United States Patent: 6,841,152
Issued: January 11, 2005
Inventors: Delovitch; Terry L. (London, CA)
Assignee: The Wellesley Hosp. Foundation (Toronto, CA)
Appl. No.: 341407
Filed: October 12, 1999
PCT Filed: January 12, 1998
PCT NO: PCT/CA98/00015
371 Date: October 12, 1999
102(e) Date: October 12, 1999
PCT PUB.NO.: WO98/30232
PCT PUB. Date: July 16, 1998
Methods are provided for preventing the development of autoimmune diseases in susceptible subjects and for prolonging acceptance of tissue transplants by administration of an agonist of the T cell CD28 co-stimulatory receptor.
Description of the Invention
This invention relates to methods and compositions for preventing the development of autoimmune disease in susceptible subjects. More particularly, the invention relates to treatment with an agonist of CD28 to prevent autoimmune disease development.
BACKGROUND OF THE INVENTION
Insulin-dependent diabetes mellitus (IDDM) or autoimmune diabetes is a polygenic, multifactorial, autoimmune disease heralded by T cell infiltration of the pancreatic islets of Langerhans (insulitis) and the .beta. progressive T cell-mediated destruction of insulin-producing cells (Bach, 1994; Atkinson and Maclaren, 1994; Tisch and McDevitt, 1996).
Non-obese diabetic (NOD) mice are susceptible to the development of IDDM and are an accepted model for the development of autoimmune IDDM in humans.
CD4+ T helper cells are required for the adoptive transfer of IDDM into recipient neonatal NOD mice or immunodeficient NOD.Scid mice (Bendelac et al., 1987, Christianson et al., 1993; Rohane et al., 1995). Cooperation between CD4+ and CD8+ T cells is required to initiate IDDM, and islet .beta. cell destruction is CD4+ T cell-dependent (Haskins and McDuffie, 1990; Wang et al., 1991). Current evidence suggests that the CD4+ effector cells of IDDM in NOD mice at Th1 cells which secrete IL-2, IFN-.gamma. and TNF-.alpha. and that the regulatory CD4+ cells are Th2 cells which secrete IL-4, IL-5, IL-6, IL-10 and IL-13 (Rabinovitch, 1994; Liblau et al., 1995; Katz et al., 1995).
NOD mouse T cells show proliferative hyporesponsiveness to T cell receptor (TCR) stimulation and this hyporesponsiveness may be causal to the development of IDDM.
It has been shown that, beginning at 3-5 weeks of age, T cell receptor (TCR) ligation in NOD mice induces the proliferative hyporesponsiveness of NOD thymic and peripheral T cells, which is mediated by reduced IL-2 and IL-4 production (Zipris et al., 1991; Rapoport et al., 1993a; Jaramillo et al., 1994).
Decreased IL-4 production by human T cells from patients with new onset IDDM has also been demonstrated recently (Berman et al., 1996). Whereas addition of IL-4, a Th2-type cytokine, potentiates Il-2 production and completely restores NOD T cell proliferative responsiveness, addition of IL-2, a Th1-type cytokine, even at high concentrations, only partially restores NOD T cell responsiveness. These findings suggest that Th2 cells may be compromised in function to a greater extent than Th1 cells in NOD mice, and raise the possibility that Th2 cells require a higher threshold of activation than Th1 cells in these mice. IL-4 not only restores NOD T cell responsiveness in vitro, but prevents insulitis and IDDM when administered in vivo to prediabetic NOD mice (Rapoport et al., 1993a) or when transgenically expressed in pancreatic .beta. cells (Mueller et al., 1996).
The proliferative hyporesponsiveness of regulatory Th2 cells in NOD mice may favour a Th1 cell-mediated environment in the pancreas of these mice, and lead to a loss of immunological tolerance to islet .beta. cell autoantigens. This is consistent with the notion that restoration of the balance between Th1 and Th2 cell function may prevent IDDM (Rabinovitch, 1994; Liblau et al., 1995; Arreaza et al., 1996).
