Adoptive Transfer Of Diabetes Research Articles

CD19+IgM+ Cells from NOD Mice Demonstrate Enhanced Therapeutic Efficacy in Type 1 Diabetes Mellitus

We describe a protective effect on autoimmune diabetes and a reduced destructive insulitis in NOD.scid recipients following splenocyte injections from diabetic NOD donors and sorted CD19+ cells compared to NOD.scid recipients receiving splenocytes alone. This protective effect was age-specific (only CD19+ cells from young NOD donors exerted this effect; p < 0.001). Furthermore, we found that CD19+IgM+ cells is the primary cell subpopulation of B cells that delayed adoptive transfer of diabetes mediated by diabetogenic T cells from NOD mice (p = 0.002). In fact, removal of IgM+ cells from the CD19+ pool did not result in diabetes protection. Blockade of IL-10 with neutralizing antibodies at the time of CD19+ cell co-transfers also abrogated the therapeutic effect, suggesting that IL-10 secretion was an important component of the protective effect. These results were strengthened by ex vivo incubation of CD19+ cells with IL-5, which resulted in enhanced IL-10 production and significantly delayed diabetes progression compared to controls (p = 0.0005). To the best of our knowledge, this is the first study demonstrating that CD19+IgM+ cells is the primary cell subpopulation of regulatory B cells delaying diabetes mediated by diabetogenic T cells from NOD mice. The potential to expand CD19+IgM+ cells, especially in response to increased stimulation using IL-5 or by pharmacologic agents, may be a new therapeutic option for type 1 diabetes. Disclosure A.D. Vonberg: None. M. Pietropaolo: None.

Vaccination with a co-expression DNA plasmid containing GAD65 fragment gene and IL-10 gene induces regulatory CD4(+) T cells that prevent experimental autoimmune diabetes.

The non-obese diabetic (NOD) mouse is a commonly used animal model for studying type 1 diabetes (T1D). The aims of our study were to explore the diabetes-preventive effect in NOD mice and the potential mechanisms of an optimized co-expression DNA vaccine containing GAD65 fragment gene with the IL-10 gene (SGAD65190-315 /IL-10). Female NOD mice at the age of 3-4weeks old were randomly divided into two groups and received intra-muscular injection of either blank pBudCE4.l vector (n = 34) or pBudCE4.l carrying the SGAD65190-315 /IL-10 (n = 32). The incidence of diabetes was monitored up to 30weeks of age. The severity of insulitis, apoptosis rate of β cells and relevant mechanisms were examined. Administration with SGAD65190-315 /IL-10 blocked the onset of autoimmune diabetes in NOD mice, significantly suppressed islet inflammation, inhibited the apoptosis of islet β cells, induced immune tolerance to autoantigen GAD65 and proinsulin and shifted the Th1/Th2 balance towards Th2. More importantly, the frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) in the spleen and pancreatic lymph nodes in vaccine-immunized mice were significantly increased, and these Tregs were GAD65-reactive. In addition, Treg depletion by anti-CD25 mAb administration abolished the protective effects of SGAD65190-315 /IL-10 on diabetes and insulitis. Moreover, depletion of CD4(+) CD25(+) T cells using magnetic-activated cell sorting impaired the protective effect of SGAD65190-315 /IL-10 vaccination on adoptive transfer of diabetes. Our data suggested that SGAD65190-315 /IL-10 DNA vaccine had protective effects on T1D by upregulating autoantigen-reactive Tregs. Our findings may provide a novel preventive therapy for T1D. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibition of Ca2+-independent phospholipase A2β (iPLA2β) ameliorates islet infiltration and incidence of diabetes in NOD mice.

