U.S. patent application number 09/735422 was filed with the patent office on 2003-01-23 for methods for the diagnosis and treatment of insulin-dependent diabetes mellitus.
This patent application is currently assigned to The Regents of The University of California.. Invention is credited to Kaufman, Daniel L., Tian, Jide.
Application Number | 20030017509 09/735422 |
Document ID | / |
Family ID | 27116528 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017509 |
Kind Code |
A1 |
Kaufman, Daniel L. ; et
al. |
January 23, 2003 |
Methods for the diagnosis and treatment of insulin-dependent
diabetes mellitus
Abstract
Methods for detecting the status of an insulin-dependent
diabetes mellitus (IDDM)-associated autoimmune response in a mammal
are provided. Specifically, the ratio of the frequency of T helper
1 cells to T helper 2 cells specific for a pancreatic .beta.-cell
associated antigen is indicative of the status of the autoimmune
response. The methods may be employed prior to the onset of the
clinical symptoms of the disease, thereby allowing identification
of those at risk for developing clinical symptoms of IDDM, or
subsequent to pancreatic tissue transplantation, for example, to
measure the efficacy of treatment directed to enhancing the
lifetime of the tissue transplant. Methods for prolonging the
survival of tissue transplants are also provided. Specifically, a
tissue-associated antigen is administered to the mammal which
serves to shift the pathogenic Th1 response associated with
pathological immunity toward a protective Th2 response.
Inventors: |
Kaufman, Daniel L.; (Santa
Monica, CA) ; Tian, Jide; (Los Angeles, CA) |
Correspondence
Address: |
Todd A. Lorenz
FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Assignee: |
The Regents of The University of
California.
|
Family ID: |
27116528 |
Appl. No.: |
09/735422 |
Filed: |
December 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09735422 |
Dec 12, 2000 |
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09119922 |
Jul 21, 1998 |
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6207159 |
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09119922 |
Jul 21, 1998 |
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08890152 |
Jul 9, 1997 |
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6022697 |
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08890152 |
Jul 9, 1997 |
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08758414 |
Nov 29, 1996 |
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Current U.S.
Class: |
435/7.21 ;
424/93.7 |
Current CPC
Class: |
A61K 39/0008 20130101;
A61P 3/10 20180101; G01N 2800/24 20130101; Y10S 514/866 20130101;
G01N 33/6854 20130101; A61P 37/06 20180101; Y10S 424/81 20130101;
G01N 2800/52 20130101; A61K 38/00 20130101; G01N 2800/042 20130101;
G01N 33/564 20130101; G01N 2800/245 20130101 |
Class at
Publication: |
435/7.21 ;
424/93.7 |
International
Class: |
G01N 033/567; A61K
045/00 |
Claims
What is claimed is:
1. A method for detecting the status of an IDDM-associated
autoimmune response in a mammal, said method comprising determining
the ratio of T helper 1 to T helper 2 cells in said mammal each of
which are specific for a pancreatic .beta.-cell-associated antigen,
wherein said ratio is indicative of the status of said autoimmune
response in said mammal.
2. The method according to claim 1, wherein the number of T helper
1 cells specific for said pancreatic .beta.-cell-associated antigen
is determined by detecting the frequency of interferon-.gamma.
secreting T-cells in said mammal that are specific for said
antigen.
3. The method according to claim 1, wherein the number of T helper
2 cells specific for said pancreatic .beta.-cell-associated antigen
is determined by detecting the frequency of interleukin-4 and
interleukin-5 secreting T-cells in said mammal that are specific
for said antigen.
4. The method according to claim 3, wherein the number of T helper
1 cells specific for said pancreatic .beta.-cell-associated antigen
is determined by detecting the frequency of interferon-.gamma.
secreting T-cells in said mammal that are specific for said
antigen.
5. The method according to claim 1, wherein said determining is
performed prior to the onset of clinical symptoms of IDDM in said
mammal.
6. The method according to claim 1, wherein said determining is
performed subsequent to a pancreatic tissue transplant in said
mammal.
7. The method according to claim 6, wherein said method measures
the efficacy of a treatment for enhancing the lifetime of said
transplant.
8. The method according to claim 1, wherein said pancreatic
.beta.-cell-associated antigen is selected from the group
consisting of GAD65, hsp65 and insulin B-chain.
9. The method according to claim 8, wherein said pancreatic
.beta.-cell-associated antigen is GAD65.
10. A method for detecting the status of an IDDM-associated
autoimmune response in a mammal, said method comprising determining
the isotype of antibodies in said mammal specifically directed
against a pancreatic .beta.-cell-associated antigen, wherein the
ratio of the amount of said antibodies having an isotype which is
associated with the presence of T helper 1 cells specific for said
.beta.-cell-associated antigen to the amount of said antibodies
having an isotype which is associated with the presence of T helper
2 cells specific for said .beta.-cell-associated antigen is
indicative of the status of the IDDM-associated autoimmunity in
said mammal.
11. The method according to claim 10, wherein said determining is
performed prior to the onset of clinical symptoms of IDDM in said
mammal.
12. The method according to claim 10, wherein said determining is
performed subsequent to a pancreatic tissue transplant in said
mammal.
13. The method according to claim 12, wherein said method measures
the efficacy of a treatment for enhancing the lifetime of said
transplant.
14. The method according to claim 10, wherein said pancreatic
.beta.-cell-associated antigen is selected from the group
consisting of GAD65, hsp65 and insulin B-chain.
15. The method according to claim 14, wherein said pancreatic
.beta.-cell-associated antigen is GAD65.
16. A method for prolonging the survival of a tissue transplant in
a mammal, said method comprising: administering to said mammal a
tissue transplant protecting amount of an antigen which is
associated with the donor tissue, wherein said antigen
administration results in an increase in the ratio of T helper 2
cells specific for said antigen to the number of T helper 1 cells
specific for said antigen.
17. The method according to claim 16, wherein said tissue
transplant is a pancreatic tissue transplant.
18. The method according to claim 16, wherein said administering is
performed prior to transplantation of said tissue into said
mammal.
19. The method according to claim 16, wherein said administering is
performed after transplantation of said tissue into said
mammal.
20. The method according to claim 16, wherein said antigen is an
autoantigen.
21. The method according to claim 20, wherein said autoantigen is a
pancreatic .beta.-cell-associated autoantigen.
22. The method according to claim 21, wherein said pancreatic
.beta.-cell-associated autoantigen is GAD65.
23. A method for reducing the severity of an IDDM-associated
autoimmune response in a mammal, said method comprising
administering to said mammal a therapeutically effective amount of
a pancreatic .beta.-cell-associated antigen, wherein said
administering results in an increase in the ratio of the number of
T helper 2 cells specific for said antigen to the number of T
helper 1 cells specific for said antigen.
24. The method according to claim 23, wherein said pancreatic
.beta.-cell-associated antigen is selected from the group
consisting of GAD65, hsp65 and insulin B-chain.