Optimal T cell activation requires signalling through the TCR and the T cell CD28 costimulatory receptor (CD28) (June et al., 1994; Bluestone, 1995; Thompson, 1995). Crosslinking of the TCR/CD3 complex in the absence of a CD28-mediated costimulatory signal induces a proliferative unresponsiveness that is mediated by the inability of T cells to produce IL-2 (Jenkins et al., 1991). CD28 costimulation prevents proliferative unresponsiveness in Th1 cells by augmenting the production of IL-2, which in turn promotes IL-4 secretion by T cells (Seder et al., 1994). The costimulatory pathway of T cell activation involves the interaction of CD28 with its ligands B7-1 and B7-2 on an antigen presenting cell (APC), with B7-2 considered as the primary ligand for CD28 (Linsley et al.,1990; Freeman et al., 1993; Lenschow et al., 1993; Freeman et al., 1995). When costimulation is blocked by either CTLA4-Ig or by anti-B7-1 or anti-B7-2 monoclonal antibodies (mAbs), differential effects on the incidence of various autoimmune diseases (e.g. IDDM) and on the development of Th1 and Th2 cells are observed (Kuchroo et al., 1995; Lenschow et al., 1995). Furthermore, in vivo studies have demonstrated that the generation of Th2 cells is more dependent upon the CD28-B7 pathway than the priming of Th1 cells, and suggest that the development of Th subsets in vivo may be influenced by limited CD28-B7 costimulation (Corry et al., 1994; Lu et al., 1994). Analyses of the development of human Th2 cells have yielded results similar to those observed in the mouse (King et al., 1995; Kalinski et al., 1995; Webb and Feldman, 1995). Interactions between CD28 and its B7-2 ligand are essential for the costimulation of an IL-4-dependent CD4+ T cell response, and IL-4 increases B7-1 and B7-2 surface expression on certain professional APCs (eg. Langerhans cells) and B cells (Freeman et al., 1995; Kawamura et al., 1995; Stack et al., 1994). Thus, failure to activate NOD thymocytes and peripheral T cells sufficiently may be due to functional and/or differentiation defects in NOD APCs, which remain able to optimally activate islet .beta. cell autoreactive CD4+ effector T cells, but not regulatory CD4+ T cells (Serreze et al., 1988; Serreze et al., 1993). Functional defects that compromise antigen presentation by NOD APCs, such as deficient CD28 costimulation, may lower their ability to stimulate regulatory Th2 cells without compromising their ability to stimulate autoreactive effector Th1 cells.
Proliferative hyporesponsiveness of T cells has been observed in other autoimmune diseases such as multiple sclerosis and myasthenia gravis.
If proliferative hyporesponsiveness of T cells in autoimmune disease could be overcome, it might be possible by that means to prevent the development of autoimmune diseases.
SUMMARY OF THE INVENTION
It has been shown that administration of an agonist of the T cell CD28 costimulatory receptor, referred to herein as CD28, can protect a susceptible subject from development of an autoimmune disease.
The treatment appears to stimulate the production of protective T cells in the treated subject.
The invention is illustrated by demonstration of the prevention of the development of autoimmune diabetes in the NOD mouse, an accepted model for human autoimmune diabetes.
In accordance with one embodiment of the invention, a method is provided for preventing the development of an autoimmune disease in a susceptible subject comprising administering to the subject an effective amount of a T cell CD28 costimulatory receptor (CD28) agonist.
In accordance with a further embodiment of the invention, a method is provided for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising administering to the mammalian recipient an effective acceptance-prolonging amount of a CD28 agonist.
In accordance with a further embodiment of the invention, a pharmaceutical composition is provided for preventing the development of an autoimmune disease in a susceptible subject comprising an effective amount of a CD28 agonist.
In accordance with a further embodiment of the invention, a pharmaceutical composition is provided for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising an effective amount of a CD28 agonist.
DESCRIPTION OF THE INVENTION
The present invention provides a method for preventing the development of an autoimmune disease in a susceptible subject by treating the subject with an agonist of CD28.
Subjects susceptible to autoimmune diseases including autoimmune diabetes, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, Hashimoto's thyroiditis, Sjogren syndrome and systemic lupus erythematosus may be treated by the method of the invention.
In accordance with a preferred embodiment, the method of the invention may be used to prevent the development of autoimmune diabetes in a susceptible human subject.
Suitable CD28 agonists include anti-CD28 agonist antibodies and B7-2 ligand protein extracellular domain polypeptide or effective fragments thereof.
B7-2 ligand protein is a transmembrane protein expressed on several types of haematopoietic cells, including antigen preventing cells and macrophages. The extracellular domain of B7-2 is involved in binding with CD28 (Peach et al., (1994), J. Exp. Med., v. 180, pp. 2049-2058). Human B7-2 protein, a polypeptide corresponding to the extracellular domain of human B7-2, or an effective fragment thereof, may be used as a CD28 agonist in the method of the invention. An "effective fragment" is a fragment which acts as a CD28 agonist.
