Cutting edge: vasostatin-1-derived peptide ChgA29-42 is an antigenic epitope of diabetogenic BDC2.5 T cells in nonobese diabetic mice

Mechanistic and therapeutic insights in autoimmune diabetes would benefit from a more complete identification of relevant autoantigens. BDC2.5 TCR transgenic NOD mice express transgenes for TCR Vα1 and Vβ4 chains from the highly diabetogenic BDC2.5 CD4(+) T cell clone, which recognizes pancreatic β cell membrane Ags presented by NOD I-A(g7) MHC class II molecules. The antigenic epitope of BDC2.5 TCR is absent in β cells that do not express chromogranin A (ChgA) protein. However, characterization of the BDC2.5 epitope in ChgA has given inconclusive results. We have now identified a ChgA29-42 peptide within vasostatin-1, an N-terminal natural derivative of ChgA as the BDC2.5 TCR epitope. Having the necessary motif for binding to I-A(g7), it activates BDC2.5 T cells and induces an IFN-γ response. More importantly, adoptive transfer of naive BDC2.5 splenocytes activated with ChgA29-42 peptide transferred diabetes into NOD/SCID mice.     

Detection and characterization of T cells specific for BDC2.5 T cell-stimulating peptides

Nonobese diabetic (NOD) mice expressing the BDC2.5 TCR transgene are useful for studying type 1 diabetes. Several peptides have been identified that are highly active in stimulating BDC2.5 T cells. Herein, we describe the use of I-Ag7 tetramers containing two such peptides, p79 and p17, to detect and characterize peptide-specific T cells. The tetramers could stain CD4(+) T cells in the islets and spleens of BDC2.5 transgenic mice. The percentage of CD4(+), tetramer(+) T cells increased in older mice, and it was generally higher in the islets than in the spleens. Our results also showed that tetAg7/p79 could stain a small population of CD4(+) T cells in both islets and spleens of NOD mice. The percentage of CD4(+), tetramer(+) T cells increased in cells that underwent further cell division after being activated by peptides. The avidity of TCRs on purified tetAg7/p79(+) T cells for tetAg7/p79 was slightly lower than that of BDC2.5 T cells. Although tetAg7/p79(+) T cells, like BDC2.5 T cells, secreted a large quantity of IFN-gamma, they were biased toward being IL-10-producing cells. Additionally, <3% of these cells expressed TCR Vbeta4. In vivo adoptive transfer experiments showed that NOD/scid recipient mice cotransferred with tetAg7/p79(+) T cells and NOD spleen cells, like mice transferred with NOD spleen cells only, developed diabetes. Therefore, we have generated Ag-specific tetramers that could detect a heterogeneous population of T cells, and a very small number of NOD mouse T cells may represent BDC2.5-like cells.     

Human DQ8 can substitute for murine I-Ag7 in the selection of diabetogenic T cells restricted to I-Ag7

The strong association of type 1 diabetes with specific MHC class II genes, such as I-A(g7) in nonobese diabetic mice and HLA-DQ8 in humans, suggests that MHC class II molecules play an important role in the development of the disease. To test whether human DQ8 molecules could cross the species barrier and functionally replace their murine homolog I-A(g7), we generated DQ8/BDC2.5 transgenic mice. We have shown that BDC2.5 transgenic T cells are selected on DQ8 in the thymus and cause diabetes in a manner similar to that seen when the T cells are selected on H2(g7). Splenocytes from DQ8/BDC2.5 mice also showed reactivity toward islets in vitro as seen in H-2(g7)/BDC2.5 mice. We conclude that DQ8 molecules not only share structural similarity with the murine homolog I-A(g7), but also can cross the species barrier and functionally replace I-A(g7) molecules to stimulate diabetogenic T cells and produce diabetes.     

Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice

Nonobese diabetic (NOD) mice carrying a transgenic TCR from an islet Ag-specific CD4 T cell clone, BDC2.5, do not develop diabetes. In contrast, the same transgenic NOD mice on the SCID background develop diabetes within 4 wk after birth. Using a newly developed mAb specific for the BDC2.5 TCR, we examined the interaction between diabetogenic T cells and regulatory T cells in NOD.BDC transgenic mice. CD4 T cells from NOD.BDC mice, expressing high levels of the clonotype, transfer diabetes to NOD.SCID recipients. In contrast, CD4 T cells expressing low levels due to the expression of both transgenic and endogenous TCR alpha-chains inhibit diabetes transfer. The clonotype-low CD4 T cells appear late in the ontogeny in the thymus and peripheral lymphoid organs, coinciding with resistance to cyclophosphamide-induced diabetes. These results demonstrate that diabetic processes in NOD.BDC mice are regulated by a balance between diabetogenic T cells and regulatory T cells. In the absence of specific manipulation, regulatory T cell function seems to be dominant and mice remain diabetes free. Understanding of mechanisms by which regulatory T cells inhibit diabetogenic processes would provide means to prevent diabetes development in high-risk human populations.     

Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors

The MHC determines susceptibility and resistance to type 1 diabetes in humans and nonobese diabetic (NOD) mice. To investigate how a disease-associated MHC molecule shapes the T cell repertoire in NOD mice, we generated a series of tetramers from I-A(g7)/class II-associated invariant chain peptide precursors by peptide exchange. No CD4 T cell populations could be identified for two glutamic acid decarboxylase 65 peptides, but tetramers with a peptide mimetic recognized by the BDC-2.5 and other islet-specific T cell clones labeled a distinct population in the thymus of young NOD mice. Tetramer-positive cells were identified in the immature CD4(+)CD8(low) population that arises during positive selection, and in larger numbers in the more mature CD4(+)CD8(-) population. Tetramer labeling was specific based on the use of multiple control tetramers, including one with a single amino acid analog peptide in which a critical TCR contact residue was substituted. The T cell population was already present in the thymus of 2-wk-old NOD mice before the typical onset of insulitis and was detected in B10 mice congenic for the NOD MHC locus, but not B10 control mice. These results demonstrate that a T cell population can expand in the thymus of NOD mice to levels that are at least two to three orders of magnitude higher than estimated for a given specificity in the naive T cell pool. Based on these data, we propose a model in which I-A(g7) confers susceptibility to type 1 diabetes by biasing positive selection in the thymus and later presenting peptides from islet autoantigens to such T cells in the periphery.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Cutting edge: interactions through the IL-10 receptor regulate autoimmune diabetes.

BDC2.5/nonobese diabetic (NOD) transgenic mice express a TCR from a diabetogenic T cell clone yet do not spontaneously develop diabetes at high incidence. Evidence exists showing that in the absence of endogenous TCR alpha-chain rearrangements this transgenic mouse spontaneously develops diabetes and that CTLA-4 negatively regulates diabetes onset. This strongly suggests that onset of diabetes in BDC2.5/NOD mice is governed by T cell regulation. We addressed the mechanism of immune regulation in BDC2.5/NOD mice. We find that activated spleen cells from young, but not old, BDC2.5/NOD mice are able to transfer diabetes to NOD-scid recipients. We have used anti-IL-10R to show that the failure of splenocytes from older mice to transfer diabetes is due to dominant regulation. We furthermore found that diabetes developed following anti-IL-10R treatment of 6-wk old BDC2.5/NOD mice indicating that endogenous IL-10 plays a key role in the regulation of diabetes onset in this transgenic mouse.     

Impaired CD4 T cell activation due to reliance upon B cell-mediated costimulation in nonobese diabetic (NOD) mice

Diabetes in nonobese diabetic (NOD) mice results from the activation of I-A(g7)-restricted, islet-reactive T cells. This study delineates several characteristics of NOD CD4 T cell activation, which, independent of I-A(g7), are likely to promote a dysregulated state of peripheral T cell tolerance. NOD CD4 T cell activation was found to be resistant to antigenic stimulation via the TCR complex, using the progression of cell division as a measure. The extent of NOD CD4 T cell division was highly sensitive to changes in Ag ligand density. Moreover, even upon maximal TCR complex-mediated stimulation, NOD CD4 T cell division prematurely terminated. Maximally stimulated NOD CD4 T cells failed to achieve the threshold number of division cycles required for optimal susceptibility to activation-induced death, a critical mechanism for the regulation of peripheral T cell tolerance. Importantly, these aberrant activation characteristics were not T cell-intrinsic but resulted from reliance on B cell costimulatory function in NOD mice. Costimulation delivered by nonautoimmune strain APCs normalized NOD CD4 T cell division and the extent of activation-induced death. Thus, by disrupting the progression of CD4 T cell division, polarization of APC costimulatory function to the B cell compartment could allow the persistence and activation of diabetogenic cells in NOD mice.     

