Role of TGF-ß in Self-Peptide Regulation of Autoimmunity.

Transforming growth factor (TGF)-ß has been implicated in regulation of the immune system, including autoimmunity. We have found that TGF-ß is readily produced by T cells following immunization with self-peptide epitopes that downregulate autoimmune responses in type 1 diabetes (T1D) prone nonobese diabetic (NOD) mice. These include multiple peptide epitopes derived from the islet ß-cell antigens GAD65 (GAD65 p202-221, GAD65 p217-236), GAD67 (GAD67 p210-229, GAD67 p225-244), IGRP (IGRP p123-145, IGRP p195-214) and insulin B-chain (Ins. B:9-23) that protected NOD mice from T1D. Immunization of NOD mice with the self-MHC class II I-Ag7 ß-chain-derived peptide, I-Aßg7 p54-76 also induced large amounts of TGF-ß and also protected these mice from diabetes development. These results indicate that peptides derived from disease related self-antigens and MHC class II molecules primarily induce TGF-ß producing regulatory Th3 and Tr1-like cells. TGF-ß produced by these cells could enhance the differentiation of induced regulatory iTreg and iTreg17 cells to prevent induction and progression of autoimmune diseases. We therefore suggest that peripheral immune tolerance could be induced and maintained by immunization with self-peptides that induce TGF-ß producing T cells.

Vasostatin-1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice.

The chromogranin A (ChgA) 29-42 sequence is the antigenic epitope for the BDC2.5 CD4(+) T-cell receptor in NOD mice (H-2(g7) ). We have now characterized the binding register of the ChgA 29-42 peptide for the I-A(g7) molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34-42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti-ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno-staining pattern was observed for the ChgA 17-38 peptide antibodies that overlap with the ChgA 29-42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29-42 peptide. These findings indicate that peptides from the N-terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.

The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes.

BACKGROUND: In Type 1 diabetes, the insulin-producing ß-cells within the pancreatic islets of Langerhans are destroyed. We showed previously that immunotherapy with Bacillus Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) of non-obese diabetic (NOD) mice can prevent disease process and pancreatic ß-cell loss. This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas. RESULTS: We hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas. We therefore quantified the Reg1, Reg2, and Reg3δ (INGAP) mRNA expression in isolated pre-diabetic NOD islets treated with IL-22. We measured IL-22, and IL-22 receptor(R)-α mRNA expression in the pancreas and spleen of pre-diabetic and diabetic NOD mice. Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22Rα following CFA treatment; 4) a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet ß-cell DNA synthesis in vitro in the presence of IL-22. CONCLUSIONS: We conclude that IL-22 may contribute to the regeneration of ß-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

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 Vß4 chains from the highly diabetogenic BDC2.5 CD4(+) T cell clone, which recognizes pancreatic ß cell membrane Ags presented by NOD I-A(g7) MHC class II molecules. The antigenic epitope of BDC2.5 TCR is absent in ß 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.

Adjuvant immunotherapy increases beta cell regenerative factor Reg2 in the pancreas of diabetic mice.

Insulin-producing ß cells can partially regenerate in adult pancreatic tissues, both in human and animal models of type 1 diabetes (T1D). Previous studies have shown that treatment with mycobacterial adjuvants such as CFA and bacillus Calmette-Guérin prevents induction and recurrence of T1D in NOD mice with partial recovery of ß cell mass. In this study, we investigated factors involved in the regeneration of ß cells in the pancreas of NOD mice during diabetes development and after treatment with adjuvants. The Regeneration (Reg) gene family is known to be involved in regeneration of various tissues including ß cells. Reg2 expression was found to be upregulated in pancreatic islets both during diabetes development and as a result of adjuvant treatment in diabetic NOD mice and in C57BL/6 mice made diabetic by streptozotocin treatment. The upregulation of Reg2 by adjuvant treatment was independent of signaling through MyD88 and IL-6 because it was not altered in MyD88 or IL-6 knockout mice. We also observed upregulation of Reg2 in the pancreas of diabetic mice undergoing ß cell regenerative therapy with exendin-4 or with islet neogenesis-associated protein. Reg2 expression following adjuvant treatment correlated with a reduction in insulitis, an increase in insulin secretion, and an increase in the number of small islets in the pancreas of diabetic NOD mice and with improved glucose tolerance tests in streptozotocin-treated diabetic C57BL/6 mice. In conclusion, adjuvant immunotherapy regulates T1D in diabetic mice and induces Reg2-mediated regeneration of ß cells.

Th17 polarized cells from nonobese diabetic mice following mycobacterial adjuvant immunotherapy delay type 1 diabetes.

IL-17-producing T cells are regarded as potential pathogenic T cells in the induction of autoimmune diseases. Previously, we have shown that injection of adjuvants containing Mycobacterium, such as CFA or bacillus Calmette-Guérin, can prevent type 1 diabetes in NOD mice. We injected NOD mice with mycobacterial products s.c. and analyzed the IL-17-producing cells from the draining lymph nodes and spleen by restimulating whole-cell populations or CD4(+) T cells in vitro with or without IL-17-polarizing cytokines. Mice receiving CFA had a concomitant rise in the level of IL-17, IL-22, IL-10, and IFN-gamma in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-beta plus IL-6 or IL-23 delayed the development of diabetes in recipient mice. IL-17-producing cells induced by CFA maintained their IL-17-producing ability in the recipient mice. Injection of CFA also changed the cytokine profile of cells in pancreatic tissue by increasing IL-17, IL-10, and IFN-gamma cytokine gene expression. We suggest that the rise in the level of IL-17 after adjuvant therapy in NOD mice has a protective effect on type 1 diabetes development.