Fragments may be screened for CD28 agonist activity, for example, using the T cell proliferation assay described in Example 1.
Alternatively, B7-2 protein may be expressed recombinantly as a soluble B7-2 immunoglobulin fusion protein. For example, a fusion protein comprising human B7-2 protein fused to the Fc domain of human IgG1 may be prepared. DNA encoding B7-2 protein may be cloned by previously described methods (Linsley et al., (1993); Peach et al., (1994)).
In accordance with a preferred embodiment of the invention, development of an autoimmune disease is prevented by treating a susceptible subject with an anti-CD28 agonist antibody.
As will be understood by those skilled in the art, not all antibodies to CD28 may function as agonists to activate CD28. An "anti-CD28 agonist antibody" is an antibody which binds selectively to CD28 and whose binding leads to activation of CD28.
Anti-CD28 agonist antibodies may be raised against the full length CD28 protein or against portions of CD28 which contain epitopes involved in the binding of CD28. For example, antibodies may be raised to the complementarity determining region 1 (CDR1)- and CDR3-portions of CD28 which mediate the B7 ligand binding of CD28. The binding of CD28 is described in Peach et al. (supra), and Linsley et al., (1993), Ann. Rev. Immunol., v. 11, pp. 191-212, the contents of which are incorporated herein by reference.
Anti-CD28 antibodies can readily be screened for CD28 agonist activity by a T cell proliferation assay, as described herein.
Human subjects susceptible to the development of autoimmune diabetes may be identified by screening based on a subject's HLA genetic make-up (Undlien et al., (1997)) or based on detection of predictive serum autoantibodies such as anti-insulin or anti-GAD antibodies (Verge et al., (1996)).
Treatment in accordance with the method of the invention should be administered in the neonatal period, from about 6 months to about 2 or 3 years of age. A series of treatments may be required over the 6 month to 2 year period of life.
Human subjects susceptible to other autoimmune diseases may be identified by screening tests appropriate for each disease, as is understood by those skilled in the art. For example, susceptibility to multiple sclerosis can be screened by MRI.
In accordance with a further embodiment, the invention provides pharmaceutical compositions for preventing the development of an autoimmune disease, the compositions comprising an effective amount of an agonist of CD28 and, optionally, a pharmaceutically acceptable carrier.
In accordance with a further embodiment, the invention provides a method for reducing the rejection of, and prolonging the acceptance of, an engrafted tissue in a mammal, by administering to the mammal an effective amount of a CD28 agonist. As used herein "engrafted tissue" includes allotransplants and xenotransplants of mammalian cells, tissues or organs. Transplantation of tissues or organs in humans is becoming ever more common, but there is always a need for control or suppression of the transplant recipient's immune system. Potential transplant recipients who may benefit from the method of the invention include recipients of kidney transplants, heart transplants, pancreas transplants, pancreatic islet transplants and liver transplants.
Treatment with the CD28 agonist may be, for example, by intravenous administration prior to, at the time of and after transplantation. For example, a suitable regime may be one treatment from one day to one week prior to transplant, one treatment on the day of the transplant and treatments at intervals of 2 to 4 days for as long as required after the transplant.
Dosage would be adjusted as required, in the judgment of the attending physician, but injections of 1 to 10 mg of agonist/kg body weight would be a suitable range for initial trial.
In order to prepare polyclonal antibodies, human CD28 co-receptor protein may be purified from T cells by conventional methods. The purified protein, coupled to a carrier protein if desired, is mixed with Freund's adjuvant and injected into rabbits or other suitable laboratory animals.
Alternatively, fusion proteins containing all or a portion of the CD28 co-receptor protein can be synthesised in a suitable host, such as a bacterium, by expression of an appropriate DNA sequence inserted in a suitable cloning vehicle. DNA encoding CD28 may be cloned by previously described methods (Linsley et al., (1993), Ann. Rev. Immunol., v. 11, pp. 191-212; Peach et al., (1994), J. Exp. Med., v. 180, pp. 2049-2058).
Two widely used expression systems for producing recombinant fusion proteins in E. coli are glutathione-S-tranferase or maltose binding protein fusions using the pUR series of vectors and trpE fusions using the pATH vectors. Human CD28 may also be produced from CHO cells transfected with human CD28 cDNA.
