. In a preferred
embodiment, treatment of the cells with the regulatory composition
is followed by immediate transplantation back into the patient.
Accordingly, in a preferred embodiment, the cells are incubated
with the regulatory composition for 12 hours to about 7 days. The
time will vary with the suppressive activity desired. For
suppression of antibody production 48 hours is especially preferred
and 5 days is especially preferred for suppression of
cytotoxicity.
[0078] In one embodiment, the cells are treated for a period of
time, washed to remove the regulatory composition, and may be
reincubated to expand the cells. Before introduction into the
patient, the cells are preferably washed as outlined herein to
remove the regulatory composition. Further incubations for testing
or evaluation may also be done, ranging in time from a few hours to
several days. If evaluation of antibody production prior to
introduction to a patient is desirable, the cells will be incubated
for several days to allow antibody production (or lack thereof) to
occur.
[0079] Once the cells have been treated, they may be evaluated or
tested prior to autotransplantation back into the patient. For
example, a sample may be removed to do: sterility testing; gram
staining, microbiological studies; LAL studies; mycoplasma studies;
flow cytometry to identify cell types; functional studies, etc.
Similarly, these and other lymphocyte studies may be done both
before and after treatment.
[0080] In a preferred embodiment, the quantity or quality, i.e.
type, of antibody production, may be evaluated. Thus, for example,
total levels of antibody may be evaluated, or levels of specific
types of antibodies, for example, IgA, IgG, IgM, anti-DNA
autoantibodies, anti-nucleoprotein (NP) antibodies, etc. may be
evaluated. Regulatory T cells may also be assessed for their
ability to suppress T cell activation or to prevent T cell
cytotoxicity against specific target cells in vitro.
[0081] In a preferred embodiment, the levels of antibody,
particularly IgG, are tested using well known techniques, including
ELISA assays, as described in Abo et al. (1987), Clin. Exp.
Immunol. 67:544 and Linker-Israeli et al. (1990), Arthritis Rheum
33:1216, both of which are hereby expressly incorporated by
reference. These techniques may also be used to detect the levels
of specific antibodies, such as autoantibodies.
[0082] In a preferred embodiment, the treatment results in a
significant decrease in the amount of IgG and autoantibodies
produced, with a decrease of at least 10% being preferred, at least
25% being especially preferred, and at least 50% being particularly
preferred. In many embodiments, decreases of 75% or greater are
seen.
[0083] In a preferred embodiment, prior to transplantation, the
amount of total or active TGF-.beta. can also be tested. As noted
herein, TGF-.beta. is made as a latent precursor that is activated
post-translationally.
[0084] After the treatment, the cells are transplanted or
reintroduced back into the patient. This is generally done as is
known in the art, and usually comprises injecting or introducing
the treated cells back into the patient, via intravenous
administration, as will be appreciated by those in the art. For
example, the cells may be placed in a 50 ml Fenwall infusion bag by
injection using sterile syringes or other sterile transfer
mechanisms. The cells can then be immediately infused via IV
administration over a period of time, such as 15 minutes, into a
free flow IV line into the patient. In some embodiments, additional
reagents such as buffers or salts may be added as well.
[0085] After reintroducing the cells into the patient, the effect
of the treatment may be evaluated, if desired, as is generally
outlined above. Thus, evaluating immunological manifestations of
the disease may be done; for example the titers of total antibody
or of specific immunoglobulins, renal function tests, tissue damage
evaluation, etc. may be done. Tests of T cells function such as T
cell numbers, phenotype, activation state and ability to respond to
antigens and/or mitogens also may be done.
[0086] The treatment may be repeated as needed or required. For
example, the treatment may be done once a week for a period of
weeks, or multiple times a week for a period of time, for example
3-5 times over a two week period. Generally, the amelioration of
the autoimmune disease symptoms persists for some period of time,
preferably at least months. Over time, the patient may experience a
relapse of symptoms, at which point the treatments may be
repeated.
[0087] In a preferred embodiment, the invention further provides
kits for the practice of the methods of the invention, i.e., the
incubation of the cells with the regulatory compositions. The kit
may have a number of components. The kit comprises a cell treatment
container that is adapted to receive cells from a patient with an
antibody-mediated or cell-mediated autoimmune disorder. The
container should be sterile. In some embodiments, the cell
treatment container is used for collection of the cells, for
example it is adaptable to be hooked up to a leukophoresis machine
using an inlet port. In other embodiments, a separate cell
collection container may be used.
[0088] In a preferred embodiment, the kit comprises a cell
treatment container that is adapted to receive cells from a patient
with a cell mediated disorder. The kit may also be adapted for use
in a automated closed system to purify specific T cell subsets and
expand them for transfer back to the patient.
[0089] The form and composition of the cell treatment container may
vary, as will be appreciated by those in the art. Generally the
container may be in a number of different forms, including a
flexible bag, similar to an IV bag, or a rigid container similar to
a cell culture vessel. It may be configured to allow stirring.
Generally, the composition of the container will be any suitable,
biologically inert material, such as glass or plastic, including
polypropylene, polyethylene, etc. The cell treatment container may
have one or more inlet or outlet ports, for the introduction or
removal of cells, reagents, regulatory compositions, etc. For
example, the container may comprise a sampling port for the removal
of a fraction of the cells for analysis prior to reintroduction
into the patient. Similarly, the container may comprise an exit
port to allow introduction of the cells into the patient; for
example, the container may comprise an adapter for attachment to an
IV setup.
[0090] The kit further comprises at least one dose of an regulatory
composition. "Dose" in this context means an amount of the
regulatory composition such as cytokines, that is sufficient to
cause an effect. In some cases, multiple doses may be included. In
one embodiment, the dose may be added to the cell treatment
container using a port; alternatively, in a preferred embodiment,
the dose is already present in the cell treatment container. In a
preferred embodiment, the dose is in a lyophilized form for
stability, that can be reconstituted using the cell media, or other
reagents.
[0091] In some embodiments, the kit may additionally comprise at
least one reagent, including buffers, salts, media, proteins,
drugs, etc. For example, mitogens, monoclonal antibodies and
treated magnetic beads for cell separation can be included.
[0092] In some embodiments, the kit may additional comprise written
instructions for using the kits.
[0093] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference in their entirety.
EXAMPLES
Example 1
Treatment of PBMCs with a Mixture of IL-2 and TFG-.beta.