Dendritic cell differentiation induced by a self-peptide derived from apolipoprotein E.

Dendritic cells (DCs) are professional APCs and potent stimulators of naive T cells. Since DCs have the ability to immunize or tolerize T cells they are unique candidates for use in immunotherapy. Our laboratory has discovered that a naturally processed self-peptide from apolipoprotein E, Ep1.B, induces DC-like morphology and surface marker expression in a murine monocytic cell line (PU5-1.8), human monocytic cell line (U937), murine splenocytes, and human peripheral blood monocytes. Microscopy and flow cytometric analysis revealed that Ep1.B-treated cells display decreased adherence to plastic and increased aggregation, dendritic processes, and expression of DC surface markers, including DEC-205, CD11c, B7.1, and B7.2. These effects were observed in both PU5-1.8 cells and splenocytes from various mouse strains including BALB/c, C57BL/6, NOD/Lt, and C3H/HeJ. Coadministration of Ep1.B with OVA antigenic peptide functions in dampening specific immune response to OVA. Ep1.B down-regulates proliferation of T cells and IFN-gamma production and stimulates IL-10 secretion in immunized mice. Ep1.B-induced differentiation resulted in the activation of PI3K and MAPK signaling pathways, including ERK1/2, p38, and JNK. We also found that NF-kappaB, a transcription factor essential for DC differentiation, is critical in mediating the effects of Ep1.B. Ep1.B-induced differentiation is independent of MyD88-dependent pathway of TLR signaling. Cumulatively, these findings suggest that Ep1.B acts by initiating a signal transduction cascade in monocytes leading to their differentiation into DCs.

Exogenous CLIP localizes into endocytic compartment of cells upon internalization: implications for antigen presentation by MHC class II molecules.

We have previously shown that exogenous CLIP (class II associated invariant chain peptide) downregulated MHC class II expression on antigen presenting cells (APC) and modulated T cell mediated immune responses. The present study was undertaken to investigate the mechanism of uptake of exogenously added CLIP peptide by APC. We found that exogenous CLIP is rapidly internalized by APC and it co-localize with MHC class II in intracellular compartments including early-, late-endosomes and lysosomes. We suggest that exogenous CLIP acts as an in vivo regulator of immune response by internalization and passage through the intracellular compartments where it interferes in peptide loading and recycling of MHC class II molecules to the APC surface. Therefore, exogenous CLIP regulates immune responses by modulation of antigen presentation by the APC.

A novel mechanism of regulatory T cell-mediated down-regulation of autoimmunity.

We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non-specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN.

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.

Mechanism of modulation of T cell responses by N-palmitoylated peptides.

Small structural changes in the antigenic peptides recognized by TCR can alter the biological properties of those peptides and convert them into weak agonists, partial agonists, or antagonists of these receptors. These altered peptide ligands (APL) are usually generated by conservative amino acid substitutions at TCR contact residues. Here, we show that APL with therapeutic properties can also be generated by attachment of palmitic acid at the N terminus of the peptide without the need to modify the peptide's primary sequence. Using N-palmitoylated pigeon cytochrome-c peptide 81-104 (PALPCC(81-104)), we were able to induce T cell hyporesponsiveness to the wild-type peptide in vitro. More importantly, administration of the PALPCC(81-104 )to mice reduced the responsiveness to the native peptide when tested ex vivo. Biochemical and functional experiments indicated that the action of N-palmitoylated peptides was due to the conversion of the native peptide into a weak agonist that could then induce T cell anergy. Our results demonstrate that N-palmitoylation of antigenic peptides is a feasible strategy to generate APL, as it avoids the need to screen multiple amino acid variants of each specific antigen to identify those with therapeutic properties.

Type 1 diabetes alters anti-hsp90 autoantibody isotype.

The 90-kDa chaperon family includes heat shock protein (hsp) 90 and glucose-regulated protein (grp) 94. These proteins play an important role in normal cellular architecture, in the etiology of some autoimmune and infectious diseases and in antigen presentation to T cells. Owing to its role in autoimmunity, we explored anti-hsp90 autoantibody (hsp90AA) response in the sera of persons with type 1 diabetes, first-degree relatives (FDR) and in normal subjects. Significant high level of hsp90AA was found in FDR, but there was no significant difference between the normal and diabetic persons. The IgG1 and IgG3 isotypes of hsp90AA were higher in persons with type 1 diabetes and FDR than in normal subjects. We found a good correlation between hsp90AA measured by ELISA and RIA. A positive correlation between serum hsp90AA and glutamic acid decarboxylase (GAD65) autoantibody (GAA) was also observed. Hsp90AA positive sera from diabetic persons immunoblotted recombinant hsp90, GAD65 and corresponding proteins in islet lysates. Our study suggests that hsp90AA are present in normal, FDR and diabetic persons. However, there is a higher level of IgG1 and IgG3 isotypes of hsp90AA in FDR and type 1 diabetic subjects. Thus, autoimmunity leading to type 1 diabetes significantly alters the autoantibody isotype to autoantigens, such as hsp90.

International Workshop on Lessons from Animal Models for Human Type 1 Diabetes: analyzing target autoantigens of humoral immunity in nonobese diabetic mice.

The aim of this workshop was to identify autoantibody assays that could provide useful markers of autoimmunity in the NOD mouse model for type 1 diabetes. It was demonstrated that insulin autoantibodies measured by sensitive radiobinding assay are a marker of autoimmunity in NOD mice and draw into question the true nature of GADA and IA-2A in this animal model.