25. The method according to claim 24, wherein said pancreatic
.beta.-cell-associated antigen is GAD65.
26. The method according to claim 23, wherein said pancreatic
.beta.-cell-associated antigen is administered in conjunction with
an adjuvant.
27. The method according to claim 26, wherein said adjuvant is
selected from the group consisting of incomplete Freund's adjuvant,
aluminum hydroxide, interleukin-4 and interleukin-10.
28. The method according to claim 23, wherein said administration
is parenteral.
29. The method according to claim 28, wherein said parenteral
administration is by subcutaneous, intramuscular, intraperitoneal,
intracavity, transdermal, intranasal, or intravenous injection.
30. The method according to claim 23, wherein said administration
is at a dosage of about 0.01 mg/kg/dose to about 2000
mg/kg/dose.
31. A method for increasing the frequency of T helper 2 cells
specific for a tissue-associated antigen relative to T helper 1
cells specific for said tissue-associated antigen in a mammal
comprising administering to said mammal said tissue-associated
antigen in an amount sufficient to induce a T helper 2 cell
response to said antigen.
32. The method according to claim 31, wherein said
tissue-associated antigen is a pancreatic .beta.-cell-associated
autoantigen.
33. The method according to claim 32, wherein said pancreatic
.beta.-cell-associated autoantigen is selected from the group
consisting of GAD65, hsp65 and insulin B-chain.
34. The method according to claim 32, wherein said pancreatic
.beta.-cell-associated autoantigen is GAD65.
35. The method according to claim 31, wherein said
tissue-associated antigen is administered in conjunction with an
adjuvant.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to improved methods for
diagnosing and treating insulin-dependent diabetes mellitus in
mammals.
BACKGROUND
[0002] Insulin-dependent diabetes mellitus (IDDM; type I diabetes)
is one of the most commonly occurring metabolic disorders in the
world. In the United States, IDDM affects approximately one in 300
to 400 people, and epidemiological studies suggest that its
incidence is continuing to increase. IDDM is caused by an
autoimmune response that results in the T lymphocyte-mediated
destruction of the insulin-producing .beta.-cells of the pancreas.
Castrano et al., Ann. Rev. Immunol. 8:647-679 (1990).
Unfortunately, however, although the general mechanism by which
IDDM occurs is known, IDDM becomes clinically evident only after
the vast majority (approximately 80% or more) of the pancreatic
.beta.-cells have been irrevocably destroyed and the individual
becomes dependent upon an exogenous source of insulin. In other
words, at the time that the disease becomes clinically evident, the
autoimmune response is well established and has already caused
irreparable damage to the insulin-producing pancreatic tissue.
[0003] Because autoimmune-induced pancreatic damage is far
progressed by the time that clinical symptoms of IDDM become
evident, successful treatment of the autoimmune response ideally
should be initiated well before the patient begins to exhibit overt
symptoms of diabetes and requires insulin replacement for his or
her own lost capability to produce insulin. In fact, any form of
therapy would be expected to be more effective if persons at risk
could be identified before the onset of clinical symptoms and the
concomitant destruction of pancreatic .beta.-cells. Moreover,
techniques useful for tracking progression of the IDDM-associated
autoimmune response in patients would allow one to assess the
effectiveness of therapies which could be employed after the onset
of disease. There is, therefore, a need for quick and reliable
techniques for diagnostically identifying persons at risk for
developing the clinical symptoms of IDDM and for monitoring the
progression of the autoimmune response in those persons.
[0004] Prior to the onset of clinical symptoms of IDDM but after
the onset of the IDDM-associated autoimmune response, attempts have
been made to control the established diverse autoreactive T cell
population, thereby effectively inhibiting progression of the
disease. For example, immunosuppressants and antibodies which are
specifically directed against autoimmune T cells may be useful for
delaying the onset of disease. However, such treatments lack
specificity and often significantly debilitate immune system
function. Moreover, immunotherapeutics directed at blocking T
cell-receptor/major histocompatibility complex (MHC) interactions
can be highly specific, but may also be confounded by the
complexity of the autoreactive T cell population and the genetic
diversity of MHCs within the patient population.
[0005] Once the clinical symptoms of IDDM become evident, numerous
different therapies have been employed for treating the
debilitating effects of the disease. For example, by far the most
commonly employed therapy for the clinical symptoms of IDDM is
exogenous insulin replacement. However, while insulin replacement
therapy allows most IDDM patients to lead somewhat normal lives,
insulin replacement is also imperfect and does not completely
restore metabolic homeostasis. As a result, severe complications
including dysfunctions of the eye, kidney, heart, and other organs
are common in diabetic patients undergoing insulin replacement
therapy.
[0006] Another common treatment for the clinical symptoms of IDDM
is pancreatic or .beta.-islet cell transplantation. However, the
insulin-producing .beta.-cells of transplanted tissues are often
rapidly destroyed by the same autoimmune response which had
previously destroyed the patient's own pancreatic tissue.
Therefore, the use of immunosuppressants after transplantation is
common, carrying with it the adverse side effects described
above.
[0007] As such, in addition to the urgent need for improved methods
for the early diagnostic identification of persons who are at risk
for developing the clinical symptoms of IDDM and for monitoring the
progression of the autoimmune response in those at risk persons,
there is also an urgent need for improved methods for
therapeutically treating those persons who already exhibit clinical
symptoms of the disease. Specifically, there exists a need for
methods effective in prolonging the lifetime of tissue transplants,
for example pancreatic tissue transplants, in mammals without the
use of immunosuppressants and for inhibiting the T cell-mediated
autoimmune mechanism underlying the disease.
[0008] The autoimmune response underlying IDDM is thought to be
mediated by proinflammatory T helper 1 (Th1) cells; cells that are
known to secrete interferon-.gamma. (IFN-.gamma.) and promote the
production of murine IgG2a isotype antibodies that are directed
against pancreatic .beta.-cell-associated autoantigens. In contrast
to Th1 cells, T helper 2 (Th2) cells are known to secrete
interleukin-4 (IL-4) and interleukin-5 (IL-5) and promote the
production of murine IgG1 isotype antibodies directed against
pancreatic .beta.-cell-associated autoantigens. We have herein
tested whether at advanced stages of the IDDM-associated autoimmune
response, the diverse Th1 cell pool could be downregulated by the
induction of an anti-inflammatory Th2 response to a single
.beta.-cell-associated antigen, thereby effectively reducing the
autoimmune-mediated destruction of the pancreatic tissue.
[0009] We have previously shown in an animal model of human IDDM,
the nonobese diabetic (NOD) mouse, that a pathogenic Th1 response
to the .beta.-cell-associated autoantigen glutamic acid
decarboxylase (GAD) arises at 4 weeks of age, concurrent with the
onset of insulitis in these animals. Kaufman et al., Nature
366:69-72 (1993). GAD is a mammalian protein which serves to
catalyze the rate-limiting step in the synthesis of
.gamma.-aminobutyric acid (GABA), a major inhibitory
neurotransmitter of the mammalian central nervous system. Spink et
al., J. Neurochem. 40:1113-1119 (1983), Huang et al., Proc. Natl.
