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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. Copyright © 2010 by The American Association of Immunologists, Inc.

Immunization with mycobacterial preparation such as Bacille Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. We found that the potential of splenocytes from BCG- immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-a, IFN-c and IL-4 by splenocytes, increased the expression of Fas high (Apo-1/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-c and TNF-a in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anti-cytokine antibodies, (ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-a and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells. Our results suggest that BCG down-regulates destructive autoimmunity by TNF-a/IFN-c-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. These studies provide a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.

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.

We have utilized the NOD islet beta-cell line NIT-1 to monitor beta-cell specific autoantibodies and to investigate the modulation of IDDM in NOD mice by NIT-1 membrane associated antigens. The sera from diabetic but not from pre-diabetic or protected NOD mice strongly stained NIT-1 cells in FACS analysis. The cell surface antigens on NIT-1 cells were trypsin-sensitive. NIT-1 cells could not be stained by anti-mouse GAD67 antibody; however, we could demonstrate the presence of GAD65 and GAD67 mRNA by RT-PCR. Longitudinal analysis of anti-NIT-1 antibodies showed that these antibodies were present in the neonates but disappeared after weaning. Sonicated NIT-1 cell membrane preparations protected NOD mice from diabetes when injected intravenously in 5 week old mice. The protection was associated with reduced cytotoxic activity and elevated Th2-like responses as indicated by IgG1 antibodies against the NIT-1 cells. Subcutaneous injection of sonicated NIT-1 membranes or the injection of control red blood cell membranes failed to induce protection. We conclude that NIT-1 cell membranes do not express GAD but contain other antigens that are important in the development and prevention of IDDM. These antigens could be useful for the diagnosis of diabetes by monitoring autoantibody levels and for the modulation of IDDM by immunotherapy.

Immunomodulatory azaspirane compounds have immunosuppressive activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis. The mechanism of action of azaspiranes appears to be the induction of antigen non-specific (natural) suppressor cell activity. In this study, we tested the azaspirane, SK&F 106610 in an animal model of autoimmune (type 1) diabetes, the BB rat. Oral administration of SK&F 106610 (15 mg/kg/day) to diabetes-prone BB rats, from age 30 days, significantly decreased diabetes incidence at 100 days from 80% (24 of 30 control rats) to 32% (10 of 31 drug-treated rats, P < 0.001). Protection from diabetes by SK&F 106610 was accompanied by decreased lymphocytic infiltration of the pancreatic islets (insulitis). No changes occurred in splenic T cell, B cell or macrophage subsets, or in proliferative responses to the mitogens lipopolysaccharide and concanavalin A (Con-A). Cell mixing experiments in vitro, however, revealed increased antigen non-specific suppressor activity (suppression of splenic lymphoproliferative response to Con-A) in spleens of SK&F 106610-treated rats. The suppressor cell activity was enriched in a low density fraction of splenic cells relatively depleted of T cells, B cells, macrophages and natural killer cells. These results indicate that the azaspirane compound, SK&F 106610 can prevent insulitis and autoimmune diabetes in BB rats and that these actions may be related to the activation of non-specific (natural) suppressor cells.

The nonobese diabetic (NOD) mouse is an excellent model of insulin-dependent (type I) human diabetes mellitus. We report that a single injection of complete Freund's adjuvant (CFA) given at an early age (5 wk) prevented the appearance of diabetes and greatly increased the life span of NOD mice without additional therapy. No treated mouse developed hyperglycemia by the age of 12 mo (n = 13), whereas all untreated mice died of diabetes before 8 mo of age (n = 38). All CFA-treated mice were alive and healthy at 12 mo of age. Some CFA-treated NOD mice that were monitored for long-term survival are still alive with no sign of disease at 18 mo of age (n = 5). Administration of CFA resulted in decreased in vitro splenic lymphocyte proliferative responses to alloantigen and mitogen. Cell-mixing experiments indicated that antigen-nonspecific inhibitory cells were elicited in the spleen and increased in the bone marrow. These regulatory cells were Thy-1− ; and nonadherent to nylon wool, as has been described for natural suppressor (NS) cells. These data lend support to a relationship between the boosting of endogenous NS activity and the establishment of tolerance to self in the context of autoimmunity. Our results suggest that early nonspecific immunotherapy of genetically predisposed individuals could prevent the development of autoimmune diabetes.