[0094] Example 1 shows that the relatively brief treatment of PBMCs
from SLE patients with IL-2 and TFG-.beta. can result in the marked
inhibition of spontaneous polyclonal IgG and autoantibody
production. As discussed below, PBMC from 12 patients with active
SLE were exposed to IL-2 with or without TGF-.beta. for 3 days,
washed and cultured seven more days. The mean decrease in IgG
secretion was 79%. The strongest inhibitory effect was observed in
cases with the most marked B cell hyperactivity. Spontaneous
production of anti-nucleoprotein (NP) antibodies was observed in 4
cases and cytokine treatment of PBMC decreased autoantibody
production by 50 to 96%. IL-2 inhibited antibody production by
either TGF-.beta.-dependent or independent mechanisms in individual
patients. In a study of anti-CD2 stimulated IgG production in a
patient with active SLE, we documented that IL-2 and TGF-.beta. can
reverse the enhancing effects of CD8+ T cells on IgG production and
induce suppressive activity instead.
[0095] 1. Methods
[0096] Study Subjects for Spontaneous Antibody Synthesis
[0097] Twelve subjects were chosen with a diagnosis of SLE that
fulfilled ARA criteria for the classification of SLE (Arnett, F. C.
et al. (1998), Arthritis Rheum 31: 315-324). These patients were
all women, 8 Hispanic, 2 African American, and 2 Asian. The age of
each patient and duration of disease is shown in Table 1. Five
patients were hospitalized and 7 were outpatients. Those patients
who were receiving corticosteroids or antimalarials are also
indicated. 8 patients were untreated. Disease activity was assessed
with SLAM (Liang, M. H. et al. (1989), Arthritis Rheum
32:1107-1118) and SLEDAI (Bombardier, C. et al. (1992), Arthritis
Rheum 35:630-640) indices with mean values of 16.5 and 13.4
respectively.
TABLE-US-00001 TABLE 1 Profile of SLE Patients Case SEX Age
Ethnicity Duration Medications SLAM SLEDAI IgG(.mu./ml) 1 F 18 AA 3
yr Nil 13 9 13.7 2 F 37 H 6 mo Nil 23 13 13.0 3 F 29 H 1 yr Nil 15
6 2.6 4 F 32 AA 4 yr Pred 5 mg 9 6 2.5 Ohchlor 400 mg 5 F 57 A 5 mo
Nil 24 19 2.2 6 F 55 H 5 mo Nil 23 22 1.5 7 F 27 H 3 yr Pred 20 mg
13 17 1.0 Ohchlor 400 mg 8 F 21 H 2 yr Nil 18 13 1.0 9 F 36 H 15 yr
Pred 20 mg 14 8 0.8 Ohchlor 400 mg Aza 25 mg 10 F 41 A 4 yr Nil 15
16 0.5 11 F 20 H 6 yr Pred 25 mg 11 16 0.4 12 F 25 H 1 yr Nil 21 16
0.4
Reagents
[0098] Recombinant TGF-.beta. and monoclonal anti-TGF-.beta.
(1D11.16) antibody, a murine IgG1, were kindly provided by Dr.
Bruce Pratt (Genzyme Pharmaceuticals, Farmington, Mass.).
Recombinant IL-10 and monoclonal anti-IL-10 (JES3-19F1) antibody,
and control rat IgG2a, were kindly provided by Dr. Satwant Narula
(Schering Plough Pharmaceuticals, Kenilworth, N.J.). Control murine
IgG1 myeloma protein was purchased from Calbiochem, San Diego,
Calif. Recombinant human IL-2 was purchased from Chiron,
Emmeryville, Calif. Anti-CD2 secreting hybridomas antibodies used
OKT11 were obtained from the American Type Culture Collection
(ATCC), Rockville, Md. and GT2 was generously provided by A.
Bernard, Nice, France). Other antibodies included: anti-CD4 (OKT4,
ATCC), anti-CD8 (OKT8, ATCC; CD8, Dako, Carpenteria, Calif.),
anti-CD11b (OKM1, ATCC), anti-CD16 (3G8), kindly provided by J.
Unkeless, New York, N.Y.); anti-CD.sub.20 (Leu 16, Becton
Dickinson, San Jose, Calif.) and anti-CD74 (L243, ATCC).
Isolation of Blood Mononuclear Cells
[0099] Peripheral blood mononuclear cells (PBMC) were prepared from
heparinized venous blood by Ficoll-Hypaque (Pharmacia, Piscataway,
N.J.) density gradient centrifugation. The mononuclear cells were
washed in PBS with 5 mM EDTA (Life Technologies, Grand Island,
N.Y.) to remove platelets, which are a rich source of
TGF-.beta..
Cell Culture Procedures
[0100] Procedures for cell cultures have been described previously
(Wahl, S. M. (1994), J Exp Med 180:1587-1590; Gray, J. D. et al.
(1998), J Immunol 160:2248-2254). In brief, 2.times.10.sup.5 of
PBMC were cultured in serum-free AIM-V culture medium (Life
Technologies) in the wells of 96-well flat bottom microtiter plate
with or without the indicated cytokines After three days of
culture, the PBMC were washed three times then fresh serum-free
medium was added. After a further 7 days at 37.degree. C.,
supernatants were harvested and assayed for total IgG and
autoantibodies reactive with calf thymus nucleoprotein (NP) by a
solid phase enzyme-linked immunoadsorbant assay (ELISA), as
described previously (Linker-Israeli, M. et al. (1990), Arthritis
Rheum 33:1216-1225). The optical density (OD) readings were
transformed into units/ml (U/ml) from a standard curve using
positive and negative standards. Supernatants from PBMC culture of
SLE patients (with high titers of anti-NP antibodies) and normal
individuals were used as controls.
Statistical Analysis
[0101] The data were analyzed using Graph Pad, Prism software (San
Diego, Calif.). We used analysis of variance (ANOVA) after log
transformation of the data and the non-parametric Mann-Whitney
test.
Anti-CD2 Induced IgG Synthesis.
[0102] The effects of CD8+ T cells cultured with or without NK
cells on anti-CD2 stimulated CD4+ T cells and B cells was examined
in a patient with SLE in a normal control. CD4+ and CD8+ cells were
prepared from nylon non-adherent lymphocytes by negative selection
using immunomagnetic beads as described previously (Gray, J. D. et
al. (1998), J Immunol 160:2248-2254). For CD4+ cells the nylon
non-adherent cells were stained with antibodies to CD8, CD16, CD11b
and CD74. The same antibodies were used to obtain CD8+ cells except
that CD4 was substituted for CD8. Purity of CD4+ cells was 95% and
CD8+ cells 89%. To obtain NK cells, PBMC were added to a nylon wool
column and the eluted, non-adherent cells were immediately rosetted
with AET treated sheep red blood cells. The non-rosetting fraction
was then stained with anti-CD3 and anti-CD74 (anti-HLA-DR)
antibodies and depleted of reacting cells using immunomagnetic
beads (Dynal). This resultant population contained 98% CD56+ and
<0.5% CD3+ and <0.5% CD20+ lymphocytes. Since SLE B cells
spontaneously secrete large amounts of IgG and because of the large
amount of blood needed to prepare sufficient numbers of B cells for
these studies, we substituted resting B cells from a healthy donor
for patient B cells in this study. To obtain B cells, nylon wool
adherent cells were immediately rosetted with SRBC to remove any T
cells, and treated with 5 mM L-leucine methyl ester for complete
removal of monocytes and functional NK cells. The resulting
population was >92% CD20+ and <0.5% CD3+.