L-selectin is not required for T cell-mediated autoimmune diabetes

Administration of anti-L-selectin (CD62L) mAb to neonatal nonobese diabetic (NOD) mice mediates long term protection against the development of insulitis and overt diabetes. These results suggested that CD62L has a key role in the general function of beta cell-specific T cells. To further examine the role of CD62L in the development of type 1 diabetes, NOD mice lacking CD62L were established. The onset and frequency of overt diabetes were equivalent among CD62L(+/+), CD62L(+/-), and CD62L(-/-) NOD littermates. Furthermore, patterns of T cell activation, migration, and beta cell-specific reactivity were similar in NOD mice of all three genotypes. Adoptive transfer experiments with CD62L(-/-) CD4(+) T cells prepared from BDC2.5 TCR transgenic mice revealed no apparent defects in migration to pancreatic lymph nodes, proliferation in response to beta cell Ag, or induction of diabetes in NOD.scid recipients. In conclusion, CD62L expression is not essential for the development of type 1 diabetes in NOD mice.     

IFN-gamma-dependent regulatory circuits in immune inflammation highlighted in diabetes

We demonstrate diverse roles of IFN-gamma in the induction and regulation of immune-mediated inflammation using a transfer model of autoimmune diabetes. The diabetogenic CD4(+)BDC2.5 (BDC) T cell clone upon transfer into NOD.scid mice induced destruction of islets of Langerhans leading to diabetes. Administration of a neutralizing Ab to IFN-gamma (H22) resulted in long-term protection (LTP) from diabetes, with inflammation but persistence of a significant, albeit decreased, number of beta cells. BDC T cells were a mixture of cells expressing high, intermediate, and low levels of the TCR. Clonotype(low) BDC T cells were required for LTP. Furthermore, islet-infiltrating leukocytes in the LTP mice contained Foxp3(+)CD4 T cells. Islet inflammation in both diabetic and LTP mice was characterized by heavy infiltration of macrophages. Gene expression profiles indicated that macrophages in diabetic mice were M1 type, while LTP mice contained M2 differentiated. The LTP was abolished if mice were treated with either Ab-depleting CD4 T cells or a neutralizing Ab to CTLA-4, in this case, only at a late stage. Neutralization of IL-10, TGF-beta, glucocorticoid-induced TNF receptor (GITR), or CD25 had no effect. Transfer of only clonotype(high)-expressing BDC T cells induced diabetes; in contrast, H22 Abs did not inhibit diabetes. While clonotype(high) T cells induced diabetes even when IFN-gamma was neutralized, paradoxically there was reduced inflammation and no diabetes if host myeloid cells lacked IFN-gamma receptor. Hence, using monoclonal CD4 T cells, IFN-gamma can have a wide diversity of roles, depending on the setting of the immune process.     

Diabetes incidence is unaltered in glutamate decarboxylase 65-specific TCR retrogenic nonobese diabetic mice: generation by retroviral-mediated stem cell gene transfer

TCR transgenic mice are valuable tools for dissecting the role of autoantigen-specific T cells in the pathogenesis of type 1 diabetes but are time-consuming to generate and backcross onto congenic strains. To circumvent these limitations, we developed a new approach to rapidly generate mice expressing TCR using retroviral-mediated stem cell gene transfer and a novel picornavirus-like 2A peptide to link the TCR alpha- and beta-chains in a single retroviral vector. We refer to these as retrogenic (Rg) mice to avoid confusion with conventional transgenic mice. Our approach was validated by demonstrating that Rg nonobese diabetic (NOD)-scid mice expressing the diabetogenic TCRs, BDC2.5 and 4.1, generate clonotype-positive T cells and develop diabetes. We then expressed three TCR specific for either glutamate decarboxylase (GAD) 206-220 or GAD 524-538 or for hen egg lysozyme 11-25 as a control in NOD, NOD-scid, and B6.H2(g7) mice. Although T cells from these TCR Rg mice responded to their respective Ag in vitro, the GAD-specific T cells exhibited a naive, resting phenotype in vivo. However, T cells from Rg mice challenged with Ag in vivo became activated and developed into memory cells. Neither of the GAD-reactive TCR accelerated or protected mice from diabetes, nor did activated T cells transfer or protect against diabetes in NOD-scid recipients, suggesting that GAD may not be a primary target for diabetogenic T cells. Generation of autoantigen-specific TCR Rg mice represents a powerful approach for the analysis of a wide variety of autoantigens.     