The expressed CD28 protein can then be purified, coupled to a carrier protein if desired, and mixed with Freund's adjuvant (to help stimulate the antigenic response of the animal) and injected into rabbits or other appropriate laboratory animals. Alternatively, the protein can be isolated from CD28-expressing cultured cells. Following booster injections at weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or purified prior to use by various methods including affinity chromatography employing Protein A-Sepharose, antigen Sepharose or Anti-mouse-Ig-Sepharose.
Monoclonal anti-CD28 antibodies may also be produced by conventional methods after injecting into mice human CD28 co-receptor protein purified from T cells or produced by recombinant expression, or portions of CD28, as described above.
The protein is injected into mice in Freund's adjuvant, for example for nine times over a three week period. The mice spleens are removed and resuspended in phosphate buffered saline (PBS). The spleen cells serve as a source of lymphocytes, some of which are producing antibody of the appropriate specificity. These are then fused with a permanently growing myeloma partner cell, and the products of the fusion, hybridomas, are plated into a number of tissue culture wells in the presence of a selective agent such as HAT. The wells are then screened by ELISA to identify those containing cells making antibody specific for CD28. These cells are then plated and after a period of growth, these wells are again screened to identify antibody-producing cells. Several cloning procedures are carried out until over 90% of the wells contain single clones which are positive for antibody production. From this procedure a stable line of clones which produce the antibody is established. The monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose or ion-exchange chromatography, as well as variants and combinations of these techniques.
Alternatively, mice may be injected with human T cells, splenic lymphocytes are harvested and hybridomas prepared, screened and cloned as described above, to obtain hybridomas producing monoclonal antibodies specific for human CD28.
For example, mice may receive an initial injection of about 1-10x106 T cells in complete Freund's adjuvant, followed at weekly intervals for about 4 to 6 weeks with further injections of T cells in incomplete Freund's adjuvant, until high anti-CD28 serum antibody titers are detected.
Anti-CD28 antibodies with a binding affinity, Kd, of at least 10-8 are preferred and antibodies with Kd.gtoreq.10-9. are especially preferred.
In view of the high degree of conservation of amino acid sequence among mammalian CD28 proteins, anti-CD28 antibodies may also be raised against non-human CD28 (Linsley et al., supra; Peach et al., supra; June et al., (1994), Immunol. Today, v. 15, pp. 321-331).
Anti-CD28 antibodies are screened for CD28 agonist activity as described above.
Techniques are available and well known to those in the art to prepare humanised antibodies which have a variable region, specific for the CD28 co-receptor, synthesised in a non-human mammal, combined with a human constant region. Such humanised antibodies may be preferable for treatment of human subjects.
For example, the antibody may be genetically engineered to be a hamster/human chimeric monoclonal antibody of IgG1K isotype, the hamster constant regions being replaced with human counterparts while retaining the hamster antigen binding regions specific for human CD28, as described by Knight et al., (1993), Mol. Immunol., v. 30, pp. 1443-1453. Portions of the antigen binding regions may also be genetically engineered to express the human counterpart antigen binding (Fv) regions.
Truncated versions of monoclonal antibodies may also be produced by recombinant techniques in which plasmids are generated which express the desired monoclonal antibody fragment(s) in a suitable host. Antibodies specific for mutagenic epitopes can also be generated.
In a further embodiment, this invention provides pharmaceutical compositions for the prevention of autoimmune diseases in mammals.
Administration of a therapeutically active amount of a pharmaceutical composition of the present invention means an amount effective, at dosages and for periods of time necessary to achieve the desired result. This may also vary according to factors such as the autoimmune disease involved, the age, sex, and weight of the subject, and the ability of the CD28 agonist to elicit a desired response in the subject. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
By pharmaceutically acceptable carrier as used herein is meant one or more compatible solid or liquid delivery systems. Some examples of pharmaceutically acceptable carriers are sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, collagen, talc, stearic acids, magnesium stearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids, pyrogen-free water, isotonic saline, phosphate buffer, and other suitable non-toxic substances used in pharmaceutical formulations. Other excipients such as wetting agents and lubricants, tableting agents, stabilizers, anti-oxidants and preservatives are also contemplated.
The compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers and formulations adapted for particular modes of administration are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
The CD28 agonist antibodies and proteins described herein may be sterilised by filtration and administered to a subject in need of treatment in a sterile saline solution.
The pharmaceutical compositions of the invention may be administered therapeutically by various routes such as by injection or by oral, nasal, buccal, rectal, vaginal, transdermal or ocular routes in a variety of formulations, as is known to those skilled in the art.