Results
[0103] In 12 patients studied, spontaneous IgG ranged from 0.4 to
13.7 .mu.g/ml (FIG. 1). Exposure of PBMC to IL-2.+-.TGF-.beta. for
72 hours decreased IgG synthesis in 8 of 12 cases studied by at
least 50% (mean decrease 79%, p=0.008, Mann Whitney). The most
dramatic decreases were observed in cases with the most marked B
cell hyperactivity. The correlation between the amount of IgG
secreted and percent inhibition by IL-2 and TGF-.beta. was r=0.647,
p=0.02.
[0104] We compared the effects of IL-2 and TGF-.beta. alone to the
combination of IL-2 and TGF-.beta.. FIG. 2 shows that each of these
cytokines also inhibited IL-2 production. However, after log
transformation to achieve a normal distribution of the data and
applying the Bonnferoni correction for multiple comparisons,
analysis of variance revealed that only the combination of IL-2 and
TGF-.beta. resulted in significant inhibition (p=0.05).
[0105] IL-10 production is increased in SLE (Llorente, L. et al.
(1993), Eur Cytokine Network 4:421-427) and this cytokine can
inhibit production of both IL-2 and TGF-.beta.. In 9 cases we also
assessed the effect of anti-IL-10, but only a modest decrease of
IgG synthesis was observed in some subjects and this difference was
not statistically significant. Similarly, TNF.alpha. production is
also decreased in a subset of patients with SLE (Jacob, C. O. et
al. (1990), Proc Natl Acad Sci 87:1233-1237). Although this
cytokine also increases the production of active TGF-.beta.
(Ohtsuka, K. et al. (198), J Immunol 160:2539-2545), the addition
of TNF.alpha. to the cultures had minimal effects (results not
shown).
[0106] We also examined SLE PBMC for spontaneous production of
anti-nucleoprotein (NP) autoantibodies and found significant titers
in 4 cases. In all cases exposure of PBMC to either IL-2 or IL-2
and TGF-.beta. inhibited anti-NP production by at least 50 percent.
TGF-.beta. by itself was ineffective (Table 2). In these cases the
effects of IL-2 by itself was equivalent to that the combination of
IL-2 and TGF-.beta..
TABLE-US-00002 TABLE 2 Effect of treating PBMC with IL-2 and
TGF-.beta. on Spontaneous Autoantibody production in SLE
Anti-nucleoprotein antibody (U/ml) Cytokine treatment Case A: Case
B: Case C: Case D: Nil 308 312 25 73 TGF-.beta. (10 pg/ml) 282 298
26 ND IL-2 & TGF-.beta. 29 14 12 35 IL-2 23 10 11 ND *Percent
of baseline values
[0107] PBMC from SLE patients were exposed to IL-2 (10 u/ml) and
TGF-.beta. (10 pg/ml) for 72 hours. The cells were washed and
cultured for seven additional days. Anti-NP released into the
supernatants was measured by an ELISA.
[0108] Previously we have reported that IL-2 increases the
production of biologically active TGF-.beta. (Ohtsuka, K. et al.
(1998), J Immunol 160:2539-2545). It was, therefore, possible that
al least some of the effects of IL-2 on spontaneous antibody
synthesis were mediated by TGF-.beta.. This possibility was
investigated by determining whether the effects of IL-2 could be
reversed by an anti-TGF-.beta. neutralizing antibody. In the
example shown in FIG. 3A, the addition of anti-TGF-.beta. did not
affect spontaneous IgG synthesis. Antagonism of TGF-.beta.,
however, did abolish the inhibitory effects of IL-2 on IgG
synthesis. PBMC from this patient (Case C in Table 2) also
spontaneously produced anti-NP antibody. Here also anti-TGF-.beta.
abolished the inhibitory effects of IL-2 on anti-NP production
(FIG. 3B). In this subject, therefore, the inhibitory effects of
IL-2 on spontaneous IgG and autoantibody synthesis were mediated by
TGF-.beta.. This effect of anti-TGF-.beta. was documented in 4 of 8
cases studied. Thus, the inhibitory effects of IL-2 could either be
TGF-.beta.-dependent or independent. Examples of each effect are
shown in Table 3.
TABLE-US-00003 TABLE 3 Effect of IL-2 and TGF-.beta. on Spontaneous
IgG Synthesis in SLE Patient A: Patient B: TGF-.beta. dependent
inhibition TGF-.beta. independent inhibition Cytokines Added
G(.mu.gm/ml) IgG (.mu.gm/ml) Medium only 2.5 (100) * 2.6 (100)
TGF-.beta. (10 pg/ml) 1.4 (56) 2.5 (96) IL-2 & TGF- .beta. 0.4
(16) 0.5 (19) IL-2 & anti-TGF- .beta. 11.6 (464) 0.5 (19) IL-2
& IgG1 3.6 (144) 0.6 (23) * Percent of baseline IgG
synthesis
[0109] We had the opportunity to repeat the study of on SLE patient
28 days after initiation of steroid therapy (Table 4). Before
treatment spontaneous IgG synthesis was greater than 2 .mu.g/ml of
IgG. Exposure of PBMC to IL-2 markedly inhibited IgG production and
TGF-.beta. had a moderate effect. Following corticosteroid therapy,
spontaneous IgG production decreased by 75%. As before, exposure of
PBMC to IL-2.+-.TGF-.beta. decreased IgG production by 50%.
However, this inhibition was reversed by anti-TGF-.beta.. Here
again, this effect of IL-2 could be explained by upregulation of
endogenous active TGF-.beta..
TABLE-US-00004 TABLE 4 Effect of Corticosteroid Therapy on
Spontaneous IgG Synthesis in SLE Before Treatment After Treatment
Cytrokine Added Day 0 Day 28 Nil 2.2 0.6 TGF-.beta. (10 pg/ml) 1.2
0.4 IL-2 (10 U/ml) 0.4 0.3 IL-2 & TGF-.beta. 0.7 0.3 IL-2 &
anti-TGF-.beta. ND 0.8 IL-2 & IgG1 ND 0.6 *Percent of baseline
IgG synthesis
[0110] In view of our previous studies in healthy subjects that
IL-2 and TGF-.beta. can induce activated CD+ T cells to
down-regulate antibody production, we attempted to isolate and
treat CD8+ T cells from SLE patients in this study. These
experiments were unsuccessful because of the marked variability of
spontaneous antibody synthesis and the large amount of blood
required from patients with active SLE for cell separation
procedures. However, we were able to obtain enough blood from one
patient with active SLE to investigate the effect of IL-2 and
TGF-.beta. on CD8+ T cell modulation of anti-CD2 induced IgG
synthesis. We have recently reported that unlike anti-CD3, a
mitogenic combination of anti-CD2 monoclonal antibodies did not
induce PBL to produce IgG (Gray, J. D. et al. (1998), J Immunol
160:2248-2254). An example is shown in FIG. 4A. This was because
anti-CD2 stimulated NK cells to produce TGF-.beta., which in turn
induced CD8+ T cells to down-regulate antibody production (Gray, J.