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.

Immunization with mycobacterial preparation such as Bacille Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. We found that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-alpha, IFN-gamma and IL-4 by splenocytes, increased the expression of Fas(high) (Apo-1/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-gamma and TNF-alpha in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anti-cytokine antibodies, (ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-alpha and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells. Our results suggest that BCG down-regulates destructive autoimmunity by TNF-alpha/IFN-gamma-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. These studies provide a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.

NOD mice have a relative deficiency of CD4 + CD25 + regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4 + CD25 + regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4 + CD25 + regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4 + CD25 + regulatory T cells. These cells secreted large amounts of TGF-β and TNF-α with little or no IFN-γ and IL-10. Adoptive transfer of these CD4 + CD25 + regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4 + CD25 + T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4 + CD25 + regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.

Oral administration of protein can induce antigen-specific immune hyporesponsiveness. However, the utility of oral tolerance to autoantigens in the treatment of autoimmune diseases may be limited when candidate autoantigens cannot be produced by conventional systems in quantities sufficient for clinical studies. Plants may be ideally suited for this purpose, as they can synthesize, glycosylate and assemble mammalian proteins to provide huge quantities of relatively low cost soluble proteins. Furthermore, edible transgenic plants could provide a simple and direct method of autoantigen delivery for oral tolerance. Therefore, the aim of this study was to determine whether a transgenic plant expression system was capable of synthesizing the diabetes-associated autoantigen, glutamic acid decarboxylase (GAD) in an immunogenic form and whether the oral administration of an autoantigen expressed by a plant could directly induce protective immune responses in a mouse model of diabetes. We show that a GAD-expressing transgenic plant, given as a dietary supplement, inhibits the development of diabetes in the non-obese diabetic (NOD) mouse.

We have previously shown that immunotherapy with complete Freund's adjuvant (CFA) or BCG is highly effective in the prevention of spontaneous insulin-dependent diabetes mellitus (IDDM) and in circumventing the rejection of syngeneic islet grafts in diabetic NOD mice. This protection is reversed by treatment with cyclophosphamide (Cy). The present study was undertaken to determine the effect of BCG vaccination on the progression of Cy-accelerated diabetes in NOD mice and to understand the mechanism of BCG immunotherapy. The time course of Cy and BCG administration showed that the progression of Cy-induced diabetes can only be blocked when BCG vaccination is given within 3 days of Cy administration. Mice given BCG 3 days before (-3 days) or 7 days after Cy treatment were not protected. BCG immunization 1 day after Cy treatment almost completely prevented insulitis in the islets of Cy-treated mice. Cy treatment reduced the endogenous production of anti-GAD67 antibody, whereas BCG vaccination 1 day after Cy treatment restored the production of anti-GAD67 antibody of IgG1 isotype. The comprehensive effect of BCG vaccination on cytokine production in Cy-treated mice was to increase IL-4 production and change the IL-4/IFN-gamma ratio in both serum and supernatant of spleen cell cultures. We found that BCG-induced protection was associated with increased splenic CD4+CD45 RB(high) T cells. Taken together, our results indicate that BCG treatment counteracts the effect of Cy on autoimmune process in IDDM. However, BCG immunotherapy has a narrow window of up to 3 days after Cy treatment to block the progression of Cy-induced diabetes and to allow for the induction of regulatory cells which may effectively downregulate the diabetogenic cells. In summary, our results suggest that BCG vaccination prevents IDDM if given in the prediabetic state. After the induction of diabetes, disease progression can only be prevented within a narrow window of opportunity by this treatment.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD67) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD67 prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD67 immune responses in naive and GAD-immunized NOD mice. Anti-GAD67 antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD67 at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD67. These T-cell proliferative responses were blocked by anti-I-ANOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD67, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD67. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD67. These results suggest that GAD67 is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD67 can prevent the onset of diabetes.