Administration by intravenous injection or infusion is preferred.
Optimal T cell activation requires signalling through both the TCR and the CD28 costimulatory receptors of the T cell. The T cells of NOD mice have been shown to be hyporesponsive to T cell receptor-stimulation of proliferation.
The present inventor has shown that this hyporesponsiveness of NOD T cells is associated with defective CD28 co-receptor costimulation.
It has been shown by the inventor that treatment of NOD mice with a CD28 agonist prevented the development of autoimmune diabetes. Treatment of neonatal NOD mice with an anti-CD28 antibody which gave CD28 costimulation completely restored the proliferative responsiveness of NOD thymocytes and peripheral T cells by augmenting their levels of secretion of IL-2 and IL-4. The stimulated increase in IL-4 secretion was predominant.
The antibody treatment effectively prevented the development of destructive insulitis in NOD mice and prevented the expected development of diabetes.
Treatment of neonatal NOD mice with anti-IL4 antibodies simultaneously with treatment with anti-CD28 antibodies abrogated the protective effect of the anti-CD28 antibodies against development of autoimmune diabetes.
The 37.51 anti-CD28 mAb used has been shown to block CD28/B7-1 interactions and thereby prevent tumour immunity (Allison, J. P. et al., Curr. Opin. Immunol, v. 7, pp. 682-686). Thus, this anti-CD28 mAb normally functions as an antagonist in animal model systems of tumour immunity. By contrast, in the NOD mouse model of IDDM, it has been demonstrated that the 37.51 anti-CD28 mAb acts as an agonist and activates rather than blocks CD28 signalling.
It is postulated that the antigen presenting cell (APC)-derived costimulatory signal transduced by the CD28 co-receptor on NOD mouse T cells may be insufficient to stimulate optimum T cell activation and that such CD28-signalled activation of IL-4-producing Th2 cells is necessary for protection from IDDM. The work of the inventor suggests that anti-CD28 antibody prevents IDDM in NOD mice by activating the CD28 signalling pathway in NOD T cells rather than by blocking the interaction between CD28 and its ligand, B7.
Prevention of IDDM by CD28 costimulation may be mediated by the activation of a subset of CD4+ regulatory T cells that confer protection against IDDM. This subset of CD4+ regulatory T cells may be hyporesponsive in NOD mice and may not receive a sufficient amount of the CD28/B7 costimulatory signal required for clonal expansion and effector function in NOD mice.
It has been proposed that precursor CD4+ Th2 cells require a strong initial T-cell stimulation, and that the amount of IL-4 produced is proportional to the magnitude of the initial T cell stimulation. In the absence of CD28 costimulation, the production of IL-4 remains below the threshold required for the optimal development of Th2 cells (Seder and Paul, 1994; Thompson, 1995; Bluestone, 1995). It is of interest that B7-1 and B7-2 ligation of CD28 mediate distinct outcomes in CD4+ T cells. B7-2 costimulation signals naive T cells to become IL-4-producing T cells, and thereby directs an immune response towards Th0 and Th2 cells (Freeman et al., 1995; Kuchroo et al., 1995). B7-1 costimulation seems to be a more neutral differentiative signal, and initiates the development of both Th1 and Th0/Th2 cells. Presumably, B7-2 plays a dominant role in the production of IL-4 due to its early expression during T cell activation (Freeman et al., 1995; Thompson, 1995). Thus, an insufficient or inappropriate signal resulting from a CD28/B7-2 interaction may be delivered to a subset of regulatory CD4+ T cells in NOD mice, and this subset may not differentiate properly into functional IL-4 producing Th2 cells.
The inventor has examined whether anti-CD28 mAb treatment of NOD mice provides the costimulation required for the expansion of and cytokine production by regulatory IL-4 producing Th2-like cells. FIG. 4 shows that anti-CD3 stimulated (in vitro) NOD thymocytes obtained at 8 weeks, peripheral splenic T cells obtained at 8 weeks and 25 weeks and islet infiltrating T cells examined at 25 weeks of age produce significantly higher levels of IL-4 compared with the same subpopulations of cells isolated from control mice treated with a hamster Ig. Shortly after termination of treatment with anti-CD28 mAb, thymic and splenic T cells showed a higher basal (no stimulation) production of IL-4 compared to cells obtained from age-matched (8 week-old) control mice. With the exception of higher splenic T cell basal responses in 8 week-old mice, no differences were detected between the proliferative responses of thymocytes, splenic T cells and islet infiltrating cells from 8 and 25 week-old anti-CD28 treated NOD mice and those of the age matched controls (FIG. 5). The increase in basal T cell proliferation and IL-4 production may reflect the preferential costimulation of Th2 cells by anti-CD28 treatment in vivo. It has been found that anti-CD28 treatment in vivo leads to an increased production of IgG1 (which reflects increased IL-4 production by T cells) rather than IgG2a anti-GAD67 antibodies (FIG. 6B). Moreover, the total number of splenic lymphocytes was increased about 1.9-fold at 8 weeks of age and 1.7-fold at 25 weeks of age in anti-CD28 treated NOD mice relative to that of control treated mice (data not shown). These findings support the idea that anti-CD28 treatment elicits the expansion and survival of IL-4 producing Th2 cells in NOD mice.