D. et al. (1998), J Immunol/60:2248-2254). In this patient, as we
have reported previously (Gray, J. D. et al. (1994), J Exp Med
180:1937-1942), CD8+ T cells enhanced IgG synthesis and this
enhancement was markedly potentiated by the combination of NK cells
and CD8+ T cells (FIG. 4B). By contrast IL-2 and TGF-.beta.
abolished the helper effects of SLE CD8+ T cells and enabled these
cells to suppress IgG production. This inhibitory effect of IL-2
and TGF-.beta. was dependent upon the presence of CD8+ T cells.
(FIG. 4C). Thus, evidence has been obtained that the effects of
IL-2 and TGF-.beta. can be mediated by CD8+ T cells.
[0111] These studies demonstrate that a short exposure of PBMC to
IL-2 and TGF-.beta. can greatly decrease subsequent spontaneous
polyclonal IgG and autoantibody production in SLE, especially in
patients with severe disease and marked B cell hyperactivity. This
study confirms previous reports indicating that IL-2 can inhibit
antibody production (Hirohata, S. et al. (1989), J Immunol 142:
3104-3112 and Fast, L. D. (1992), J Immunol 149:1510-1515) and
reveals that picomolar concentrations of TGF-.beta. can contribute
to this down-regulation. In the group of 12 patients studied, the
inhibitory effect of IL-2 and TGF-.beta. on polyclonal IgG
synthesis was greater than the effect of IL-2 alone. However, the
inhibitory effects of IL-2 were heterogeneous. In 4 of 8 cases
studied, the inhibition was TGF-.beta.-dependent in that a
neutralizing anti-TGF-.beta. mAb abolished the effect. In the
remaining cases the down-regulatory effects of IL-2 were
TGF-.beta.-independent. Similarly, both TGF-.beta.-dependent and
independent inhibition of spontaneous anti-NP autoantibody
production was documented. We also investigated the effects of
antagonizing the IL-10 and adding TNF-.alpha. because of previously
described abnormalities in the production of these cytokines in SLE
(Llorente L. et al. (1993), Eur Cytokine Network 4:421-427; Jacob,
C. O. et al. (1990), Proc Natl Acad Sci 87:1233-1237). These
procedures, however, had minimal effects on spontaneous antibody
synthesis where lymphocytes had been activated previously.
[0112] Others have reported that the degree of B cell hyperactivity
in SLE correlates with disease activity (Blaese, R. M. et al.
(1980), Am J Med 69:345-350; Klinman, D. M. et al. (1991),
Arthritis Rheum 34:1404-1410). This was not the case in the present
study, possibly because of concurrent drug therapy. In general,
those patients with marked spontaneous antibody synthesis were
untreated whereas those with less B cell activity were currently
receiving prednisone. We presented one case where spontaneous IgG
synthesis decreased markedly after corticosteroid therapy was
begun. This patient's B cells had also been secreting anti-NP
antibody before treatment, and production of this autoantibody
became undetectable after steroid therapy (result not shown).
[0113] TGF-.beta.s consist of a multifunctional family of cytokines
important in tissue repair, inflammation and immunoregulation
(Massague, J. (1990), Annu Rev Cell Biol 6597-641). TGF-.beta. is
different from most other cytokines in that it is secreted as an
inert precursor molecule and converted to its biologically active
form extracellularly (Massague, J. (1990), Annu Rev Cell Biol
6597-641; Flaumenhaft, R. et al. (1993), Adv Pharmacol 24:51-76).
Regulatory T cells in various experimental autoimmune models such
as experimental autoimmune encephalitis (Weiner, H. L. et al.
(1994), Annu Rev Immunol 12:809-837) and colitis (Neurath, M. F. et
al. (1996), J Exp Med/83:2605-2516) produce this cytokine
TGF-.beta. is immunosuppressive in nanomolar concentrations and can
inhibit T and B cell proliferation, NK cell cytotoxic activity and
the generation of T cell cytotoxicity (Letterio, J. J. et al.
(1998), Ann Rev Immunol 16:137-162). By contrast, TGF-.beta. has
been reported to promote the growth of murine CD4+ cells and CD8+
cells (Kehrl, J. H. et al. (1986), J Exp Med 163:1037-1050; Lee, H.
M. et al. (1993), J Immunol 151:668-677) and can promote B cell
differentiation (Van Vlasselaer, P. et al. (1992), J Immunol
148:2062-2067).
[0114] In our previous studies with lymphocytes from healthy
subjects to generate regulatory T cells, the picomolar
concentrations of TGF-.beta. used were smaller than that required
for inhibition of T or B cell function (Gray, J. D. et al. (1998),
J Immunol 160:2248-2254; Gray, J. D. et al. (1994), J Exp Med
180:1937-1942). Similar concentrations were used in the present
studies with SLE patients and TGF-.beta. by itself had modest
inhibitory effects on antibody synthesis. As before, a combination
of IL-2 of TGF-.beta. produced the most potent inhibition. In our
previous studies, this effect was mediated by CD8+ T cells.
[0115] IL-2 has well established effects on the induction of T
suppressor cell activity (Hirohata, S. et al. (1989), J Immunol
142:3104-3112; Fast, L. D. J Immunol 149:1510-1515), but whether
these effects are direct or indirect is unclear. In mice deletion
of the IL-2 gene results in massive lymphoproliferation and
autoimmune disease (Sadlack, B. et al. (1995), Eur J Immunol
25:3053-3059). In SLE, a negative correlation was reported between
IL-2 levels and B cell hyperactivity (Huang, Y. P. et al. (1988), J
Immunol 141:827-833). Previously, we attempted to inhibit
spontaneous antibody production in SLE with IL-2, but the results,
however were extremely variable. While we observed strong
inhibition in some cases, in others IL-2 markedly increased
antibody production. We believe that the timing and the cytokine
milieu explains the more consistent inhibition observed in this
study. Here the IL-2 and TGF-.beta. were present only during the
initial 72 hours of culture rather than the entire culture period.
Enhancement of antibody synthesis in the latter case could be
explained by the positive effects of IL-2 on B cell differentiation
(Coffman, R. L. et al. (1988), Immunol Rev 102:5-28). IL-2 can
down-regulate antibody production by several mechanisms. In
addition to the TGF-.beta. circuit described in the report, IL-2
induced inhibition can occur by up-regulation of IFN-.gamma.