Acad. Sci. USA 87:8491-8495 (1990), Kobayashi et al., Neurosci.
7:2768-2772 (1987), Chang et al., J. Neurosci. 8:2123-2130 (1988),
Bu et al., Proc. Natl. Acad. Sci. USA 89:2115-2119 (1992), Karlsen
et al., Diabetes 41:1355-1359 (1992) and U.S. Pat. No. 5,475,086,
issued Dec. 12, 1995. The GAD protein is present on various tissues
and exists in multiple isoforms, one of which is GAD65, an antigen
found to be associated with pancreatic .beta.-cells. Subsequent to
the anti-GAD Th1 response described above, T-cell autoimmunity
appears to spread to other .beta.-cell antigens such as a 65 kD
heat shock protein (hsp65), insulin B-chain, carboxypeptidase H and
peripherin in a cascade of autoimmune responses that ultimately
leads to IDDM. Kaufman et al., supra and Tisch et al., Nature
366:72-75 (1993).
[0010] Thus, methods for shifting a pathogenic Th1-associated
autoimmune response to a protective anti-inflammatory Th2 response
would be useful for inhibiting the destructive effects of the
autoimmune response and, therefore, for inhibiting and/or delaying
the onset of clinical symptoms of IDDM and for prolonging the
survival of pancreatic tissue transplants. Moreover, an analysis of
the Th1/Th2 profiles of any particular subject will provide
information as to risk for developing the clinical symptoms of IDDM
and for determining progression of the autoimmune response and the
effect of therapies after the onset of the autoimmune response.
SUMMARY OF THE INVENTION
[0011] It is an object of the present invention to provide methods
for detecting the status of insulin-dependent diabetes mellitus
(IDDM)-associated autoimmunity in a mammal who is at risk of
developing the clinical symptoms of IDDM or who has undergone a
pancreatic tissue transplant. Accordingly, in one aspect, the
present invention is directed to methods for detecting the status
of IDDM-associated autoimmunity in a mammal which comprises
determining the frequency of T helper 1 and T helper 2 cells in the
subject which are specific for a pancreatic .beta.-cell-associated
antigen. IDDM-associated autoimmune destruction of
insulin-producing pancreatic .beta.-cells is a result of the
presence of .beta.-cell-associated antigen-specific T helper 1
cells; T cells which can be identified by their ability to secrete
interferon-.gamma. (IFN-.gamma.). As demonstrated herein, however,
induction of a Th2 response, thereby resulting in a relative
increase in the number of T helper 2 cells as compared to T helper
1 cells specific for the .beta.-cell-associated antigen, is
indicative of protection against further destruction of pancreatic
.beta.-cells. In contrast to T helper 1 cells, the presence of T
helper 2 cells can be detected by their ability to secrete
interleukin-4 (IL-4) and interleukin-5 (IL-5). Preferably, the
.beta.-cell-associated antigens are autoantigens. Illustrative
.beta.-cell-associated autoantigens include GAD65, hsp65 and
insulin B-chain. This diagnostic method can be employed prior to
the onset of clinical symptoms, for the purposes of detecting the
status of the IDDM-associated autoimmune response and determining
whether the subject is at risk for developing IDDM, or subsequent
to the onset of clinical symptoms, for the purposes of detecting
the status of the autoimmune response against a pancreatic tissue
transplant.
[0012] In another aspect, the status of IDDM-associated
autoimmunity in a mammal who is at risk of developing the clinical
symptoms of IDDM or who has undergone a pancreatic tissue
transplant can be detected by determining the isotype of antibodies
present in the subject which are directed against a specific
.beta.-cell-associated antigen. As demonstrated herein, the
presence of pathogenic T helper 1 cells specific for a
.beta.-cell-associated antigen in mice induces the production of
IgG2a isotype antibodies against that antigen whereas the presence
of protective T helper 2 cells specific for the antigen induces the
production of IgG1 isotype antibodies against that antigen. As
such, by measuring the relative numbers of IgG2a and IgG1 isotype
antibodies against a specific .beta.-cell-associated antigen (or
the corresponding isotype antibodies from other mammals which are
indicative of the presence in the mammal of T helper 1 and T helper
2 cells directed against the .beta.-cell-associated antigen), one
may determine the status of the autoimmune response in the
subject.
[0013] It is a further object of the present invention to provide
an improved method for prolonging the survival of a tissue
transplant in a mammal. Specifically, the present invention is
directed to a method for prolonging the survival of a tissue
transplant in a mammal by administering to that mammal a tissue
transplant protecting amount of an antigen which is associated with
the donor transplanted tissue. As demonstrated herein, the
administration of a pancreatic .beta.-cell-associated antigen to a
mammal either prior to or shortly after pancreatic tissue
transplantation results in an increased ratio of the number of
protective T helper 2 cells specific for that antigen to the number
of pathogenic T helper 1 cells specific for that antigen. As such,
this shift from a pathogenic Th1 response toward a protective Th2
response results in inhibition of the immune response and an
effective increase in the lifetime of the tissue transplant. The
method may be practiced with antigens derived from virtually any
tissue which is transplanted and which results in the induction of
a T cell-mediated immune response.
[0014] A further object of the present invention is to provide a
method for reducing the severity of an IDDM-associated autoimmune
response in a mammal comprising administering to the mammal a
therapeutically effective amount of a pancreatic
.beta.-cell-associated antigen, wherein the administration of the
antigen results in an increase in the ratio of the number of T
helper 2 cells specific for that antigen to the number of T helper
1 cells specific for that antigen. The antigen may be administered
alone or in combination with an adjuvant for the purpose of
increasing the T helper 2/T helper 1 ratio.
[0015] Another embodiment of the present invention is directed to a
method for increasing the frequency of T helper 2 cells specific
for a tissue-associated antigen relative to T helper 1 cells
specific for the tissue-associated antigen which comprises
administering to the mammal the tissue-associated antigen in an
amount sufficient to induce a T helper 2 cell response to the
antigen.
[0016] These and other aspects of the invention will become evident
upon reference to the following detailed description and the
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A-1D show that antigen administration to nonobese
diabetic (NOD) mice induces antigen-specific IgG1 antibodies.
Specifically, animals were treated with .beta.-galactosidase
(.tangle-solidup.) or glutamic acid decarboxylase 65 (GAD65)
(.circle-solid.) and antibodies to .beta.-galactosidase (FIGS. 1A
and 1C) and GAD65 (FIGS. 1B and 1D) were characterized using
isotype-specific ELISA assays. The data are represented as the mean
absorbance values over background of triplicate samples from
individual mice (n=10 for each group). The background absorbance
ranged between 0.06 and 0.1. Experimental and control serum samples
were tested simultaneously in two separate assays. In FIGS. 1C and
1D, data is presented as the levels of IgG1 versus the levels of
IgG2a in individual mice.
