Edwin Lee-Chan

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-γ in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-β 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-γ 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.

Identification of CD4+ T Cell-Specific Epitopes of Islet-Specific Glucose-6-Phosphatase Catalytic Subunit-Related Protein: A Novel β 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-Ag7) 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-Ag7) Ab, confirming that the response is indeed I-Ag7 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-γ and IL-10 cytokines, whereas P2-primed cells secreted only IFN-γ. 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-Ag7-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.

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.

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

BackgroundIn Type 1 diabetes, the insulin-producing β-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 β-cell loss. This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas.ResultsWe hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas. We therefore quantified the Reg1, Reg2, and Reg 3δ (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-22R α following CFA treatment; 4) a down-regulation in Reg1 and Reg 2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet β-cell DNA synthesis in vitro in the presence of IL-22.ConclusionsWe conclude that IL-22 may contribute to the regeneration of β-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 Vβ4 chains from the highly diabetogenic BDC2.5 CD4+ T cell clone, which recognizes pancreatic β cell membrane Ags presented by NOD I-Ag7 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-Ag7, 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.

Preventative role of interleukin-17 producing regulatory T helper type 17 (Treg 17) cells in type 1 diabetes in non-obese diabetic mice.

T helper type 17 (Th17) cells have been shown to be pathogenic in autoimmune diseases; however, their role in type 1 diabetes (T1D) remains inconclusive. We have found that Th17 differentiation of CD4+ T cells from BDC2·5 T cell receptor transgenic non‐obese diabetic (NOD) mice can be driven by interleukin (IL)‐23 + IL‐6 to produce large amounts of IL‐22, and these cells induce T1D in young NOD mice upon adoptive transfer. Conversely, polarizing these cells with transforming growth factor (TGF)‐β + IL‐6 led to non‐diabetogenic regulatory Th17 (Treg17) cells that express high levels of aryl hydrocarbon receptor (AhR) and IL‐10 but produced much reduced levels of IL‐22. The diabetogenic potential of these Th17 subsets was assessed by adoptive transfer studies in young NOD mice and not NOD.severe combined immunodeficient (SCID) mice to prevent possible transdifferentiation of these cells in vivo. Based upon our results, we suggest that both pathogenic Th17 cells and non‐pathogenic regulatory Treg17 cells can be generated from CD4+ T cells under appropriate polarization conditions. This may explain the contradictory role of Th17 cells in T1D. The IL‐17 producing Treg17 cells offer a novel regulatory T cell population for the modulation of autoimmunity.

Preventative role of IL‐17 producing regulatory Th17 (Treg17) cells in type 1 diabetes in NOD mice

T helper type 17 (Th17) cells have been shown to be pathogenic in autoimmune diseases; however, their role in type 1 diabetes (T1D) remains inconclusive. We have found that Th17 differentiation of CD4+ T cells from BDC2·5 T cell receptor transgenic non‐obese diabetic (NOD) mice can be driven by interleukin (IL)‐23 + IL‐6 to produce large amounts of IL‐22, and these cells induce T1D in young NOD mice upon adoptive transfer. Conversely, polarizing these cells with transforming growth factor (TGF)‐β + IL‐6 led to non‐diabetogenic regulatory Th17 (Treg17) cells that express high levels of aryl hydrocarbon receptor (AhR) and IL‐10 but produced much reduced levels of IL‐22. The diabetogenic potential of these Th17 subsets was assessed by adoptive transfer studies in young NOD mice and not NOD.severe combined immunodeficient (SCID) mice to prevent possible transdifferentiation of these cells in vivo. Based upon our results, we suggest that both pathogenic Th17 cells and non‐pathogenic regulatory Treg17 cells can be generated from CD4+ T cells under appropriate polarization conditions. This may explain the contradictory role of Th17 cells in T1D. The IL‐17 producing Treg17 cells offer a novel regulatory T cell population for the modulation of autoimmunity.

A Self MHC Class II β-Chain Peptide Prevents Diabetes in Nonobese Diabetic Mice

We explored T cell responses to the self class II MHC (I-Ag7) β-chain-derived peptides in diabetic and prediabetic nonobese diabetic (NOD) mice. We found that one of these immunodominant epitopes of the β-chain of I-Ag7 molecule, peptide 54–76, could regulate autoimmunity leading to diabetes in NOD mice. T cells from prediabetic young NOD mice do not respond to the peptide 54–76, but T cells from diabetic NOD mice proliferated in response to this peptide. T cells from older nondiabetic mice or mice protected from diabetes do not respond to this peptide, suggesting a role for peptide 54–76-specific T cells in pathogenesis of diabetes. We show that this peptide is naturally processed and presented by the NOD APCs to self T cells. However, the peptide-specific T cells generated after immunization of young mice regulate autoimmunity in NOD mice by blocking the diabetogenic cells in adoptive transfer experiments. The NOD mice immunized with this peptide are protected from both spontaneous and cyclophosphamide-induced insulin-dependent diabetes mellitus. Immunization of young NOD mice with this peptide elicited T cell proliferation and production of Th2-type cytokines. In addition, immunization with this peptide induced peptide-specific Abs of IgG1 isotype that recognized native I-Ag7 molecule on the cell surface and inhibited the T cell proliferative responses. These results suggest that I-Aβg7(54–76) peptide-reactive T cells are involved in the pathogenesis of diabetes. However, immunization with this peptide at young age induces regulatory cells and the peptide-specific Abs that can modulate autoimmunity in NOD mice and prevent spontaneous and induced diabetes.

Adjuvant Immunotherapy Increases β Cell Regenerative Factor Regeneration Gene 2 in the Pancreas of Diabetic Mice

Insulin-producing β 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 β cell mass. In this study, we investigated factors involved in the regeneration of β 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 β 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 β 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 β cells.

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-γ 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-κB, 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.