(Noble, A. et al. (1998), J Immunol 160:566-571), or by cytolytic
mechanisms (Stohl, W. et al. (1998), J Immunol 160:5231-5238;
Esser, M. T. et al. (1997), J Immunol 158:5612-5618).
[0116] Previously, we had investigated the regulatory effects of NK
cells on antibody synthesis and reported that while the direct
effect of NK cells is to up-regulate IgG synthesis (Kinter, A. et
al. (1995), Proc Natl Acad Sci USA 92:10985-10989), these
lymphocytes have the opposite effect when cultured with CD8+ T
cells in healthy subjects (Gray, J. D. et al. (1994), J Exp Med
180:1937-1942). In SLE patients, however, the combination of CD8+ T
cells and NK cells enhanced IgG production (Linker-Israeli, M. et
al. (1990), Arthritis Rheum 33:1216-1225). This was again observed
in the present report. While in the normal subject the addition of
NK cells to CD8+ T cells markedly inhibited anti-CD2 stimulated IgG
synthesis, the opposite was observed in SLE. From studies of
normals we had learned that NK cell-derived TGF-.beta. induced
co-stimulated CD8+ T cells to down-regulate IgG and IgM production
(Gray, J. D. et al. (1998), J Immunol 160:2248-2254). In this study
IL-2 and TGF-.beta. induced moderate suppressive activity by CD8+ T
cells. It is likely, therefore, that in SLE at least one way that
IL-2 and TGF-.beta. inhibit B cell activity is by generating
regulatory T cells. In addition, other lymphocyte populations
treated with these or other cytokines may also down-regulate B
cells activity in SLE.
Example 2
1. The Correlation of TGF-.beta. Production to Disease Activity and
Severity
[0117] Having shown that the lymphocyte production of the total and
active forms of TGF-.beta. is decreased, we next asked whether
these defects correlate with disease activity and/or severity.
TGF-.beta.1 production by blood lymphocytes from 17 prospectively
studied SLE patients was compared with 10 rheumatoid arthritis (RA)
patients and 23 matched healthy controls. In RA the levels of
active TGF-.beta.1 were lower than controls, but not deceased to
the extent found in SLE. Levels of constitutive and anti-CD2
stimulated active TGF-.beta. detected in picomolar amounts were
markedly reduced in 6 untreated patients hospitalized with recent
onset, very active and severe SLE and similarly reduced in 11
patients with treated, less active disease. thus, decreased
production of active TGF-.beta.1 did not correlate with disease
activity. By contrast, decreased production of total TGF-.beta.1
inversely correlated with disease activity. Thus it appears that
although impaired lymphocyte secretion of the latent precursor of
TGF-.beta.1 may result as a consequence of disease activity, the
ability to convert the precursor molecule to its active form may be
an intrinsic cellular defect. Insufficient exposure of T cells to
picomolar concentrations amounts of TGF-.beta.1 at the time they
are activated can result in impaired down-regulation of antibody
synthesis. Thus, decreased lymphocyte production of active
TGF-.beta.1 in SLE can contribute to B cell hyperactivity
characteristic of this disease.
2. Methods
Study Subjects
[0118] Seventeen subjects with a diagnosis of SLE who fulfilled the
American College of Rheumatology criteria for the classification of
SLE (Tan, E. M. et al. (1982), Arthritis Rheum 25:1271-1277), 10
subjects with RA who fulfilled the ACR 1987 revised criteria for
the classification of RA (Arnett, F. C. et al. (1988), Arthritis
Rheum 31:315-324), and 23 healthy donors were studied. The SLE
group consisted of 15 women and 2 men (15 Hispanic, 1 African
American, 1 Asian). The mean age was 34.5 years (range, 20-75
years). Six patients were hospitalized, and 11 were attending an
outpatient clinic. All of the hospitalized patients were untreated
before admission and were studied before they received their first
dose of corticosteroids. Outpatients were receiving less than 20 mg
of prednisone, and none were receiving cytotoxic drugs. Disease
activity was assessed with the SLAM (Liang, M. H. et al. (1989),
Arthritis Rheum 32:1107-1118) and SLEDAI (Bombardier, C. et al.
1992), Arthritis Rheum 35:630-640) indices with mean values of 6.6
and 7.6, respectively. The RA group consisted of 9 women and 1 man
(9 Hispanic, 1 Asian). The mean age was 50.9 years (range, 39-67
years). All of the patients were attending the outpatient clinic
and had mild to moderately active disease. The mean duration of
disease was 9.5 years. One patient received myochrysine, 3 patients
received prednisone (1, 1 and 20 mg), 3 patients received
methotrexate, and one patient received sulphasalazine. Healthy
donors served as controls and were matched as closely as possible
for age, sex, and ethnic groups.
TABLE-US-00005 TABLE 5 Clinical Characteristics of Two Groups of
SLE Patients Hospitalized Outpatient Clinical Data (n = 6) (n = 11)
p Value Age 26.8 38.6 1.037 Sex (F/M) 6/0 9/2 Ethnic Group (H/AA/A)
5/0/1 10/1/0 Disease Duration (yr) 0.71 8.25 0.051 Disease Activity
SLAM 13.3 2.9 0.014 SLEDAI 15.7 4.1 0.006 Prednisone Dose (mg/day)
41.2 7.8 0.008 Active Renal Disease 83% 9% 0.028 Hemolytic Anemia
67% 9% 0.064 Anti-DNA (titer) 466.7 33.0 0.064 C3 47.5 98.6 0.008
C4 13.7 18.6 0.127
Reagents
[0119] Antibodies used were supernatants of hybridomas secreting
anti-CD2 (OKT11, American Type Culture Collection (ATCC),
Rockville, Md., and GT2 made available by Dr. Alain Bernard, Nice,
France). A monoclonal antibody recognizing TGF-.beta. isoforms 1, 2
& 3 (1D11), an antibody against TGF-.beta. isoforms 2&3
(3C7), and rTGF-.beta.2 were kindly provided by Dr. Bruce Pratt
(Genzyme Pharmaceuticals, Farmington, Mass.).
Isolation of Blood Lymphocytes
[0120] Peripheral blood mononuclear cells (PBMC) were prepared from
heparinized venous blood by Ficoll-Hypaque (Pharmacia, Piscataway,
N.J.) density gradient centrifugation using methods described
previously (Ohtsuka, K. et al. (1998), J Immunol 160:2539-2545).
The mononuclear cells were washed in PBS with 5 mM EDTA (Life
Technologies, Grand Island, N.Y.) to remove platelets, which are a
rich source of TGF-.beta.. Peripheral blood lymphocytes (PBL) were
separated from PBMC by centrifugation through a continuous Percoll
(Pharmacia) density gradient. The percentage of monocytes remaining
in the high density, lymphocyte-enriched fraction was somewhat
higher in SLE (8.5% vs 4.3%).