Autoimmune β-cell death leads to type 1 diabetes, and with findings that Ca2+-independent phospholipase A2β (iPLA2β) activation contributes to β-cell death, we assessed the effects of iPLA2β inhibition on diabetes development. Administration of FKGK18, a reversible iPLA2β inhibitor, to NOD female mice significantly reduced diabetes incidence in association with 1) reduced insulitis, reflected by reductions in CD4+ T cells and B cells; 2) improved glucose homeostasis; 3) higher circulating insulin; and 4) β-cell preservation. Furthermore, FKGK18 inhibited production of tumor necrosis factor-α (TNF-α) from CD4+ T cells and antibodies from B cells, suggesting modulation of immune cell responses by iPLA2β-derived products. Consistent with this, 1) adoptive transfer of diabetes by CD4+ T cells to immunodeficient and diabetes-resistant NOD.scid mice was mitigated by FKGK18 pretreatment and 2) TNF-α production from CD4+ T cells was reduced by inhibitors of cyclooxygenase and 12-lipoxygenase, which metabolize arachidonic acid to generate bioactive inflammatory eicosanoids. However, adoptive transfer of diabetes was not prevented when mice were administered FKGK18-pretreated T cells or when FKGK18 administration was initiated with T-cell transfer. The present observations suggest that iPLA2β-derived lipid signals modulate immune cell responses, raising the possibility that early inhibition of iPLA2β may be beneficial in ameliorating autoimmune destruction of β-cells and mitigating type 1 diabetes development.

SDF-1–CXCR4 differentially regulates autoimmune diabetogenic T cell adhesion through ROBO1–SLIT2 interactions in mice

We had previously reported that stromal cell-derived factor 1 (SDF-1) mediates chemorepulsion of diabetogenic T cell adhesion to islet microvascular endothelium through unknown mechanisms in NOD mice. Here we report that SDF-1-mediated chemorepulsion occurs through slit homologue (SLIT)2-roundabout, axon guidance receptor, homologue 1 (Drosophila) (ROBO1) interactions. C-X-C receptor (CXCR)4 and ROBO1 protein expression was measured in mouse and human T cells. Parallel plate flow chamber adhesion and detachment studies were performed to examine the molecular importance of ROBO1 and SLIT2 for SDF-1-mediated T cell chemorepulsion. Diabetogenic splenocyte transfer was performed in NOD/LtSz Rag1(-/-) mice to examine the effect of the SDF-1 mimetic CTCE-0214 on adoptive transfer of diabetes. CXCR4 and ROBO1 protein expression was elevated in diabetic NOD/ShiLtJ T cells over time and coincided with the onset of hyperglycaemia. CXCR4 and ROBO1 expression was also increased in human type 1 diabetic T cells, with ROBO1 expression maximal at less than 1 year post diagnosis. Cell detachment studies revealed that immunoneutralisation of ROBO1 prevented SDF-1-mediated chemorepulsion of NOD T cell firm adhesion to TNFα-stimulated islet endothelial cells. SDF-1 increased NOD T cell adhesion to recombinant adhesion molecules, a phenomenon that was reversed by recombinant SLIT2. Finally, we found that an SDF-1 peptide mimetic prevented NOD T cell adhesion in vitro and significantly delayed adoptive transfer of autoimmune diabetes in vivo. These data reveal a novel molecular pathway, which regulates diabetogenic T cell recruitment and may be useful in modulating autoimmune diabetes.

Induction of tolerance in the NOD mouse by WE14, a peptide of Chromogranin A (167.14)

Abstract Type 1 diabetes (T1D) is a chronic autoimmune disease mediated by autoreactive T cells. We recently reported that a new autoantigen, chromogranin A (ChgA), is targeted by diabetogenic T cell clones in the NOD mouse (Stadinski, Delong et al, Nat Immunol 2010). To investigate the induction of antigen-specific tolerance by WE14, a natural proteolytic cleavage product of ChgA, we tested whether this peptide could protect against adoptive transfer of diabetes in the NOD mouse model. Our data indicate that the production of inflammatory cytokines is less in both CD4 and CD8 T cells from NOD mice after WE14 immunization. Furthermore, co-transfer of spleen cells from NOD mice immunized with WE14 delayed disease transfer by spleen cells from diabetic NOD mice. We also demonstrated that when the WE14 peptide is coupled to antigen-presenting cells (APCs) by a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI), transfer of disease into NOD recipients by the pathogenic Th1 T cell clone BDC-2.5 was totally prevented. These results suggest that WE14 may be a tolerogenic peptide in T1D. We are currently investigating the mechanism of this antigen-specific tolerance through analysis of T cells from the pancreas after adoptive transfer.