We have previously reported that a single injection of complete Freund's adjuvant (CFA) can prevent diabetes appearance in diabetes-prone (DP) BB rats. In this study, we investigated further the mechanism of CFA-induced protection from diabetes. We found that adoptive transfer of splenic cells from CFA-treated DP rats into young DP rats protected the latter from diabetes development. This suggested that CFA-induced protection from diabetes resulted from activation of regulatory (suppressor) cells. Cell mixing experiments in vitro indicated that CFA activated splenic cells with antigen-nonspecific suppressor activity (suppression of lymphoproliferative responses to lipopolysaccharide and to allogeneic splenic cells). Fractionation of splenic cells on Percoll revealed that the suppressor activity resided in low density cells relatively depleted of T-cells, B-cells, macrophages and NK cells. These results suggest that non-specific (natural) suppressor cells in CFA-treated BB rats may be responsible for suppressing autoimmune responses and preventing insulitis and diabetes development.

The BB rat spontaneously develops an insulin-dependent diabetes mellitus (IDDM) that closely resembles this disease in man. The pathogenesis involves autoimmune destruction of pancreatic islet beta-cells. In the present study, a single intraperitoneal injection of complete Freund's adjuvant (CFA) in diabetes-prone (DP) BB/Wor rats between 9 and 28 days of age reduced the incidence of diabetes at 120 days from 89% to 10-28%, whereas injection of CFA after 40 days of age was ineffective. The CFA-injected, diabetes-free DP rats had normal levels of pancreatic insulin and little or no mononuclear leukocyte infiltration in the islets. These protective effects of CFA were not associated with any changes in peripheral blood leukocyte counts or monoclonal antibody-defined T cell, B cell, or macrophage subsets in the spleen of the DP rats. These results suggest that it is possible to prevent diabetes by adjuvant treatment in early life without general immunosuppression or sustained therapy.

BALB/c mice rendered tolerant to alloantigens by neonatal injection of semi-allogeneic (C57BL/6 x BALB/c)F1 spleen cells develop a thrombocytopenia in association with an autoimmune lupus-like syndrome. The possible mechanisms involved in the thrombocytopenia were investigated. The development of thrombocytopenia was first detected at 3 weeks of age coinciding with the start of the other autoimmune manifestations and was always related to a state of tolerance and B cell chimerism. There was a significant increase of megakaryocytes in bone marrow and spleens from thrombocytopenic tolerant mice and radiolabeled platelets from these mice were more rapidly eliminated from the bloodstream than normal platelets when injected into normal recipients. A significant correlation between the spleen weight and the decrease of the circulating platelets was observed, although some mice with severe thrombocytopenia had only a moderate spleen enlargement. Thrombocytopenia significantly correlates with the levels of platelet-associated IgG (PAIgG) but not with anti-single-stranded DNA antibodies or circulating immune complexes. Platelets from mice with high levels of PAIgG had a shorter life-span when injected into normal mice than those from mice with low or normal PAIgG. The possibility that PAIgG are partially due to antibodies reacting specifically with platelet membrane components was analyzed. First, F(ab')2 Ig fragments from tolerant mice were shown to bind to normal platelets, in contrast to F(ab')2 Ig fragments from normal mice. Second, some monoclonal antibodies produced by hybridomas derived from tolerant mice reacted in vitro with platelets and induced a transient thrombocytopenia after i.v. injection into normal mice. These data suggest that the thrombocytopenia observed in tolerant mice is the result of a peripheral hyperdestruction of platelets associated with (a) hypersplenism, (b) nonspecific fixation of immunoglobulins, probably as immune complexes and (c) with autoantibodies reacting specifically with platelets. It may represent an interesting model for human chronic idiopathic thrombocytopenia.

Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.