Nondepleting anti-CD4 has an immediate action on diabetogenic effector cells, halting their destruction of pancreatic beta cells

The induction of tolerance in a primed immune system is a major aim for therapy in autoimmunity and transplant rejection. In this paper, we investigate the action of the nondepleting anti-CD4 Ab, YTS 177. Although this Ab is nondepleting, we have demonstrated a direct action in vivo on activated effector cells. We show that the Ab inhibits transfer of insulin-dependent diabetes mellitus by the CD4+ Th1 clone BDC2.5 to nonobese diabetic mice. Furthermore, we show that this Ab acts directly on diabetogenic effector cells because it prevented BDC2.5-induced insulin-dependent diabetes mellitus in nonobese diabetic-scid recipients in the absence of other T cells. The Ab halts the diabetic process even when it is administered after the BDC2.5 cells have infiltrated the pancreas and destruction of islets is already underway. This is accompanied by an immediate decrease in proinflammatory cytokine production with cessation of beta cell destruction and disappearance of infiltrating cells from the pancreas, leaving any remaining beta cells intact. These data suggest that Abs such as this may be effective not only because they induce regulatory T cells but also because they are able to directly prevent effector cell function.     

Vaccination with Single Chain Antigen Receptors for Islet-Derived Peptides Presented on I-Ag7 Delays Diabetes in NOD Mice by Inducing Anergy in Self-Reactive T-Cells

To develop a vaccination approach for prevention of type 1 diabetes (T1D) that selectively attenuates self-reactive T-cells targeting specific autoantigens, we selected phage-displayed single chain antigen receptor libraries for clones binding to a complex of the NOD classII MHC I-A^g7 and epitopes derived from the islet autoantigen RegII. Libraries were generated from B-cell receptor repertoires of classII-mismatched mice immunized with RegII-pulsed NOD antigen presenting cells or from T-cell receptor repertoires in pancreatic lymph nodes of NOD mice. Both approaches yielded clones recognizing a RegII-derived epitope in the context of I-A^g7, which activated autoreactive CD4⁺ T-cells. A receptor with different specificity was obtained by converting the BDC2.5 TCR into single chain form. B- but not T-cells from donors vaccinated with the clones transferred protection from diabetes to NOD-SCID recipients if the specificity of the diabetes inducer cell and the single chain receptor were matched. B-cells and antibodies from donors vaccinated with the BDC2.5 single chain receptor induced a state of profound anergy in T-cells of BDC2.5 TCR transgenic NOD recipients while B-cells from donors vaccinated with a single chain receptor specific for I-A^g7 RegII peptide complexes induced only partial non-responsiveness. Vaccination of normal NOD mice with receptors recognizing I-A^g7 RegII peptide complexes or with the BDC2.5 single chain receptor delayed onset of T1D. Thus anti-idiotypic vaccination can be successfully applied to T1D with vaccines either generated from self-reactive T-cell clones or derived from antigen receptor libraries.     

Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.     

Regulation of type 1 diabetes by a self-MHC class II peptide: role of transforming growth factor beta (TGF-beta).

The present study was undertaken to analyze the regulatory T cells generated in response to class I derived self-I-A beta(g7) (54-76) peptide. It was observed T cells from young unprimed type 1 diabetes (T1D) prone NOD mice did not respond to self-I-A beta(g7) (54-76) peptide although T cells from primed young NOD mice showed a strong response. T cells from young unprimed BALB/c mice responded to self-I-A beta(d) (62-78) peptide. However, a breakdown of tolerance to these peptides was observed with age in both the strains. Culture supernatant from I-A beta(g7) (54-76) peptide-primed cells secreted large amounts of TGF-beta and inhibited T cell responses in allogeneic-MLR. Further, I-A beta(g7) (54-76) peptide specific T cell lines from young (I-A.Y) and diabetic (I-A.D) NOD mice were established. I-A.Y secreted IL-4, TGF-beta and IL-10 while I-A.D T cell line secreted IL-10 and IFN-gamma. We found that I-A.D T cell line induced diabetes when transferred in NOD/SCID mice but I-A.Y T cell line did not induce disease. These results show that immunization of NOD mice with I-A beta(g7) (54-76) peptide at a younger age induces a regulatory T cell response suggesting that correcting the defects in immunoregulatory mechanisms using self-MHC peptides may be one of the approaches to prevent autoimmune diseases like T1D.     

Treatment of experimental autoimmune prostatitis in nonobese diabetic mice by the vitamin D receptor agonist elocalcitol