[0018] FIGS. 2A-2B show that antigen administration to NOD mice
stimulates the expansion of antigen-specific Th2 cells.
Specifically, the frequency of .beta.-galactosidase-specific (FIG.
2A) and GAD65-specific (FIG. 2B) interferon-.gamma.- (IFN-.gamma.),
interleukin-4- (IL-4) and interleukin-5- (IL-5) secreting splenic T
cells in .beta.-galactosidase- (.box-solid.) and GAD65-
(.quadrature.) treated mice were determined by ELISPOT. Data are
represented as the mean number of spot-forming colonies (SFC) per
10.sup.6 splenic T cells .+-. s.e.m. Samples were tested in
triplicate. Experimental and control mice were tested
simultaneously in two separate experiments (n=5 for each
group).
[0019] FIG. 3 shows that adoptive cotransfer of splenic T cells
from GAD65-treated mice protects recipient mice from IDDM.
Specifically, splenic mononuclear cells from mice treated with
.beta.-galactosidase (.tangle-solidup.) or GAD65 (.largecircle.)
were cotransferred with T cells from diabetic NOD mice to
5-week-old irradiated female NOD mice. A positive control group
received cells only from untreated-diabetic NOD mice (.diamond.).
Blood glucose levels were monitored frequently, and animals were
considered diabetic after two consecutive blood glucose levels
of>13 mmol/l (n=10 for all groups).
[0020] FIG. 4 shows that administration of GAD65 after the
establishment of .beta.-cell autoimmunity inhibits disease
progression. Specifically, female NOD mice were treated at 8 weeks
of age with IFA alone (.diamond.), .beta.-galactosidase (A) or
GAD65 (.largecircle.). The mice were followed for the onset of
hyperglycemia (n=10 for each group).
[0021] FIG. 5 shows that administration of GAD65 prolongs survival
of syngeneic islet grafts in diabetic NOD mice. Islets were
transplanted into diabetic NOD mice that had been treated with
.beta.-galactosidase (A), GAD65 (.largecircle.), insulin B-chain
(.quadrature.), or hsp65 peptide (.diamond.), and insulin
administration was discontinued. Data are presented as time post
transplantation at which hyperglycemia recurred (blood glucose
levels>13 mmol/l). (n=14 for .beta.-galactosidase, n=17 for
GAD65, n=9 for insulin B chain and n=11 for hsp65).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0022] The present invention is based at least in part upon the
surprising finding that a shift from a T helper 1 (Th1) response
towards a T helper 2 (Th2) response specific for a pancreatic
.beta.-cell-associated antigen results in protection against the
pancreatic .beta.-cell destruction that is associated with
IDDM-associated autoimmunity. As such, the correlation between a
shift from a Th1 response to a Th2 response and protection against
the autoimmune-induced destruction of pancreatic .beta.-cells can
be exploited for both diagnostic and therapeutic purposes.
[0023] Accordingly, in one embodiment, the present invention is
directed to a method for detecting the status of an IDDM-associated
autoimmune response in a mammal. The term "detecting the status"
when used in reference to IDDM-associated autoimmunity means
determining the degree to which the autoimmune response has
progressed in the subject or, in the case where the method is
performed subsequent to a pancreatic tissue transplant, to
determine if a therapy is working to enhance the lifetime of the
tissue transplant by lessening the pathogenic anti-.beta.-cell
autoimmune response. It is well known that the autoimmune mechanism
underlying IDDM is far progressed by the time that clinical
symptoms of the disease become evident and, therefore, one may
determine the degree to which the autoimmune response has
progressed in a subject even before the onset of clinical symptoms
of the disease.
[0024] A mammalian subject is "diagnosed as having" IDDM once
clinical symptoms of the disease become evident. For the most part,
this occurs at the time that the subject requires an exogenous
source of insulin to survive. Detecting the status of an
IDDM-associated autoimmune response in subjects who have been
diagnosed as being at risk for IDDM is useful, for example, for
monitoring or measuring the efficacy of a treatment to inhibit
disease progression. Prior to the onset of clinical symptoms of the
disease, a subject may also be "at risk of having" IDDM. This term
is intended to define those persons who have not yet developed
clinical symptoms of IDDM but who exhibit IDDM-associated
autoimmunity. As such, one may detect the status of an
IDDM-associated autoimmune response in a subject prior to onset of
the clinical symptoms of the disease.
[0025] One may detect the status of an IDDM-associated autoimmune
response in a subject in a number of ways. For example, as
demonstrated herein, because the characteristics and clinical
symptoms of the disease are associated with the presence of T
helper 1 cells specific for various pancreatic
.beta.-cell-associated antigens, and because a shift from a
pathogenic Th1 response toward a Th2 response, thereby resulting in
an increase in the ratio of the number of T helper 2 cells for a
specific pancreatic .beta.-cell-associated antigen to the number of
T helper 1 cells specific for that antigen, is indicative of a
protective effect and a lessening of the severity of the
destructive IDDM-associated autoimmune response, determining the
frequency of T helper 1 and T helper 2 cells in the subject that
are specific for a pancreatic .beta.-cell-associated antigen is
indicative of the status of the autoimmune response. For example,
an increase in the ratio of T helper 1 cells as compared to T
helper 2 cells specific for a particular pancreatic
.beta.-cell-associated antigen is indicative of a worsening of the
destructive autoimmune response, whereas a decrease in that ratio
is indicative of a reduction in the severity of the destructive
autoimmune response. As such, the ratio of T helper 1 cells
specific for a pancreatic .beta.-cell-associated antigen to T
helper 2 cells specific for that antigen is indicative of the
status of the autoimmune response.
[0026] The frequency or number of T helper 1 and T helper 2 cells
specific for a pancreatic .beta.-cell-associated antigen that are
present in the subject may be determined using techniques that are
available and well known in the art. For example, T helper 1 cells
are known to secrete IFN-.gamma. whereas T helper 2 cells are known
to secrete IL-4 and IL-5. One may determine the frequency of
IFN.gamma.-, IL-4- and IL-5-secreting T cells that are specific for
a particular pancreatic .beta.-cell-associated antigen by employing
the ELISPOT assay described by Forsthuber et al., Science
271:1728-1730 (1996), by cytokine quantitation by ELISA or by
quantitative PCR analysis of the mRNAs encoding the cytokines.
Other T helper 1 and/or T helper 2 cell specific markers may also
find use as predictive markers of the presence of these cells.
