Low-dose warfarin functions as an immunomodulator to prevent cyclophosphamide-induced NOD diabetes.

Warfarin has been used as an anticoagulant for a long time. Recently, the pleiotropic effect of warfarin has been investigated. As low-dose warfarin has been reported to have anti-inflammatory effect through suppression of IL-6 secretion and inhibit the immune-associated signal between Tyro3 and its ligand, Gas6, the effect of low-dose warfarin on autoimmune diabetes in NOD mice was examined. To investigate the anti-inflammatory effect of warfarin, IL-6 secretion by splenocytes was examined in the presence of various concentrations of warfarin. Low concentration of warfarin inhibited IL-6 secretion. mRNA expression of Rse, one of the Tyro3 receptor family members, and Gas6 were analyzed in NOD mice. It was detected in islets, splenocytes and bone-marrow derived dendritic cells. 0.25 mg/l or 0.50 mg/l of warfarin was orally administered to NOD mice as a cyclophosphamide-induced diabetes model. Oral administration of warfarin at much lower doses than those clinically used as an anticoagulant significantly reduced the degree of insulitis and diabetes incidence in this model. We previously demonstrated that anti-FasL Ab-treatment led to complete prevention of autoimmune diabetes in NOD mice. As Fas/FasL signaling is reported to be essential for cyclophosphamide-induced diabetes model, we extracted RNA from lymphocytes of the inguinal lymph nodes of anti-FasL Ab-treated NOD mice and performed real-time PCR to determine expression of Rse gene. Interestingly, the expression of Rse gene related to the blockade of Fas/FasL signaling was reduced to less than half the level of untreated mice. In conclusion, low-dose warfarin is a potential immunomodulator which can prevent autoimmune diabetes.

Prevention of recurrent but not spontaneous autoimmune diabetes by transplanted NOD islets adenovirally transduced with immunomodulating molecules.

Pancreatic islet transplantation has the potential to maintain normoglycemia in patients with established type 1 diabetes, thereby obviating the need for frequent insulin injections. Our previous study showed that recombinant IL-12p40-producing islets prevented the recurrence of NOD diabetes. First, to see which immunomodulating molecule-secreting islet grafts can most powerfully prevent diabetes development in NOD mice without immunosuppressant, NOD islets were transfected with one of the following adenoviral vectors: Ad.IL-12p40, Ad.TGF-beta, Ad.CTLA4-Ig, or Ad.TNF-alpha after which they were transplanted under the renal capsule of acutely diabetic NOD mice. The immunomodulating molecules produced by these adenovirus-transfected islets in vitro were 74+/-19ng, 50+/-4ng, 821+/-31ng, and 77+/-18ng/100 islets, respectively. Transplantation of IL-12p40, TNF-alpha, and CTLA4-Ig but not TGF-beta-secreting islets displayed enhanced survival and delayed diabetes recurrence in recent-onset diabetic recipients. IL-12p40-producing islet grafts most powerfully prevented recurrent diabetes in NOD mice. In addition, local production of TNF-alpha and CTLA4-Ig significantly prolonged islet graft survival. In second series of experiment, these manipulated islets were transplanted under the renal capsule of 10-week-old NOD recipients and were also transplanted subcutaneously into 2-week-old NOD recipients. Transplantation of these islets into 2- or 10-week-old pre-diabetic mice failed to protect them from developing diabetes; in fact, transplantation of Ad.TNF-alpha-transfected islets into 2-week-old mice actually accelerated diabetes onset. Taken together, this approach was ineffectual as a prophylactic protocol. In conclusion, this study showed comparisons of the immunomodulating effects of 4 different adenoviral vectors in the same transplantation model and local production of IL-12p40, TNF-alpha and CTLA4-Ig significantly prevented recurrent diabetes in NOD mice.

Autoreactive T cell response in CD25-negative fraction of peripheral blood mononuclear cells in established type 1 diabetes.

CD4(+)CD25(+) T cells (Tregs) play a critical role in maintaining dominant peripheral tolerance, and pathogenic autoreactive T cells may be frequent in the CD25-negative fraction of peripheral blood mononuclear cells (PBMCs) from patients with autoimmune disease. We therefore investigated whether T cell autoimmune responses to recombinant GAD65 can be detected by the use of ELISPOT assay in the CD25-negative fraction of PMBCs from Japanese type 1 diabetes (T1D) patients. The frequency of CD4(+)CD25(+) T cells was not different among patients with newly developed T1D, established T1D, and healthy controls. The CD25 positive cell-depleted fraction was obtained by negative selection with antihuman CD25 magnetic beads, reducing the number of CD4(+)CD25(+) T cells from 4-5% to less than 1%. In whole PBMC fraction, there was a significant elevation of IFN-gamma spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN-gamma spots from patients with established T1D was not significant. In the CD25-negative fraction, unlike whole PBMCs, we observed the significant IFN-gamma spots to GAD65 in the fraction from patients with established T1D (P < 0.05), but not in those with recently diagnosed disease. The phenomena were not observed for IL-4 spots. Our data suggest a possible role of Tregs maintaining dominant peripheral tolerance in T1D and application of further improved T cell assay detecting autoimmunity even in established T1D.