Effects of Autoimmunity and Immune Therapy on β-Cell Turnover in Type 1 Diabetes

beta-Cell mass can expand in response to demand: during pregnancy, in the setting of insulin resistance, or after pancreatectomy. It is not known whether similar beta-cell hyperplasia occurs following immune therapy of autoimmune diabetes, but the clinical remission soon after diagnosis and the results of recent immune therapy studies suggest that beta-cell recovery is possible. We studied changes in beta-cell replication, mass, and apoptosis in NOD mice during progression to overt diabetes and following immune therapy with anti-CD3 monoclonal antibodies (mAbs) or immune regulatory T-cells (Tregs). beta-Cell replication increases in pre-diabetic mice, after adoptive transfer of diabetes with increasing islet inflammation but before an increase in blood glucose concentration or a significant decrease in beta-cell mass. The pathogenic cells are responsible for increasing beta-cell replication because replication was reduced during diabetes remission induced by anti-CD3 mAb or Tregs. beta-Cell replication stimulated by the initial inflammatory infiltrate results in increased production of new beta-cells after immune therapy and increased beta-cell area, but the majority of this increased beta-cell area represents regranulated beta-cells rather than newly produced cells. We conclude that beta-cell replication is closely linked to the islet inflammatory process. A significant proportion of degranulated beta-cells remain, at the time of diagnosis of diabetes, that can recover after metabolic correction of hyperglycemia. Correction of the beta-cell loss in type 1 diabetes will, therefore, require strategies that target both the immunologic and cellular mechanisms that destroy and maintain beta-cell mass.

Peptide p277 of HSP60 signals T cells: inhibition of inflammatory chemotaxis

Peptide p277 is a 24-amino acid fragment of the heat shock protein 60 molecule, first discovered to be an antigen for diabetogenic T-cell clones in non-obese diabetic (NOD) mice. Therapeutic vaccination with p277 can arrest the spontaneous diabetogenic process both in NOD mice and in humans associated with a T(h)1 to T(h)2 cytokine shift specific for the autoimmune T cells. We now report that p277 can directly signal human T cells via innate toll-like receptor (TLR)-2, leading to up-regulation of integrin-mediated adhesion to fibronectin, and inhibition of chemotaxis to the chemokine SDF-1alpha in vitro. Resting CD45RA(+) T cells responded to lower concentrations of p277 than resting CD45RO(+) T cells, but activation of CD45RO(+) T cells greatly increased their sensitivity to p277. Mouse T cells, but not macrophages, were also sensitive to the innate effects of peptide p277, and adoptive transfer of diabetes by splenic T cells from NOD mice could be inhibited by p277 treatment before transfer. Thus, T cells do respond innately to p277, and signaling by soluble p277 through TLR2 could contribute to the treatment of type 1 diabetes; p277 may stop the destruction of beta cells by signaling in concert both innate and adaptive receptors on T cells.

Lymphopenia‐driven CD8+ T cells are resistant to antigen‐induced tolerance in NOD.scid mice

T cells undergoing lymphopenia-driven proliferation acquire effector and memory properties that can be pathogenic. Indeed, generalized lymphopenia is associated with a variety of autoimmune diseases such as type 1 diabetes. The current study was carried out to determine how CD8(+) T cells undergoing acute lymphopenic expansion respond to antigen under tolerizing conditions in vivo. Adoptive transfer of diabetes by TCR-transgenic CD8(+) T cells was enhanced following treatment of NOD. scid recipients with a high dose of soluble peptide. Furthermore, whereas TCR-transgenic CD8(+) T cells underwent clonal deletion and failed to differentiate into CTL in peptide-treated lymphoreplete recipient mice, TCR-transgenic CD8(+) T cells in a lymphopenic environment were resistant to clonal deletion, and CTL differentiation was enhanced by a high dose of soluble peptide. Moreover, peptide treatment had distinct effects on expression of the anti-apoptotic protein Bcl-X(L) in TCR-transgenic CD8(+) T cells under lymphopenic versus lymphoreplete conditions. These results demonstrate that CD8(+) T cells undergoing lymphopenia-driven expansion in NOD. scid recipients are resistant to antigen-induced tolerance, and readily differentiate into CTL upon stimulation with a high dose of soluble peptide.

Expression of the B7.1 costimulatory molecule on pancreatic beta cells abrogates the requirement for CD4 T cells in the development of type 1 diabetes.