[0027] One may also detect the status of an IDDM-associated
autoimmune response in a subject by determining the isotype of
antibodies present in the subject that are specifically directed
against a pancreatic .beta.-cell-associated antigen. For example,
as demonstrated herein, the presence of specific antibody isotypes
are associated with the presence of pathogenic T helper 1 cells and
protective T helper 2 cells, wherein the isotypes specific for each
cell type differ. Specifically, murine IgG2a isotype antibodies are
associated with the presence of T helper 1 cells and murine IgG1
antibodies (corresponding to IgE and IgG4 antibodies in humans) are
associated with the presence of T helper 2 cells. As such, the
ratio of the number of T helper 2 cell-associated isotype
antibodies to the number of T helper 1 cell-associated isotype
antibodies which are specifically directed against a pancreatic
.beta.-cell-associated antigen is indicative of the status of the
autoimmune response. With regard to antibodies, use of the term
"specifically directed against" means that the antibody is capable
of binding to an epitope present on the pancreatic
.beta.-cell-associated antigen. Methods for determining specific
binding of antibodies to antigenic epitopes and for determining the
isotype of antibodies are well known in the art.
[0028] The terms "pancreatic .beta.-cell-associated antigen",
".beta.-cell-associated antigen" and grammatical equivalents
thereof are intended to denote the various antigenic determininants
present on pancreatic .beta.-cells to which autoimmune responses
may be directed, thereby resulting in the destruction of
insulin-producing pancreatic .beta.-cells. Exemplary antigens
present on mammalian pancreatic .beta.-cells to which autoimmune
responses may be directed include GAD65 (see U.S. Pat. No.
5,475,086), heat shock protein 65 (hsp65; see U.S. Pat. No.
5,114,844), insulin, insulin B-chain, carboxypeptidase H and
peripherin. Other pancreatic .beta.-cell-associated antigens are
known in the art. The identification of T helper 1 and T helper 2
cells specific for these antigens and/or antibody isotypes
specifically directed against these antigens may be performed as
described above.
[0029] In another embodiment, the present invention is directed to
a method for prolonging the survival of a tissue transplant in a
mammal wherein the method comprises the step of administering to
the mammal a tissue transplant protecting amount of an antigen
which is associated with the donor tissue. Antigens which are
"associated with the donor tissue" are those antigens which
represent the MHC characteristics of the donor tissue The term
"prolonging the survival of" when used in regard to tissue
transplants means that a tissue transplant in the presence of the
administration of a tissue transplant protecting amount of a
tissue-associated antigen is maintained in an functional state for
a longer period of time than is a tissue transplant in the absence
of any associated antigen administration. By "functional state" is
meant that the transplanted tissue retains it normal physiological
function.
[0030] A "pancreatic tissue transplant" may comprise an entire
pancreas, with or without associated structures, or may comprise
only a portion of an entire pancreas capable of producing insulin.
Pancreatic .beta.-islet cells are an example of the latter type of
transplant.
[0031] Administration of a tissue transplant protecting amount of a
donor tissue-associated antigen results in an increase in the ratio
of the number of protective T helper 2 cells to pathogenic T helper
1 cells specific for that antigen, thereby effectively reducing the
pathogenic immune destruction of cells of the transplanted tissue
and, in turn, resulting in an effective increase in the lifetime of
the tissue transplant. In some cases, there will have been no
initial pathogenic T helper 1 cell response to the antigen of
interest so a mere increase in the number of protective T helper 2
cells serves to increase the T helper 2/T helper 1 ratio. By
"tissue transplant protecting amount" is meant the amount of donor
tissue-associated antigen sufficient to increase the ratio of Th2
to Th1 cells specific for that antigen, thereby reducing the
destructive autoimmune response and effectively prolonging the
lifetime of the tissue transplant in the host. This amount should
not be so high so as to cause adverse side effects, such as
unwanted cross reactions, anaphylactic reactions, and the like and
will differ depending upon the nature of the host, the nature of
the transplanted tissue, the clinical setting, the severity and
degree of the disease, the mode of administration, and the like.
The amount of donor tissue-associated antigen administered can be
determined empirically by those of ordinary skill in the art and
without undue experimentation and can be adjusted by the individual
physician in the event of any counterindications. A tissue
transplant protecting amount may vary from about 0.1 mg/m.sup.2 to
about 2000 mg/m.sup.2, preferably from about 0.1 mg/m.sup.2 to 500
mg/m.sup.2/dose, in one or more dose administrations daily, for one
or several days or boosted in regular intervals (for example, once
every two to six months).
[0032] Pancreatic .beta.-cell-associated antigens which find use
for administration to mammalian subjects include the various
antigenic molecules present on pancreatic .beta.-cells to which
immune responses may be directed. Exemplary antigens present on
mammalian pancreatic .beta.-cells which are useful for
administration include GAD65 (see U.S. Pat. No. 5,475,086), heat
shock protein 65 (hsp65; see U.S. Pat. No. 5,114,844), insulin,
insulin B-chain, carboxypeptidase H, peripherin, and other
pancreatic .beta.-cell-associated antigens and autoantigens which
are known in the art. Either the natural, intact antigen may be
administered, either unconjugated or conjugated to other molecules
such as radionuclides, and the like, or "conservative amino acid
variants" of the antigen may be employed; the phrase "pancreatic
.beta.-cell-associated antigen" being intended to encompass such
variants. Antigens derived from other non-.beta.-cell pancreatic
tissue may also find use herein.
[0033] Conservative amino acid variants of tissue-associated
antigens, including pancreatic .beta.-cell-associated antigens and
autoantigens, may be produced and screened for functionality by
techniques which are routine in the art. Conservative amino acid
variants are variant antigens which possess at least one
conservative amino acid substitution in its amino acid sequence,
the substitution(s) not substantially affecting the ability of the
molecule to prolong the survival of a tissue transplant or to shift
a Th1 response toward a Th2 response. Conservative amino acid
substitutions preferably are made in accordance with the following
Table 1 when it is desired to modulate finely the characteristics
of a tissue-associated antigen (amino acids presented in their
standard three letter amino acid codes).
1 TABLE 1 Original Residue Exemplary Substitutions Ala (A) gly; ser
Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn
Glu (E) asp Gly (G) ala; pro His (H) asn; gln Ile (I) leu; val Leu
(L) ile; val Lys (K) arg; gln; glu Met (M) leu; tyr; ile Phe (F)
met; leu; tyr Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe
Val (V) ile; leu
[0034] Tissue-associated antigens, including pancreatic
.beta.-cell-associated antigens, which find use may also be
"deletional" or "insertional" variants of the natural molecule
which are variants possessing deletions or insertions,
respectively, from the natural amino acid sequence. Such variants
retain a substantial amino acid sequence homology to the natural
antigen and retain the functional ability to prolong the survival
of a tissue transplant or by shifting a Th1 response toward a Th2
response. By "substantial amino acid sequence homology" is meant
that the variant retains an amino acid homology greater than 70%,
preferably at least about 80%, and more preferably at least about
90% with the natural polypeptide sequence. Techniques for producing
and screening such variants for functionality are well known in the
art and could be routinely employed without undue
experimentation.
[0035] The tissue-associated antigen of interest can be
administered parenterally by injection, inhalationally, orally or
by gradual perfusion over time. The pancreatic
.beta.-cell-associated antigen can be administered intravenously,
intraperitoneally, intramuscularly, subcutaneously, intracavity,
transdermally, intranasally, or enterally. The antigen of choice
may be administered either prior to or after the donor tissue is
transplanted in the host, preferably immediately before or
immediately after transplantation, with the only caveat being that
the administration be performed at a time which allows for the
autoimmune-induced destruction of the transplanted tissue to be
reduced.