[0121] a) Cell Culture Procedures
[0122] Procedures for cell cultures have been described previously
((Ohtsuka, K. et al. (1998), J Immunol 160:2539-2545). In brief,
1.times.10.sup.5 of the lymphocytes were added to the wells of
96-well flat bottom microtiter plate (Greiner Rocky Mountain
Scientific, Salt Lake City Utah). The cultures were carried out in
AIM-V serum free medium (Life Technologies), since serum contains
significant amount of latent TGF-.beta.. Anti-CD2 was used at the
optimal concentrations to induce TGF-.beta. production (GT2 1:40
and T11 1:80) hybridoma culture supernatants. Previous studies have
revealed that anti-CD2 strongly stimulates PBL to produce
TGF-.beta. (Gray, J. D. et al. (1998), J Immunol
160:2248-2254).
[0123] b) TGF-.beta. Assay
[0124] Mink lung epithelial cells (MLEC) which had been transfected
with an expression construct containing a plasminogen activator
inhibitor (PAI-1) promoter fused to luciferase reporter gene were
kindly provided by Dr D. B. Rifkin, New York, N.Y. MLEC at
2.times.10.sup.4/well were incubated with 200 .mu.l supernatants
for 18 h at 37.degree. C. To assay for luciferase activity, MLEC
were lysed by a cell lysis reagent (Analytical Luminescence, Ann
Arbor, Mich.). Cell lysates were then reacted with assay buffer and
luciferin solution (both from Analytical Luminescence) immediately
before being measured in a luminometer (Lumat, Berthold Analytical
Instruments Inc., Nashua, N.H.). To measure total TGF-.beta.
activity, samples were heated at 80.degree. C. for 3 minutes to
release the active cytokine from the latent complex. Active
TGF-.beta. activity was measured without heating of supernatants.
In all assays, several concentrations of rTGF-.beta. were included
to generate a standard curve. The variability of replicate cultures
was less than 10 percent (Ohtsuka, K. et al. (1998), J Immunol
160:2539-2545).
[0125] c) Statistical Analysis
[0126] The significance of the results was analyzed using the
Mann-Whitney test and Spearman rank correlation performed using
GBSTAT software (Professional Statistics and Graphics Computer
Program, Dynamic Microsystems Inc., Silver Spring, Md.).
[0127] d) Results
[0128] We measured constitutive and stimulated TGF-.beta.1 produced
by PBL from patients with SLE or RA, and compared these values with
those from normal controls. The cytokine detected in culture
supernatants was neutralized by a mAb recognizing isoforms 1, 2,
& 3, but not by one against isoforms 2&3, a result
confirming the production of TGF-.beta.1. Compared to normal
controls, constitutive production of active TGF-.beta.1 was
significantly decreased in SLE (14.+-.5 vs 56.+-.21 pg/ml, p=0.02,
FIG. 5). Anti-CD2 stimulated active TGF-.beta.1 was also decreased
(87.+-.22 vs 399.+-.103 pg/ml, p=0.003). In RA, the mean value for
constitutive TGF-.beta.1 was similar to that of SLE (19.+-.5 pg/ml)
and after stimulation by anti-CD2 was intermediate between normal
and SLE (197.+-.54 pg/ml; FIG. 5).
[0129] Constitutive total TGF-.beta.1 produced by lymphocytes was
also decreased in SLE in comparison with the normal group
(286.+-.82 vs 631.+-.185 pg/ml, p=0.05). The value in RA was
intermediate between normal and SLE (435.+-.161 pg/ml). Following
the addition of anti-CD2, total TGF-.beta.1 increased in SLE
somewhat more than in normal controls so that the differences were
not statistically significant. Values in the RA group were again
intermediate between the normal and SLE group.
[0130] To look for a possible relationship between decreased levels
of TGF-.beta.1 and disease activity, we compared hospitalized SLE
patients with those seen in the outpatient clinic. The clinical
characteristics of these two groups are summarized in Table 5.
Those that were hospitalized were younger; 5 of 6 had symptoms for
less than 3 months; they had markedly active disease; and most had
severe SLE with nephritis and/or hemolytic anemia. The outpatient
group by contrast, had chronic disease which had become less active
following treatment. Notwithstanding this marked difference in
disease heterogeneity, duration, activity, and severity, both
constitutive and stimulated active TGF-.beta.1 production were
significantly decreased in both groups in comparison with normal
controls (Table 6).
TABLE-US-00006 TABLE 6 Comparison of TGF-.beta.1 Production by
Lymphocytes from Two Groups of Patients with SLE* Normal SLE Group
1 Group 2 (n = 23) (n = 6) (n = 11) Active TGF-.beta.1 (pg/ml)
Constitutive 56 .+-. 21 21 .+-. 14.dagger. 10 .+-. 4.dagger. CD2
stimulated 399 .+-. 103 117 .+-. 52.dagger. 70 .+-. 19.dagger-dbl.
Total TGF- .beta.1 (pg/ml) Constitutive 631 .+-. 185 132 .+-.
44.dagger. 365 .+-. 120 CD2 stimulated 771 .+-. 136 226 .+-.
74.dagger. 667 .+-. 166 *PBL 1 .times. 10.sup.5/well were cultured
for 48 h, and the supernatants were tested for TGF-.beta.1.
[0131] SLE patients were divided into 2 groups. Group 1:
Hospitalized patients. Group 2: Outpatient clinic patients.
[0132] p values indicate comparison between the SLE group indicated
and the normal controls as assessed by the Mann-Whitney test;
.dagger. p<0.05, .dagger-dbl. p<0.01.
[0133] When we looked for correlations between levels of active and
total TGF-.beta.1 with disease activity, there was a significant
negative correlation between anti-CD2 stimulated production of
total TGF-.beta.1 and the SLEDAI (r=-0.55, p=0.03, but not the SLAM
index (-0.43, p=11). The SLEDAI index is weighted for central
nervous system involvement and renal disease. Thus, an impaired
capacity for lymphocytes to secrete the precursor form of
TGF-.beta.1 appears to be associated with severe disease.
[0134] e) The Levels of Active TGF-.beta.1 Did not Correlate with
Disease Activity.
[0135] The principal finding in this example is that decreased
production of active TGF-.beta.1 in SLE does not correlate with
disease activity or severity. Decreased amounts of constitutive and
stimulated active TGF-.beta.1 were found in both patients with
recent onset and established disease. Moreover, the values did not
correlate with activity, as measured by the SLAM and SLEDAI
indices, or severity as assessed by vital organ involvement.
However, while total TGF-.beta.1 production was also decreased in
SLE, this defect appeared to correlate with disease activity. It
was found chiefly in hospitalized SLE patients. The finding that
total TGF-.beta.1 production correlated most strongly with the
SLEDAI index, which is weighted for major organ system involvement,
also suggests a relationship with disease severity.