Although HLA-DQ8 has been implicated as a key determinant of genetic susceptibility to human type 1 diabetes, spontaneous diabetes has been observed in HLA-DQ8 transgenic mice that lack expression of murine MHC class II molecules (mII(-/-)) only when the potent costimulatory molecule, B7.1, is transgenically expressed on pancreatic beta cells. To study the contribution of HLA-DQ8 to the development of diabetes in this model, we crossed RIP-B7.1mII(-/-) mice with a set of transgenic mouse lines that differed in their HLA-DQ8 expression patterns on APC subpopulations, in particular dendritic cells and cortical thymic epithelial cells. Surprisingly, we found that even in the absence of HLA-DQ8 and CD4 T cells, a substantial fraction of the RIP-B7.1mII(-/-) mice developed diabetes. This disease process was remarkable for not only showing insulitis, but also inflammatory destruction of the exocrine pancreas with diffusely up-regulated expression of MHC class I and ICAM-1 molecules. Expression of HLA-DQ8 markedly increased the kinetics and frequency of diabetes, with the most severe disease in the lines with the highest levels of HLA-DQ8 on cortical thymic epithelial cells and the largest numbers of CD4 T cells. However, the adoptive transfer of diabetes was not HLA-DQ8-dependent and disease could be rapidly induced with purified CD8 T cells alone. Expression of B7.1 in the target tissue can thus dramatically alter the cellular and molecular requirements for the development of autoimmunity.

Regulation of autoimmune diabetes by complete Freund's adjuvant is mediated by NK cells.

Autoimmune (type 1) diabetes results from a loss of beta cells that is mediated by self-reactive T cells. Previous studies have shown that a single injection of CFA prevents diabetes in nonobese diabetic (NOD) mice, but the mechanism(s) of protection remain unknown. We show here that NOD mice immunized with CFA have a markedly reduced incidence of diabetes and that this reduced incidence is associated with a decrease in the number of beta cell-specific, autoreactive CTL. In addition, the adoptive transfer of diabetes into syngeneic NOD/SCID recipients was prevented by CFA immunization, and the protective effects of CFA were lost when cells expressing the NK cell marker, asialo GM1, were removed from both donor cells and recipient mice. Returning a population of CD3-DX5+ cells to the adoptive transfer restored the protective effects of CFA. Therefore, NK cells mediate the protective effects of CFA possibly through the down-regulation of autoreactive CTL and stimulation of NK cells represents a novel approach to the prevention of autoimmune diabetes.

Maternal immunization with a soluble TCR-Ig chimeric protein: long term, V beta-8 family-specific suppression of T cells by maternally transferred antibodies.

Maternal transfer of TCR clonotypic Ab protected young NOD mice against the adoptive transfer of diabetes by the BDC 2.5 T cell clone. The effect of maternal anti-TCR Vbeta-8 Ab on T cell development and function has now been investigated. SJL/J mice, which lack TCR Vbeta-8, were immunized with soluble, chimeric D10 TCR-IgG1 containing Vbeta-8.2. The (SJL/J x AKR/J) F1 offspring of immunized female SJL/J mice were severely depleted of peripheral T cells bearing Vbeta-8 until 11 to 17 wk of age. The loss of Vbeta-8 expression did not appear to be due to modulation of cell surface TCR. Since the Vbeta-8+ T cell population was unperturbed in the (AKR/J x SJL/J) F1 offspring of D10 TCR-IgG1-immunized AKR/J mothers making D10 clonotypic Ab, the effect was immunologically specific. The deletion of Vbeta-8+ T cells had functional consequences. In the in vitro response to the superantigen, staphylococcal enterotoxin B, the usually observed participation of Vbeta-8.2+ T cells was largely suppressed, whereas the recruitment of Vbeta-3+ T cells remained unaltered. In control mice, T cell responses to the 134- to 146-residue peptide of conalbumin (pCA(134-146)) were biased toward use of Valpha-2/Vbeta-8.2 TCR. In D10 TCR-IgG1 maternally immunized (SJL x AKR/J) F1 mice, the T cell responses to pCA(134-146) were suppressed, and T cell lines derived from these in vitro were devoid of Vbeta-8.2 expression. With an increased understanding of TCR V gene usage in autoimmune diseases, similar strategies for the depletion of autoreactive T cells may become feasible in humans.

Inhibition of diabetes by an insulin-reactive CD4 T-cell clone in the nonobese diabetic mouse.