[0036] Preparations for parental administration include sterile
aqueous or non-aqueous solutions, suspensions, aerosols and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solutions, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, inert gases, and the like.
[0037] In yet another embodiment, the present invention is directed
to a method for reducing the severity of an IDDM-associated
autoimmune response in a mammal comprising administering to the
mammal a therapeutically effective amount of a pancreatic
.beta.-cell-associated antigen, wherein administration of the
antigen results in an increase in the ratio of the number of T
helper 2 cells specific for the antigen to the number of T helper 1
cells specific for the antigen. The term "ameliorate" or
"ameliorating" denotes a lessening of the detrimental effect or
severity of the autoimmune response in the subject receiving
therapy, the severity of the response being determined by means
which are well known in the art. The term "therapeutically
effective amount" means that the amount of pancreatic
.beta.-cell-associated antigen used for administration is
sufficient to reduce the severity of the autoimmune response.
Therapeutically effective amounts will differ based upon the nature
of the patient, the degree and severity of the autoimmune response,
the clinical setting, the mode of administration, and the like and
can be determined empirically by the skilled practitioner without
undue experimentation. A therapeutically effective amount may vary
from about 0.1 mg/kg to about 2000 mg/kg, preferably from about 0.1
mg/kg/dose to about 500 mg/kg/dose, in one or more dose
administrations daily, for one or several days or boosted in
regular intervals.
[0038] The "clinical symptoms" of IDDM are well known to those
skilled in the art and include, for example, the requirement that
the subject be placed on an exogenous source of insulin for
survival.
[0039] For administration purposes, a tissue-associated antigen may
be administered alone or in conjunction with an adjuvant which
useful for inducing a T helper 2 response. By "T helper 2 response"
is meant a shift in the Th1-based autoimmune response toward a
Th2-based response, wherein the number of T helper 2 cells specific
for the antigen increases relative to the number of T helper 1
cells specific for the antigen, thereby reducing the severity of
the immune response. A wide variety of adjuvants find use herein
and include incomplete Freund's adjuvant (IFA), aluminum hydroxide,
the RIBI adjuvant, GMDP, peptides derived from Leshmania, BCG and
other peptides that preferentially induce Th2 responses, nucleic
acids, including for example, immunogenic mRNA (imRNA) and DNA,
cytokines, including for example, IL-4, interleukin-10 (IL-10),
molecules such as B7-2 and IL-12 p40 dimer, and the like. Other
adjuvants are known in the art and could be routinely employed by
those skilled in the art to determine if they assist in the
generation of a Th2 response.
[0040] T helper 2 responses may also be induced independent of the
use of adjuvant by adjusting the dose of antigen administered,
wherein higher doses and lower doses at frequent intervals favor
the induction of a T helper 2 response relative to a T helper 1
response, and varying the site of administration, wherein sites
such as the lung mucosa favor T helper 2 responses independent of
adjuvant.
[0041] The following examples are offered by way of illustration,
and not by way of limitation.
EXPERIMENTAL
[0042] Methods and Materials
[0043] Mice: NOD mice were purchased from Taconic Farms
(Germantown, N.Y.) and bred under specific pathogen-free
conditions. Only female NOD mice were used in this study. In our
NOD mouse colony, insulitis begins at 4 weeks of age. The average
age of disease onset is at 22 weeks, with about 80% of the mice
displaying IDDM by 30 weeks of age.
[0044] Antigens: Mouse GAD65 (Lee et al., Biochem. Biopphys. Acta
1216:157-160 (1993)) and control E. coli .beta.-galactosidase were
purified as previously described. Kaufman et al., supra. The hsp65
immunodominant peptide (Elias et al., Lancet 343:704-706 (1994))
was synthesized by standard fluorenyl methyloxycarbonyl (fmoc)
chemistry and purified by chromatography. The amino acid
composition of each peptide was verified by mass spectrometry.
Insulin B-chain was purchased from Sigma.
[0045] GAD65 Autoantibody Assays: At 8 weeks of age, NOD mice
received a single intraperitoneal (i.p.) injection of 100 .mu.g
.beta.-galactosidase or GAD65 in 50% IFA (Gibco BRL, Gaithersberg,
Md.). Four weeks later, serum samples were tested for GAD65 and
.beta.-galactosidase antibodies by ELISA. .beta.-Galactosidase or
GAD65 (Synectics Biomedical, Stockholm) at 10 .mu.g/ml was bound to
96-well plates (Nunc, Weisbaden, Germany), in 0.1 M NaHCO.sub.3, pH
9.6 (.beta.-galactosidase) or 8.5 (GAD65) at 4.degree. C.
overnight. The wells were rinsed with PBS and then blocked with 3%
BSA in PBS for 1 hour. Mouse sera was added (0.1 ml of a 1/500
dilution) and incubated 1 hour at 37.degree. C. Following washing,
bound Ig was characterized using affinity-purified horseradish
peroxidase (HRP)-coupled goat anti-mouse IgG+A+M (H+L), or
HRP-coupled goat anti-mouse isotype specific antibodies for IgG1
and IgG2a (Southern Biotech Associates and ABTS). Serum samples
from untreated BALB/c and AKR mice were used as negative
controls.
[0046] ELISPOT Analysis: At 8 weeks of age, mice received a single
i.p. injection of 100 .mu.g .beta.-galactosidase or GAD65 in 50%
IFA. Fourteen days later, splenic T cells were isolated and the
frequency of .beta.-galactosidase and GAD65-specific T cells
secreting IL-4, IL-5 and IFN-.gamma. was determined was determined
by using the ELISPOT technique as previously described (Forsthuber
et al., Science 271:1728-1730 (1996)), with the exception that
GAD65 and .beta.-galactosidase (100 .mu.g/ml) were used as
antigens, and 11B11 together with biotinylated BVD6-24G2
(PharMingen, San Diego, Calif.) was used for capture and detection
of IL-4. The resulting spots were counted manually.
[0047] Adoptive Transfer of Diabetes: Eight-week-old NOD mice were
injected i.p. with 100 .mu.g GAD65 or control .beta.-galactosidase
in 100 .mu.l of 50% IFA and reinjected at 14 weeks of age. Five
weeks later, single-cell suspensions of splenic mononuclear cells
were prepared from each group, as well as from unmanipulated
diabetic NOD mice. Ten million splenic mononuclear cells from the
unmanipulated diabetic mice were mixed with an equal number of
splenic mononuclear cells from GAD65 or
.beta.-galactosidase-treated mice and injected intravenously into
5-week-old female NOD mice that had received 500 rad
.gamma.-irradiation. Wicker et al., Diabetes 35:855-860 (1986).
Another control group received 1.times.10.sup.7 splenic mononuclear
cells obtained only from unmanipulated, diabetic mice.