Immunological aspects of 'fulminant type 1 diabetes'.

'Fulminant diabetes' has been recognized as a super-acute onset and non-autoimmune type 1 diabetes. To evaluate autoimmunity against pancreatic beta cell in fulminant diabetes, ELISPOT assay was applied to the peripheral blood of these patients. In our ELISPOT system, GAD65-reactive and insulin B9-23-reactive IFN-gamma spots were detected in 46.3 and 26.0% of autoantibody-positive type 1 diabetes. Also, in fulminant type 1 diabetic patients, IFN-gamma spots in response to GAD65 and insulin B9-23 peptide were detected in 69.2 and 25.0%, respectively. These results suggest that anti-beta cell autoimmunity contributes to develop fulminant type 1 diabetes. Fulminant type 1 diabetes is known to have IDDM-resistant HLA DR2 with similar frequency of non-T1D subjects. In a mouse model, when islet-reactive CD8 cells are transferred to young NOD mice, the recipients develop overt diabetes within 1 week with massive insulitis. In (NOD x Balb/c) F1 mice, which hold idd-resistant genes, transfer of islet-reactive CD8 cells induced diabetes to 60% F1 recipients within 1 week with the later disappearance of insulitis. This mouse model shows very similar feathers to fulminant type 1 diabetes; idd-resistant HLA and no insulitis. These results implicated that once anti-islet immunity is optimally activated, subjects with partially resistant alleles could become overt diabetes.

Insulin as a T cell antigen in type 1 diabetes supported by the evidence from the insulin knockout NOD mice.

Rodents have two functional preproinsulin genes named insulin 1 and insulin 2 on different chromosome and have two amino acid differences in insulin B chain. We have established insulin 1 or insulin 2 knockout (KO) non-obese diabetic (NOD) colonies in the animal institute of Kobe University and evaluated anti-insulin autoimmunity. Similar to the previous report, insulin 1-KO provides strong protection from insulitis (islet-infiltration of mononuclear cells) and diabetes, whereas the insulin 2-KO markedly accelerated insulitis and development of diabetes even at further backcross breeding with NOD/Shi/Kbe mice (P<0.0001). Expression of serum anti-insulin autoantibodies (IAA) was enhanced in insulin 2-KO mice at a time between 10 and 15 weeks of age (P<0.005) while the expression of insulin 1-KO NOD mice was rather reduced. Furthermore, T cell reactivity in splenocytes of insulin 2-KO NOD mice to insulin 1 B:9-23 peptide was increased (P<0.05), suggesting that expanding insulin-reactive T cells may contribute to the acceleration of diabetes in insulin 2-KO mice. Based on those observations, we hypothesize that insulin 1 is a crucial T cell antigen in murine autoimmune diabetes and modification of anti-insulin autoimmunity can be applicable to antigen-based therapy for human type 1 diabetic patients.

T cell epitope mapping study with insulin overlapping peptides using ELISPOT assay in Japanese children and adolescents with type 1 diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease. Insulin seems to be a critical antigen recognized by autoreactive T cells. In this study, we performed T cell epitope mapping of insulin using serial overlapping peptides in Japanese patients with T1D. Serial overlapping insulin peptides comprising 23 peptides, which were each 15-amino acid long, were prepared based on insulin sequence. Cytokine secretion from peripheral T cells against these peptides was studied by enzyme-linked immunospot (ELISPOT) assay in 18 patients with recent-onset T1D and 12 patients with established T1D, and compared with 17 healthy control subjects. In ELISPOT assay, IFN-gamma-secreting T cells, but not IL-4, against several insulin peptides were observed in 77.8% of patients with recent-onset T1D, 50.0% of patients with established T1D, and 0% of healthy control subjects. All epitopes recognized by T cells were identified in the B-chain of insulin. The most frequent epitope existed at the B10-24 region (9/18), followed by B1-15 and B11-25 regions (6/18, each), with B4-18, B9-23, and B12-26 identified in some patients. These data did not correlate with insulin autoantibodies or HLA-DRB1 of the patients. This is the first report of T cell epitope mapping using one amino acid serial overlapping peptides of insulin in T1D. ELISPOT assay revealed the frequent existence of insulin peptide-specific T cells in patients with recent-onset and established T1D. The T cell epitopes of insulin were similar but not identical in our cohort, which probably explains the difficulty encountered in prevention of human T1D by using insulin.

Detection of autoreactive T cells in type 1 diabetes using coded autoantigens and an immunoglobulin-free cytokine ELISPOT assay: report from the fourth immunology of diabetes society T cell workshop.