[0136] This study also included a control group of RA patients
whose disease activity was comparable to SLE patients with
established disease. Although TGF-.beta.1 values in the RA group
was somewhat less than the normal controls, with the exception of
constitutive active TGF-.beta.1, the magnitude of the defect was
not as marked as in SLE and was not statistically significant.
[0137] Previously, we have documented that NK cells are the
principal lymphocyte source of TGF-.beta. and the only lymphocyte
population to constitutively produce this cytokine in its active
form (Gray, J. D. et al. (1998), J Immunol 160:2248-2254). It was
of interest, therefore, to find that constitutive production of NK
cell-derived TGF-.beta. was decreased in SLE. We also learned that
both IL-2 and TNF-.alpha. could enhance the production of active
TGF-.beta.. Production of both of these cytokines are decreased in
SLE (Gray, J. D. et al. (1994), J Exp Med 180:1937-1942). However,
in most patients exogenous IL-2 and TNF-.alpha. could not restore
TGF-.beta. production to normal (Example 2). IL-10 production is
increased in SLE (Llorente, L. et al. (1993), Eur Cytokine Network
4:421) and correlations between elevated levels and disease
activity have been reported (Housslau, F. A. et al. (1995), Lupus
4:393-395; Haglwara, E. et al. (1996), Arthritis Rheum 39:379).
IL-10 can inhibit IL-2, TNF-.alpha. and TGF-.beta. production
(Example 2 and Moore, K. W. et al. (1993), Ann Rev Immunol
11:165-190). The findings that production of active TGF-.beta. is
decreased in patients with mild as well as active disease, and that
we could only partially reverse the production defect by
antagonizing IL-10 (Example 2), suggests that increased IL-10
production, by itself, cannot account for decreased lymphocyte
production of active TGF-.beta.1 in SLE. Several mechanisms are
probably involved. It is likely that one or more defects in the
extracellular conversion of the latent precursor to the mature,
active form may explain this abnormality.
[0138] Although TGF-.beta. has well documented inhibitory
properties on lymphocyte proliferation and effector cell function
(Letterio, J. J. et al. (1998), Ann Rev Immunol 16:137-162),
stimulatory properties have also been reported (Lee, H. M. et al.
(1991), J Immunol 151:668-677). TGF-.beta. modulates cytokine
production by stimulated T cells as well as up-regulating its
production. In mice, TGF-.beta.1 selectively activates CD8.sup.+ T
cells to proliferate (Lee, H. M. et al. (1991), J Immunol
151:668-677), and augments the maturation of naive cells to memory
T cells (Lee, H. M. et al. (1991), J Immunol 147:1127-1133). In
humans TGF-.beta.1 is a potent inducer of effector T cells
(Cerwenka, A. et al. (1994), J Immunol 153:4367-4377). While large
(nanogram/ml) quantities are required for immuno-suppressive
effects, we have shown that only small (picogram/ml) quantities are
needed to co-stimulate CD8.sup.+ T cells for down-regulatory
effects on antibody production (Gray, J. D. et al. (1998), J
Immunol 160:2248-2254).
[0139] These studies suggest, therefore, that while impaired
lymphocyte secretion of the latent precursor of TGF-.beta.1 may
result as a consequence of disease activity, decreased active
TGF-.beta.1 production in SLE is more complex and may result from
several different mechanisms. We have proposed that programming
naive T cells to down-regulate antibody production requires the
presence of pg/ml quantities of active TGF-.beta. at the time they
are activated and have evidence to support this suggestion (Gray,
J. D. et al. (1998), J Immunol 160:2248-2254). Therefore, a lack of
picomolar amounts of active TGF-.beta. in the local environment at
a critical time could possibly account for ineffective T cell
regulatory function to control B lymphocyte activity in SLE.
Example 3
1. Treating SLE with Mitogens
[0140] In this example, IgG production is down regulated by
treating the cells with an regulatory composition comprising a
mitogen such as a combination of mitogenic anti-CD2 monoclonal
antibodies. These antibodies may be added in soluble form or
immobilized on beads to cross link receptors on T cells and NK
cells. The cells are prepared as outlined in the above examples,
and then they are incubated with mitogens to augment the population
of cells that down regulate antibody production. Con A is available
from Sigma (St. Louis, Mo.).
[0141] Although it is not known how anti-CD2 works, it is believed
that these antibodies induce NK cells in the PBMC preparation to
secrete active TGF-.beta. (Ohtsuka, K. et al. (198), J Immunol
160:2539-2545); TGF-.beta. then acts on T cells to become antibody
suppressor cells.
[0142] The cells are then washed, if necessary, and transplanted
back into the patient.
Example 4
1. Treating Cells with a Mixture of Cytokines and Mitogens
[0143] Cells are prepared as outlined above, and incubated with an
regulatory composition comprising a mixture of mitogen and cytokine
to induce populations of cells that down regulate antibody
production. An example of this approach is shown in FIG. 4C. In
this example, maximum induction of suppression was obtained by
treating CD4+ cells and CD8+ cells with Con A, IL-2 and TGF-.beta.
for.
[0144] For the preparation of regulatory T cells that will be
transferred back to the patient anti-CD2 and/or anti-CD3 monoclonal
antibodies will be used instead of Con A to activate T cells. The
regulatory composition contains TGF-.beta. with or without IL-2.
The cells are incubated with the composition for 4 to 72 hours
using standard incubation techniques in a closed system such as the
Nexell 300i.Magnetic
[0145] a) Cell Selection System.
[0146] Following incubation, the cells are washed with HBBS to
remove any cytokine and mitogen in the solution. The cells are
optionally further expanded by culturing with anti-CD3.+-.anti-CD28
immobilized on beads. The cells are suspended in 200-500 ml of HBBS
and reintroduced into a patient.
Example 5
1. Treating Cells to Normalize Cell-Mediated Immunity
[0147] Contributing to autoantibody production in SLE is an
imbalance between IL-10 and TNF-.alpha. production. Levels of IL-10
are excessive and levels of TNF-.alpha. are decreased (Llorente et
al. 1995. J. Exp. Med. 181:839-44) (Houssiau, F. A. et al., 1995.
Lupus 4:393-5. (Ishida, H. et al. 1994. J. Exp. Med. 179:305-10)
(Jacob, C. O. and McDevitt, H. O., 1988. Nature 331:356-358). We
have evidence that this imbalance is corrected by strongly
activating T cells in the presence of TGF-.beta. and have recently
elucidated the mechanism of action of this effect.
[0148] Purified T cells were prepared as outlined above, and
incubated with ConA and IL-2 with or without TGF-.beta.. FIG. 6
shows that T cell stimulation in the absence of TGF-.beta.,
resulted in increased production of IL-10. However, when TGF-.beta.
was added to stimulated T cells, IL-10 production was blocked and
production of TNF-.alpha. was increased. In addition, TNFR2
expression was increased significantly. Without being bound by
theory, It is believed that accelerated TNF-.alpha. signaling via
TNFR2 induced by TGF-.beta. results in regulatory T cells that
inhibit antibody production. Our results support this
suggestion.
