Yasuyo Okumachi

Regulatory CD8+ T cells induced by exposure to all-trans retinoic acid and TGF-β suppress autoimmune diabetes

Antigen-specific regulatory CD4(+) T cells have been described but there are few reports on regulatory CD8(+) T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8(+) T cells from 8.3-NOD transgenic mice. CD8(+) T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-beta, and all-trans retinoic acid (ATRA) for 5days. CD8(+) T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-beta and ATRA had low Foxp3(+) expression (1.7+/-0.9% and 3.2+/-4.5%, respectively). In contrast, CD8(+) T cells induced by exposure to IGRP, SpDCs, TGF-beta, and ATRA showed the highest expression of Foxp3(+) in IGRP-reactive CD8(+) T cells (36.1+/-10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8(+) T cells cultured with IGRP, SpDCs, TGF-beta, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8(+) T cells suppressed the proliferation of diabetogenic CD8(+) T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-beta induces CD8(+)Foxp3(+) T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.

NO-mediated cytotoxicity contributes to multiple low-dose streptozotocin-induced diabetes but not to NOD diabetes

Type 1 diabetes (T1D) is caused mostly by autoimmune destruction of pancreatic beta-cells, the precise mechanism of which remains unclear. Two major effector mechanisms have been proposed: direct cell-mediated and indirect cytokine-mediated cytotoxicity. Cytokine-mediated beta-cell destruction is presumed mainly caused by NO production. To evaluate the role of iNOS expression in T1D, this study used a novel iNOS inhibitor ONO-1714. ONO-1714 significantly reduced cytokine-mediated cytotoxicity and NO production in both MIN6N9a cells and C57BL/6 islets in the presence of IL-1beta, TNF-alpha, and IFN-gamma. To evaluate whether NO contributes to diabetes progression in vivo, ONO-1714 was administered to four different mouse models of autoimmune diabetes: multiple low-dose STZ (MLDS)-induced C57BL/6, CY-induced, adoptive transfer and spontaneous NOD diabetes. Exposure to STZ in vitro induced NO production in MIN6N9a cells and C57BL/6 islets, and in vivo injection of ONO-1714 to MLDS-treated mice significantly reduced hyperglycemia and interestingly, led to complete suppression of cellular infiltration of pancreatic islets. In contrast, when ONO-1714 was injected into spontaneous NOD mice and CY-induced and adoptive transfer models of NOD diabetes, overt diabetes could not be inhibited in these models. These findings suggest that NO-mediated cytotoxicity significantly contributes to MLDS-induced diabetes but not to NOD diabetes.

Administration of a determinant of preproinsulin can induce regulatory T cells and suppress anti-islet autoimmunity in NOD mice.

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A(g7) molecule. Preproinsulin-1 L7-24 peptide (L7-24) emulsified in Freunds incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7-24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7-24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7-24 peptide, which has a high affinity for pockets of I-A(g7), induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for 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.

Intravenous Administration of Proinsulin 1 or 2–Expressing Fiber‐Mutant Recombinant Adenovirus Vector Protects against the Development of Diabetes in NOD Mice

Insulin has been reported as a major autoantigen in both human and murine type 1 diabetes (T1D). Insulin1‐knockout NOD mice with only insulin2 are protected against the development of autoimmune diabetes, suggesting that insulin1 has strong immunogenicity and insulin2 has weak immunogenicity or a possible protective role in the pathogenesis of type 1 diabetes. In this study, we have developed fiber‐mutant adenovirus vectors that express murine proinsulin1 or proinsulin2 (named Ad.Pins1‐RGD/Ad.Pins2‐RGD) and administered those virus vectors to the NOD mouse to evaluate modulation of autoimmune responses. The intravenous administration of either Ad.Pins1‐RGD or Ad.Pins2‐RGD at 3 and 5 weeks of age strongly suppressed the development of overt diabetes, accompanied by a significant reduction of insulin autoantibody (IAA), and suppression of disease was similar between administration of Ad.Pins1‐RGD and that of Ad.Pins2‐RGD. Our study suggests that systemic administration of fiber‐mutant adenovirus vectors, which induce transient expression of proinsulin, may be applicable to a gene therapy inducing tolerance to insulin.

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‐γ spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN‐γ spots from patients with established T1D was not significant. In the CD25‐negative fraction, unlike whole PBMCs, we observed the significant IFN‐γ 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.

One amino acid difference is critical for suppression of the development of experimental autoimmune diabetes (EAD) with intravenous injection of insulinB:9-23 peptide.

InsulinB:9-23 peptide (insB:9-23) reactive T cells has been reported as crucial for type 1 diabetes. In this study, experimental autoimmune diabetes (EAD) mice, which subcutaneous immunization of ins1 or 2B:9-23 induced autoimmune diabetes in F1(B7.1B6 x BALB/c), was investigated for antigen specific therapy to delete pathogenic T cells. Intravenous injection of ins1 or 2B:9-23 significantly delayed the development of diabetes on the corresponding peptide-induced EAD (ins1EAD or ins2EAD) concomitant with reduced insulitis and insulin autoantibodies expression. Population of Foxp3(+) CD4(+) T cell was unchanged whereas the level of anti-insB:9-23 specific IgG(2a) but not IgG(1) were specifically decreased, suggesting reduction of pathogenic insB:9-23 reactive T cells. Most interestingly, intravenous administration of ins2B:9-23, whose amino acid sequence had one amino acid difference at position 9 delayed the development of diabetes in both ins1EAD and ins2EAD whereas ins1B:9-23 administration delayed diabetes in the ins1EAD but not ins2EAD, suggesting that one amino acid difference gives critical influence on the effect of intravenous injection of antigenic peptide for type 1 diabetes.