A cloned Th1 cell line was isolated from pancreatic lymph nodes of NOD mice that carries a T-cell receptor encoding Vbeta14 and proliferates in response to NOD islets, islet supernatant, and crystalline bovine and rat insulin, specifically to a B-chain peptide bound to IA(g7). The response to islet supernatant was reduced by 75% by anti-insulin antibody treatment. The insulin-reactive clone reduced insulitis and totally blocked the development of spontaneous diabetes in NOD mice (n = 8) as well as the adoptive transfer of diabetes into irradiated NOD mice following the injection of splenocytes from diabetic mice (n = 13). Trafficking of the adoptively transferred cells was assessed by labeling the clone or diabetic splenocytes with a fluorescent marker (DiI). The labeled clone was detected in the islet periphery, whereas labeled splenocytes alone invaded the islets by 3 days. In contrast, the protective clone dramatically delayed and reduced the number of labeled diabetic splenocytes infiltrating the islet, although their appearance in the spleen was unaffected. In vitro, the clone as well as supernatant derived from the clone blocked the proliferation of diabetic NOD splenocytes to islets. This inhibitory effect was diminished by anti-transforming growth factor-beta. In conclusion, an insulin-specific Th1 cell was isolated from NOD mice that traffics to the islet and prevents the spontaneous development and the adoptive transfer of diabetes. It appears to act locally by releasing transforming growth factor-beta and/or other factors that inhibit homing to and/or proliferation of diabetic splenocytes within the islet. These findings may provide insights into and suggest mechanisms for the protective effects of insulin therapy against diabetes.

Immunization with soluble BDC 2.5 T cell receptor-immunoglobulin chimeric protein:antibody specificity and protection of nonobese diabetic mice against adoptive transfer of diabetes by maternal immunization.

The BDC 2.5 T cell clone is specific for pancreatic beta-cell antigen presented by I-Ag7, and greatly accelerates diabetes when injected into 10-21-d-old nonobese diabetic (NOD) mice. The BDC 2.5 T cell receptor (TCR) has been solubilized as a TCR-IgG1 chimeric protein. All NOD mice immunized against BDC 2.5 TCR-IgG1 produced antibodies recognizing TCR C alpha/C beta epitopes that were inaccessible on the T cell surface. 56% of the mice produced antibodies against the BDC 2.5 clonotype that specifically blocked antigen activation of BDC 2.5 cells. We have used the adoptive transfer model of diabetes to demonstrate that maternal immunization with soluble TCR protects young mice from diabetes induced by the BDC 2.5 T cell clone.

A major histocompatibility complex class II restriction for BioBreeding/Worcester diabetes-inducing T cells.

Inbred diabetes-prone (DP) BioBreeding/Worcester (BB/Wor) (RT1u) rats develop spontaneous autoimmune diabetes, which, like human insulin-dependent diabetes mellitus, is mediated by autoreactive T lymphocytes. Breeding studies have shown an absolute requirement for at least one copy of the major histocompatibility complex (MHC) RT1u haplotype for spontaneous diabetes expression. Concanavalin A-activated spleen cells from acutely diabetic DP rats adoptively transfer diabetes only to recipients that express at least one RT1u haplotype. To investigate the basis for the MHC requirement in BB/Wor autoimmunity, diabetes-inducing T cell lines were derived from the spleens of acutely diabetic DP rats. Upon activation in vitro with islet cells, the T cell lines adoptively transfer insulitis and diabetes into young DP recipients and non-diabetes-prone RT1 congenic rat strains that are class IIu. Recipients that are RT1u at only the class I A or C locus, but not at the class II B/D loci, do not develop diabetes after T cell transfer. The adoptive transfer of diabetes by Concanavalin A-activated diabetic DP spleen cells also requires that donor and recipient share class II B/Du gene products. Furthermore, the adoptive transfer of diabetes into MHC class IIu congenic rats is independent of the class I haplotype; i.e., it occurs in the presence of class I Aa Cu or Au Ca gene products. BB/Wor T cells can be activated in vitro for the transfer of diabetes with islet cell antigens and class II-positive but not class IIu-negative antigen-presenting cells. The inductive phase of BB diabetes is therefore MHC class II restricted, and this appears to operate at the level of interaction between inducing T cells and class IIu antigen-presenting cells. These results may explain the well-documented, but not yet understood, MHC class II genetic contribution to insulin-dependent diabetes mellitus pathogenesis, and they may facilitate the development of protocols designed to prevent diabetes onset in susceptible individuals.