On the basis of on the marked inhibitory activity of the vitamin D receptor agonist Elocalcitol on basal and growth factor-induced proliferation of human prostate cells and on its potent anti-inflammatory properties, we have tested its capacity to treat experimental autoimmune prostatitis (EAP) induced by injection of prostate homogenate-CFA in nonobese diabetic (NOD) mice. Administration of Elocalcitol, at normocalcemic doses, for 2 wk in already established EAP significantly inhibits the intraprostatic cell infiltrate, leading to a profound reduction in the number of CD4(+) and CD8(+) T cells, B cells, macrophages, dendritic cells, and I-A(g7)-positive cells. Immunohistological analysis demonstrates reduced cell proliferation and increased apoptosis of resident and infiltrating cells. Significantly decreased production of the proinflammatory cytokines IFN-gamma and IL-17 is observed in prostate-draining lymph node T cells from Elocalcitol-treated NOD mice stimulated by TCR ligation. In addition, Elocalcitol treatment reduces IFN-gamma production by prostate-infiltrating CD4(+) T cells and draining lymph node T cells specific for an immunodominant peptide naturally processed from prostate steroid-binding protein, a prostate-specific autoantigen. Finally, CD4(+) splenic T cells from Elocalcitol-treated NOD mice show decreased ability, upon adoptive transfer into NOD.SCID recipients, to induce autoimmune prostatitis, paralleled by a reduced capacity to produce IFN-gamma in response to prostate steroid-binding protein. The results indicate that Elocalcitol is able to interfere with key pathogenic events in already established EAP in the NOD mouse. These data show a novel indication for vitamin D receptor agonists and indicate that treatment with Elocalcitol may inhibit the intraprostatic inflammatory response in chronic prostatitis/chronic pelvic pain syndrome patients.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

CD8+ T cell tolerance in nonobese diabetic mice is restored by insulin-dependent diabetes resistance alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.     

The activity of immunoregulatory T cells mediating active tolerance is potentiated in nonobese diabetic mice by an IL-4-based retroviral gene therapy

Splenocytes from nonobese diabetic mice overexpressing murine IL (mIL)-4 upon recombinant retrovirus infection lose their capacity to transfer diabetes to nonobese diabetic-scid recipients. Diabetes appeared in 0-20% of mice injected with mIL-4-transduced cells vs 80-100% of controls injected with beta-galactosidase-transduced cells. Protected mice showed a majority of islets (60%) presenting with noninvasive peri-insulitis at variance with beta-galactosidase controls that exhibited invasive/destructive insulitis. Importantly, in all recipients, the transduced proteins were detected within islet infiltrates. Infiltrating lymphocytes from recipients of mIL-4-transduced cells produced high levels of mIL-4, as assessed by ELISA. In recipients of beta-galactosidase-transduced cells, approximately 60% of TCRalphabeta(+) islet-infiltrating cells expressed beta-galactosidase, as assessed by flow cytometry. The protection from disease transfer is due to a direct effect of mIL-4 gene therapy on immunoregulatory T cells rather than on diabetogenic cells. mIL-4-transduced purified CD62L(-) effector cells or transgenic BDC2.5 diabetogenic T cells still transferred disease efficiently. Conversely, mIL-4 transduction up-regulated the capacity of purified immunoregulatory CD62L(+) cells to inhibit disease transfer. These data open new perspectives for gene therapy in insulin-dependent diabetes using T cells devoid of any intrinsic diabetogenic potential.     

Identification of CD4+ T cell-specific epitopes of islet-specific glucose-6-phosphatase catalytic subunit-related protein: a novel beta cell autoantigen in type 1 diabetes

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) has been identified as a novel CD8(+) T cell-specific autoantigen in NOD mice. This study was undertaken to identify MHC class II-specific CD4(+) T cell epitopes of IGRP. Peptides named P1, P2, P3, P4, P5, P6, and P7 were synthesized by aligning the IGRP protein amino acid sequence with peptide-binding motifs of the NOD MHC class II (I-A(g7)) molecule. Peptides P1, P2, P3, and P7 were immunogenic and induced both spontaneous and primed responses. IGRP peptides P1-, P2-, P3-, and P7-induced responses were inhibited by the addition of anti-MHC class II (I-A(g7)) Ab, confirming that the response is indeed I-A(g7) restricted. Experiments using purified CD4(+) and CD8(+) T cells from IGRP peptide-primed mice also showed a predominant CD4(+) T cell response with no significant activation of CD8(+) T cells. T cells from P1-, P3-, and P7-primed mice secreted both IFN-gamma and IL-10 cytokines, whereas P2-primed cells secreted only IFN-gamma. Peptides P3 and P7 prevented the development of spontaneous diabetes and delayed adoptive transfer of diabetes. Peptides P1 and P2 delayed the onset of diabetes in both these models. In summary, we have identified two I-A(g7)-restricted CD4(+) T cell epitopes of IGRP that can modulate and prevent the development of diabetes in NOD mice. These results provide the first evidence on the role of IGRP-specific, MHC class II-restricted CD4(+) T cells in disease protection and may help in the development of novel therapies for type 1 diabetes.