[0048] T-Cell Proliferation Assays: Female NOD mice were injected
i.p. at 8 weeks of age with 100 .mu.g GAD65, or control
.beta.-galactosidase, in 100 .mu.l of 50% IFA. The mice were
reinjected 2 weeks later. At 12 weeks of age, splenic T cells were
tested for proliferative responses to GAD65, hsp65,
.beta.-galactosidase and the HEL peptide, as previously described.
Kaufman et al., supra.
[0049] IDDM Incidence: At 8 weeks of age, groups of 10 female NOD
mice were injected i.p. with 50 .mu.l GAD65 or control
.beta.-galactosidase in 100 l of 50% IFA. Another control group
received 100 .mu.l of 50% IFA alone. Because there may be a
requirement for continual antigen presentation (Ramsdell et al.,
Science 257:1130-1134 (1992)), the mice were reinjected every 6
weeks until 40 weeks of age. Urine glucose levels were monitored
weekly for diabetes by Tes-tape (Lilly). After we observed abnormal
glucose in the urine, blood glucose levels were monitored twice
weekly. A recording of two consecutive blood glucose levels of
>13 mmol/l was considered as IDDM onset.
[0050] Transplantation of Islets: Female NOD mice were monitored
for the onset of IDDM, after which the mice were maintained on
1.0-1.5 units insulin (Humulin U, Lilly) per day. At the time of
IDDM onset, mice were injected with either 100 .mu.g of GAD65,
hsp65, insulin B-chain or control .beta.-galactosidase i.p. in 50%
IFA. Ten days later the mice were reinjected. Ten days after the
second treatment, 3000 freshly isolated islets from newborn NOD
mice were transplanted into the space beneath the kidney capsule,
and humulin administration was discontinued. The mice were
reinjected every 2 weeks. Recurrence of diabetes is defined as two
consecutive blood glucose levels of <13 mmol/l.
EXAMPLE 1
Antigen Administration Promotes a IgG1 Response
[0051] It was unknown whether it was possible to modulate
GAD65-specific Th1/Th2 responses in prediabetic NOD mice and
whether this could interfere with disease progression. As such, NOD
mice were treated only once with GAD65 or control
.beta.-galactosidase in incomplete Freund's adjuvant (IFA) at 8
weeks of age, well after the onset of insulitis, as described in
Materials and Methods. Four weeks after administration, serum
collected from the animals was tested for the presence of
antibodies directed against GAD65 or .beta.-galactosidase by
employing isotype specific ELISA assays. The results are presented
in FIG. 1.
[0052] Specifically, the data presented in FIG. 1 demonstrate that
anti-.beta.-galactosidase immunoglobulins were readily detected in
serum samples taken from the .beta.-galactosidase-treated group,
but were only slightly above the background levels in sera from the
GAD65-treated group (FIG. 1A). The level of GAD65 antibodies in the
.beta.-galactosidase-trea- ted group was similar to that found in
untreated NOD mice (data not shown). Serial dilutions of sera
showed a linear relationship with resulting absorbance measures.
Antibodies to GAD65 in sera from untreated BALB/c and AKR mice were
at background levels.
[0053] As also shown in FIG. 1, high levels of GAD65 autoantibodies
were present in almost all serum samples from the GAD65-treated
group, whereas mice injected with .beta.-galactosidase had low
levels of GAD65 autoantibodies (FIG. 1B), as did unmanipulated NOD
mice (data not shown). The induced .beta.-galactosidase and GAD65
antibodies were predominantly of an IgG1 isotype (FIG. 1),
indicating these treatments primed antigen-specific Th2 help (as
compared to IgG2a antibodies which correlate with a pathologic Th1
response). Mosmann et al., Annu. Rev. Immunol. 7:145-173 (1989). In
individual mice, there tended to be an inverse correlation between
IgG1 and IgG2a GAD65 antibody levels (FIG. 1D). Thus, a single
administration of GAD65 to prediabetic NOD mice functioned to shift
the pathogenic Th1 response toward a protective Th2 response as
evidenced by the generation of Th2-dependent IgG1 GAD65
antibodies.
EXAMPLE 2
Antigen Administration Induces a Th2 Response
[0054] Traditionally, it has been difficult to define Th1 and Th2
activity because of the low precursor frequency of antigen-specific
T cells. We used an improved ELISPOT assay (Forsthuber et al.,
Science 271:1728-1730 (1996)) to monitor frequency of splenic
antigen-specific T cells that produce Th1- and Th2-type cytokines
following the administration of control .beta.-galactosidase or
GAD65. We observed that .beta.-galactosidase administration
predominantly primed an increase in the frequency of interleukin-4
(IL-4) and interleukin-5 (IL-5) secreting
.beta.-galactosidase-specific Th2 cells (FIG. 2A). As expected,
.beta.-galactosidase-reactive T cells were infrequently observed in
the GAD65-treated group.
[0055] Interferon-.gamma. (IFN-.gamma.)-secreting GAD65-specific
Th1 cells were frequently detected in .beta.-galactosidase-treated
mice whereas IL-4- and IL-5-secreting GAD65-specific Th2 cells were
rarely detected (FIG. 2B). Following a single treatment with GAD65,
however, there was a dramatic increase in the frequency of IL-4-
and IL-5-secreting GAD65-reactive T cells (Th2 cells), which on
average were 15- and 10-fold as frequent (respectively) as in the
control group (FIG. 2B). Furthermore, the average frequency of
GAD65-specific IFN-.gamma.-secreting T cells (Th1 cells) in
GAD65-treated mice was 65% of that found in the control group,
suggesting that this treatment limited the expansion of Th1
responses to GAD65.
[0056] Therefore, ELISPOT analysis reveals that both
.beta.-galactosidase and GAD65 administration promoted the
expansion of IL-4- and IL-5-secreting T cells (thereby confirming
the activation of a specific Th2 response) and limited the
expansion of IFN-.gamma. secreting T-cells (thereby confirming the
inhibition of the Th1 response). These data indicate that
autoantigen administration induces a shift from a Th1 response to
an antigen-specific Th2 response in recipient mice. Thus, even
after the establishment of Th1 autoimmunity, it is possible to
additionally engage Th2 cells and dramatically alter the balance of
antigen-specific Th1/Th2 cells.
EXAMPLE 3
GAD65 Treatment Prevents Adoptive Transfer of IDDM
[0057] To examine whether the induced T-cell responses could
inhibit an established pathogenic autoimmune response, we tested
the ability of T cells from .beta.-galactosidase- and GAD65-treated
NOD mice to inhibit the adoptive transfer of diabetes. We observed
that 90% of the mice receiving a mixture of splenic mononuclear
cells from .beta.-galactosidase-treated and diabetic mice developed
IDDM within 5 weeks after transfer--similar to a positive control
group that received mononuclear cells exclusively from diabetic
mice (FIG. 3). However, only 10% of the mice that received a
mixture of mononuclear cells from GAD65-treated and diabetic mice
developed IDDM (P<0.01). Thus, consistent with the induction of
an active tolerance mechanism, GAD65 (but not .beta.-galactosidase)
treatment induces potent regulatory cells in prediabetic NOD mice
that are capable of blocking target tissue destruction by a
diverse, activated effector T-cell population.