The "gold standard" for evaluation of immunoassays is blinded testing, using coded samples and antigens. Although assays for autoreactive T cells are no exception to this rule, it is nonetheless rarely applied in this context. To facilitate such investigations, we coded a panel of 10 peptides representing T cell epitopes reported to be of relevance to islet autoimmunity. These were deployed in a novel cytokine ELISPOT assay, in which the use of immunoglobulin-free medium reduces background reactivity and thus potentially enhances the specificity and sensitivity of detection of autoreactive T cells. Significant IFN-gamma production against GAD65 (554-575), insulin (B9-23), and IA-2 (709-736) peptides were observed in type 1 diabetic patients, whereas no significant changes from background were detected in healthy controls. These results confirm the utility of the blinded performance of T cell assays as the most robust platform for assessing technologies to T cell autoreactivity.

Detection of GAD65-reactive T-Cells in type 1 diabetes by immunoglobulin-free ELISPOT assays.

OBJECTIVE: To investigate the prevalence of beta-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELISPOT) assay and assessed its usefulness for diagnosing this disease. RESEARCH DESIGN AND METHODS: Cellular immune responses to beta -cell autoantigens were studied both by immunoglobulin-free proliferation assays and ELISPOT assays in 33 patients with type 1 diabetes and 15 patients with type 2 diabetes, compared with 23 healthy control subjects. Autoantibodies against GAD65 and IA-2 were measured by radioimmunoassay. RESULTS: Significant proliferative responses to GAD65 were observed in 10 of 31 (32.3%) type 1 diabetic patients (P < 0.05), whereas GAD65-reactive gamma-interferon (IFN-gamma)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay (P < 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-gamma positivity in ELISPOT and two of five (40.0%) showed significant proliferation against GAD65. CONCLUSIONS: Using a newly developed ELISPOT assay, GAD-reactive T-helper 1 cells in PBMC of type 1 diabetic patients could be identified at a higher frequency than by the proliferation assay. Therefore, the immunoglobulin-free ELISPOT assay is an excellent tool for detecting T-cell reactivity to autoantigens with greater specificity and, in combination with beta-cell autoantibody determination, will improve the diagnosis of type 1 diabetes.

Fas/Fas ligand interactions play an essential role in the initiation of murine autoimmune diabetes.

Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. To elucidate the role of Fas/FasL interactions in NOD diabetes, the effects of neutralizing anti-FasL antibody on autoimmune responses were evaluated. Islet-specific CD8(+) and CD4(+) T-cells expressed FasL upon activation and mediated FasL-dependent cytotoxicity against Fas-expressing target cells in vitro, although their cytotoxicity against islet cells was not blocked by anti-FasL antibody. Moreover, administration of anti-FasL antibody failed to inhibit diabetes in vivo in the CD8(+) T-cell adoptive transfer model. On the other hand, blockade of Fas/FasL interactions significantly inhibited CD4(+) T-cell-dependent diabetes in adoptive transfer models. These results suggest a substantial contribution of Fas/FasL interactions to CD4(+), but not CD8(+), T-cell-mediated destruction of pancreatic beta-cells. When anti-FasL antibody was administered to NOD mice between 5 and 15 weeks of age, the onset of diabetes was slightly delayed but the incidence was not decreased. However, administration of anti-FasL antibody at 2-4 weeks of age completely prevented insulitis and diabetes. These results suggest that Fas/FasL interactions contribute to CD4(+) T-cell-mediated beta-cell destruction and play an essential role in the initiation of autoimmune NOD diabetes.

Tolerance mechanisms in murine autoimmune diabetes induced by anti-ICAM-1/LFA-1 mAb and anti-CD8 mAb.

A short-term administration of antibodies against ICAM-1/LFA-1 or CD8 molecules during a critical period of younger age resulted in complete protection of autoimmune diabetes in NOD mice. In this study, we attempted to elucidate the tolerance mechanisms. Transfer of splenocytes from both antibody-treated NOD mice to NOD-SCID mice failed to develop diabetes. On the other hand, when splenocytes from diabetic mice were transferred to the antibody-treated mice, 40% of both mAb-treated recipients became diabetic. In vitro response of T cells from these protected mice exhibited strong proliferation against syngeneic islet cells or ConA. Furthermore, semiquantitative RT-PCR analysis of cytokines showed that T cells from anti-CD8-treated mice could express IFN-gamma, IL-4, IL-10 and TGF-beta1 in response to islet antigen. In contrast, T cells from anti-ICAM-1/LFA-1-treated mice expressed IFN-gamma, IL-10 and TGF-beta1 but not IL-4. These results suggest that tolerance mechanisms like clonal deletion, anergy, immunoregulatory T cells or Th1 to Th2/Th3 cytokine shifting are not responsible for the tolerance induction, indicating the presence of other unrevealed mechanism responsible for the loss of capability of autoreactive T cells to infiltrate and destroy the pancreatic beta-cells in vivo.