Anti-CD28 treatment did not significantly alter the level of IFN-.gamma. secretion by T cells from 8 week-old NOD mice compared with that observed in age-matched control mice. However, levels of IFN-.gamma. secretion by thymocytes and splenic T cells from 25 week-old anti-CD28 treated NOD mice were markedly reduced in comparison to those levels detected in control mice. These data demonstrate the long term down regulation of Th1 cell function, which may arise from the preferential activation of Th2 cells induced by CD28 costimulation during the inductive phase of the autoimmune process. The downregulation and/or functional deviation of Th1 cells towards a Th2 cell phenotype by IL-4 is more effective than and dominant over the inhibition of Th2 cell function by IL-12 (Perez et al., 1995; Szabo et al., 1995; Murphy et al., 1996). These results also agree with reports that INF-.gamma. secreting Th1 cells potentiate the effector phase of insulitis, IFN-.gamma. is directly involved in .alpha. cell destruction (Pilstrom et al., 1995; Rabinovitch et al., 1995; Herold et al., 1996; Shimada et al., 1996) and the early differentiation of naive T cells into Th2 cells is dependent on CD28 signalling (Webb and Feldman, 1995; Lenschow et al., 1996). It is noteworthy that in human T cells, CD28 costimulation is a critical requirement for the development of Th2-like cells but not Th1-like cells, and Th2 cell function remains CD28-independent after the initial costimulation (Webb and Feldman, 1995).
Although anti-CD28 mAb treatment protects from IDDM, this treatment still allows for the development of a non-destructive insulitis. Therefore, this treatment does not interfere with the migration of diabetogenic T cells to the pancreatic islets. Rather, anti-CD28 treatment appears to induce regulatory T cells in the pancreas to suppress islet .beta. cell destruction and progression to overt IDDM. Evidence in support of this is derived from assays of secretion of IL-4 and INF-.gamma. by infiltrating cells from mice treated with anti-CD28 or control Ig (FIG. 4) and from analyses of the levels of expression of these cytokines in the pancreas of anti-CD28 treated NOD mice at 25 weeks of age (FIG. 6A). The intra-pancreatic expression of IL-4 was significantly higher in anti-CD28 mAb treated mice, whereas the expression of INF-.gamma. remained essentially unaltered in these mice. Committed autoreactive cells, including Th1 cells, may accumulate in pancreatic islets but the functions of IL-4 predominate to inhibit INF-.gamma. mediated .beta. cell damage.
FACS analyses of the phenotype and surface expression of various cell adhesion molecules in anti-CD28 treated and control NOD mice at 8-25 weeks of age also indicated that anti-CD28 mAb treatment did not interfere with the migration of diabetogenic T cells to pancreatic islets (data not shown). The levels of surface expression of LFA-1, L-selectin and CD44 on the surface of splenic T cells did not suffer significantly between untreated and anti-CD28 treated NOD mice. Similarly, the levels of surface expression of markers of activation such as CD-69, ICAm-1 and B7-2 on B cells were increased only slightly in anti-CD28 treated NOD mice. The T (CD3+):B (CD19+) and CD4:CD8 T cell ratios in NOD mice were not altered by anti-CD28 treatment.
The activation of the CD4+ Th2 cells may arise from the ability of CD28 ligation to sustain the proliferative response and enhance the longer term survival of T cells by the delivery of a signal that protects from apoptosis through the upregulation of survival factors such as Bcl-XL.
Claim 1 of 7 Claims
What is claimed is:
1. A method for protecting against the development of autoimmune diabetes in a susceptible subject comprising administering to the subject an effective amount of a T cell CD28 co-stimulatory receptor (CD28) agonist wherein the agonist is an anti-CD28 agonist antibody.