EXAMPLE 4
GAD65 Administration Inhibits Proliferative T-Cell Responses
[0058] We tested splenic-T-cells for proliferative responses to
GAD65 and the immunodominant peptide of heat shock protein (hsp 65,
peptide 277, see Elias et al., supra) 4 weeks after treatment.
Splenic T cells from both mice treated with IFA (alone) and those
treated with .beta.-galactosidase displayed strong T-cell responses
to GAD65 and hsp65 (Table 2), which were similar in magnitude to
the responses by splenic T cells from age-matched unmanipulated NOD
mice. Kaufman et al., supra. In contrast, NOD mice injected with
GAD65 displayed markedly reduced proliferative T-cell responses to
GAD65 (Table 2). As ELISPOT analysis revealed that the frequency of
GAD65-reactive Th1 cells was reduced by only 32% in GAD65-treated
mice (FIG. 2b), these data suggest that the primed GAD65-specific
Th2 response actively downregulated Th1 proliferation in vitro.
However, responses to control concanavalin A (con A) and recall
responses to hen egg white lysozyme (HEL) were unaffected by GAD or
.beta.-galactosidase treatment (Table 2). It is interesting that
the inhibitory effect was not confined only to the administered
autoantigen, as proliferative responses to hsp65 were also reduced
in GAD65-treated mice. Indeed, the frequency of hsp65-reactive Th1
cells, as determined by ELISPOT analysis, was reduced by 30% in
GAD65-treated prediabetic NOD mice (data not shown), suggesting
that the induction of GAD65-specific Th2 responses limited the
inflammatory cascade of .beta.-cell reactive T-cell responses.
[0059] Thus, GAD65 administration markedly reduces proliferative
T-cell responses to not only GAD65, but to other .beta.-cell
autoantigens (such as hsp65), thereby effectively inhibiting the
autoimmune cascade which results in destruction of pancreatic
.beta.-islet cells.
2TABLE 2 GAD65 Administration Inhibits Proliferative T-Cell
Responses to .beta.- cell Autoantigens, Reduces Intraislet
IFN-.gamma. transcription and Insulitis. Treatment IFA alone
.beta.-galactosidase GAD65 A. Spleen Cells .beta.-galactosidase 1.1
.+-. 0.2 4.1 .+-. 1.9 1.3 .+-. 0.2 GAD65 10.9 .+-. 2.6 13.1 .+-.
2.9 4.2 .+-. 0.9 hsp65 10.9 .+-. 1.9 11.5 .+-. 3.4 7.3 .+-. 2.5 HEL
1.0 .+-. 0.2 1.1 .+-. 0.1 0.9 .+-. 0.3 ConA 18.6 .+-. 2.1 20.3 .+-.
1.4 18.1 .+-. 1.2 B. Lymph Node Cells HEL 47.8 .+-. 3.4 50.3 .+-.
2.9 48.7 .+-. 4.3 PPD 52.4 .+-. 4.7 50.1 .+-. 3.6 56.7 .+-. 3.1 C.
Insulitis Score 2.6 .+-. 0.7 2.1 .+-. 1.6 1.3 .+-. 1.3 The mean
antigen-induced T-cell proliferation over background was expressed
as stimulation index .+-. s.e.m. (n = 6 for each group). Primed
lymph node T-cell recall responses to HEL or purified protein
derivative (PPD) were unaffected by treatment with GAD65 or
.beta.-galactosidase. The background for medium alone ranged from
1300 to 2800 c.p.m. Mice from control and experimental groups were
tested simultaneously in two separate experiments (using triplicate
cultures). None of the #antigens induced significant proliferation
of splenic T cells from BALB/c mice (data not shown). Insulitis
score was determined as previously described (n = 6 for each
group). Kaufman et al., supra.
EXAMPLE 5
GAD65 Treatment Inhibits Disease Progression
[0060] Examination of the pancreases from 12-week-old control mice
treated with IFA (alone) or .beta.-galactosidase revealed that all
islets had infiltrating lymphocytes. The severity of insulitis in
control animals (Table 2) was similar to that observed in
unmanipulated NOD mice. Kaufman et al., supra. In contrast, in the
GAD65-treated group, 40% of the islets were free of lymphocytic
infiltrates and insulitis was less severe. Because the treatments
were begun well after the onset of insulitis, the presence of some
lymphocytic infiltrates in GAD65-treated mice was not unexpected.
Thus, administering GAD65 to prediabetic mice inhibited lymphocytic
infiltration of the islets.
[0061] Approximately 80% of the control mice developed IDDM by 35
weeks of age (FIG. 4), paralleling the disease course observed in
unmanipulated female NOD mice. In contrast, 80% of the
GAD65-treated mice showed no signs of hyperglycemia at 40 weeks of
age (P<0.03). We detected high levels of GAD65 IgG1
autoantibodies in the sera of all 40-week-old GAD65-treated mice
that remained disease-free. However, the two GAD65-treated mice
that developed IDDM had very low levels of GAD65 autoantibodies at
the time of disease onset (data not shown).
[0062] Thus, GAD65 administration significantly reduces long-term
IDDM incidence. Collectively, the data presented in Examples 1-5
demonstrate that .beta.-cell autoantigen administration, even after
the establishment of a pathogenic Th1 response, can induce a
protective Th2 response, which shifts the Th1/Th2 balance and is
associated with inhibition of disease progression.
EXAMPLE 6
GAD65 Treatment Prolongs Syngeneic Islet Graft Survival
[0063] We next examined whether this therapy could be extended to
protect transplanted islets from established autoimmune responses
in diabetic NOD mice. Immediately following the onset of diabetes,
mice were maintained on insulin and were treated with
.beta.-galactosidase control antigen, or with one of the following
.beta.-cell autoantigens; GAD65, hsp65 (peptide 277), or the
B-chain of insulin (which contains the immunodominant determinant;
see Muir et al., J. Clin. Invest. 95:628-634 (1995) and Daniel et
al., Proc. Natl. Acad. Sci. USA 93:956-960 (1996)). Following
transplantation of newborn islets, exogenous insulin administration
was discontinued and no immunosuppressants were used to block
rejection. The results of these experiments are presented in FIG.
5.
[0064] As is shown in FIG. 5, we observed that mice treated with
the .beta.-galactosidase control antigen, the hsp65 peptide or
insulin B-chain became diabetic approximately 10 days post
transplantation. In contrast, GAD65-treated mice remained
euglycemic for an average of 48 days post transplantation. The
GAD65-treated mice had residual islet cell function, which allowed
them to survive in a chronic hyperglycemic state up to 20 weeks
post transplantation (data not shown).
[0065] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0066] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
* * * * *