[0149] We have determined that upregulation of TNF-.alpha. by
TGF-.beta. is essential for the induction of regulatory T cells.
FIG. 7 shows two experiments where the addition of TGF-.beta. to
activated CD8+ T cells resulted in marked suppression of IgG
production. This suppressive activity depended upon TNF-.alpha. as
an essential intermediate. In each of these experiments, a
neutralizing anti-TNF-.alpha. antibody completely abolished the
suppressive effects of the CD8+ regulatory T cells (CD8reg).
[0150] Patients with SLE have a marked defect in cell-mediated
immunity with impaired production of IL-2, TNF-.alpha. and
IFN-.gamma.. (Horwitz, D. A. et al. (1997), Dubois' Lupus
Erythematosus, 5th Ed. (1997), pp. 83-96, D. J. Wallace et al.
eds., Williams and Wilkins, Baltimore). Without being bound by
theory, it is believed that the defect in lymphocyte production of
TGF-.beta. is partially responsible for impaired production of
IL-2, TNF-.alpha. and IFN-.gamma.. We have found that stimulation
of T cells in the presence of TGF-.beta. significantly increased
production of IL-2, TNF-.alpha. and IFN-.gamma. when these cells
were restimulated. Moreover, this result was dependent upon
upregulation of TNF-.alpha. by TGF-.beta. (see FIG. 8).
[0151] We have evidence that TGF-.beta. production is decreased in
SLE and that this defect contributes to the imbalance between IL-10
and TNF-.alpha.. Without being bound by theory, it is believed that
high levels of IL-10 in SLE sustain autoantibody production and are
responsible for decreased production of TNF-.alpha., IL-2,
IFN-.gamma.. Decreased production of these cytokines is responsible
for defective cellular immunity in SLE. We have demonstrated that
under specified conditions, TGF-.beta. down-regulates IL-10 and
enhances the production of TNF-.alpha.. Down-regulation of IL-10
and enhancement of TNF-.alpha. production by TGF-.beta. plays a
crucial role in the normalization of regulatory T cell activity in
SLE, restoration of cell-mediated immunity and remission of
disease.
Example 6
1. Generation of Regulatory T Cells that Suppress Cell-Mediated
Autoimmunity
[0152] The previous examples used regulatory compositions to treat
antibody-mediated autoimmune diseases. Similar compositions are
used to induce CD4+ as well as CD8+ T cells to suppress
cell-mediated autoimmune diseases. We show that CD8+ or CD4+ cells
conditioned by TGF-.beta. alone suppressed the generation of T cell
cytotoxicity.
[0153] Instead of using mitogens to induce regulatory T cells, the
allogeneic mixed lymphocyte reaction is used for this purpose. In
this reaction, T cells from one individual recognize and respond to
foreign histocompatibility antigens displayed by other individuals
PBMCs. These responder T cells proliferate and develop the capacity
to kill these target cells.
[0154] To develop suppressor T cells, various CD4+ and CD8+ T cell
subsets from one individual (donor A) were cultured with irradiated
T cell-depleted mononuclear cells from another individual (donor
B). The cells were cultured for 5 days with or without TGF-.beta.
(1 ng/ml) in the suspensions. After this time, TGF-.beta. was
removed and the cells added to fresh T cells from donor A and non-T
cells from donor B. FIG. 9 shows TGF-.beta. induced both CD4+ and
CD8+ T cell subsets to develop the capacity to inhibit cell
mediated cytotoxicity. FIG. 10 shows two additional experiments
with CD4+ regulatory T cells induced by TGF-.beta..
[0155] Further studies revealed that regulatory CD4+ T cells
generated in this manner have a unique mode of action. Unlike the
CD8+ and CD4+ T cells generated previously which suppress by
secreting inhibitory cytokines, these allo-specific regulatory CD4+
T cells have a contact dependent mechanism of action (FIG. 11).
Without being bound by theory, it is believed that these regulatory
T cells suppress other T cells from being activated. Addition of
these T cells to responder T cells and allo-stimulator cells
inhibited proliferation (FIG. 12) and decreased the ability of
responder CD8+ killer precursor cells to become activated (FIG.
13).
[0156] We also learned that these regulatory CD4+ cells express
IL-2 receptors (CD25) on their cell surface and were extremely
potent (FIG. 14). Decreasing the proportion of regulatory CD4+
cells to responder T cells from 1:4 (20%) to 1:32 (3%) only
minimally decreased the inhibitory effects of these cells.
[0157] Because only a few of these cells are needed for potent
down-regulatory effects, it is likely that a sufficient number can
be transferred to patients to suppress autoimmunity or other
desired immunosuppressive effects, such as inhibiting of graft
rejection.
Example 7
1. Stimulating CD4+ T Cells to Produce Immunosuppressive Levels of
TGF-.beta.
[0158] CD4+ T cells that produce immunosuppressive levels of
TGF-.beta. have been named Th3 cells, but the mechanisms involved
in their development are poorly understood. We have obtained
evidence that strong stimulation of CD4+ cells with the
superantigen, staphylococcus enterotoxin B (SEB), or repeated
stimulation of CD4+ cells stimulated with a lower concentration of
SEB induced these cells to produce immunosuppressive levels of
active TGF-.beta..
[0159] FIG. 15 shows increased production of both active and total
TGF-.beta. produced by CD4+ T cells stimulated with increasing
concentrations of SEB. FIG. 16 shows the effect of repeated
stimulation of CD4+ T cells with low doses of SEB. By the third
time these T cells were stimulated with SEB, they produced
significant amounts of the active form of TGF-.beta..
[0160] FIG. 17 shows the effects of SEB on naive (CD45RA+CD45RO--)
CD4+ and CD8+ T cells. The cells were stimulated with SEB every 5th
day for a total of three stimulations. The percentages of each T
cell subset and the cells expressing the CD25 IL-2 receptor
activation marker were determined after each stimulation. Panels A
and C show that by including TGF-.beta. 1 ng/ml in the initial
stimulation, CD4+ T cells became the predominant subset in the
cultures after repeated stimulation. Figures B and D show that CD25
expression by SEB stimulated cells decreased by the third
stimulation in control cultures. However, CD25 expression remained
very high if the T cells were primed with TGF-.beta.. Thus,
TGF-.beta. appears to have preferential effects on CD4+ cells if
these T cells are repeatedly stimulated and almost all of these
cells were CD25+ after culture for 20 days.
[0161] In summary, following T cell stimulation, the predominant
regulatory effects of TGF-.beta. are directed to CD8+ cells. Upon
repeated stimulation, this cytokine now induces CD4+ cells to
become regulatory cells and these cells are more potent than CD8+
cells in their suppressive activities.
* * * * *