Pancreatic Lymph Nodes are Early Targets of T Cells during Adoptive Transfer of Diabetes in NOD Mice

The circulation pathway of diabetogenic T lymphocytes prior to insulitis was investigated using adoptive transfer of diabetes in the non-obese diabetic (NOD) mouse model. Transferred T cells were distinguished from recipient T cells using two strains of mice congenic at the Thy1 locus. They were monitored in the pancreas and in several lymphoid organs including thymus, spleen, and lymph nodes from pancreatic, mesenteric, axillary, inguinal and lomboaortic areas, from Day 0 to Day 15 after the adoptive lymphocytic transfer. Immunohistochemical studies showed that at Day 2 post-transfer the pancreatic lymph nodes (PLN) and to a lesser extent the spleen, are the first two organs to be infiltrated. The amount of T cells of donor origin using quantitative flow cytometric analysis was 4% and 2.6% respectively. This percentage increased to 19% in the PLN at Day 15 and did not exceed 7% in the spleen. Analysis of the expression of IL-2 receptor present at the surface of activated T lymphocytes showed that 73% of donor T cells were activated in the PLN within 3 days post-transfer in contrast to 0% in the spleen. The accumulation and activation of T cells in the PLN may imply a role of these lymphoid organs in harbouring the diabetogenic T cells during the early steps of the disease.

Adoptive Transfer of Autoimmune Diabetes Mellitus to Athymic Rats: Synergy of CD4+ and CD8+ T Cells and Prevention by RT6+ T Cells

We describe the induction and prevention of autoimmune insulin dependent diabetes mellitus (IDDM), and its pathological substrate, insulitis, in congenitally athymic nude rats following injections of major histocompatibility complex (MHC) compatible lymph node T cells. The cells capable of adoptive transfer of autoimmunity were obtained from diabetes resistant (DR) BB rats that had been rendered hyperglycemic by in vivo depletion of the RT6+ regulatory T cell subset. We first established that our adoptive transfer assay system is cell dose- and time dependent and therefore amenable to quantitative analysis. It was also observed that both CD4+ and CD8+ T cells are required for efficient transfer of autoimmunity. The data indicate that, as in the NOD mouse, a synergistic interaction between CD4+ and CD8+ T cells is important for beta cell destruction. Finally, we demonstrated that the admixture of equal numbers of lymph node T cells, 60% of which were RT6+, from intact, non-diabetic DR rats prevented the adoptive transfer of IDDM mediated by diabetogenic T cells from RT6-depleted DR-BB rats. We conclude that an equilibrium between autoreactive and regulatory cells determines the expression of autoimmunity in the DR-BB rat and in the adoptive transfer of diabetes in quantitative analytical systems.

Complete Freund's adjuvant-induced T cells prevent the development and adoptive transfer of diabetes in nonobese diabetic mice.

Insulin-dependent diabetes mellitus is an autoimmune disease that is characterized by the destruction of insulin-producing beta cells in the islet of Langerhans. We have recently reported that the induction of the disease in nonobese diabetic (NOD) mice can be prevented by a single injection of CFA. In this study, we have explored the cellular basis and the time course of the disease protection. Since CFA contains a mycobacterial cell wall that has adjuvant property, we investigated the protective role of mycobacteria in young NOD mice. Mice injected with Mycobacterium tuberculosis or Mycobacterium bovis (BCG vaccine) at 4 wk of age were also found to be protected from diabetes. We have found that complete protection from diabetes is only achieved by administration of CFA between 4 and 10 wk of age. Draining lymph node cells or spleen cells from CFA-treated NOD mice transfer the protection. Adoptive transfer of spleen cells from CFA-treated mice with spleen cells from acutely diabetic mice delayed the induction of disease into irradiated recipient mice. CFA-treated old NOD mice were also resistant to passive transfer of disease by spleen cells from acutely diabetic mice. Depletion of the Thy 1.2+ cells or CD4(+)-bearing T cells abrogated the protection. However, disease can be induced in the protected mice by cyclophosphamide treatment. We also found that thymocytes from NOD mice responded only weakly to mitogen Con A. CFA treatment, however, restored the ability of these cells to respond to Con A. Finally, our results suggest that T cells induced after CFA treatment of NOD mice prevent both the induction and effector phases of the disease.

Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors.

Precise definition of the role of both CD4 and CD8 T-cell subsets from NOD mice in the adoptive transfer of diabetes has been complicated by the possibility that endogenous T-cells may be recruited. Two newly created NOD congenic stocks, NOD.NON-Thy-1a and NOD/LtSz-scid, have been used as T-cell donors and recipients, respectively, to eliminate contributions from endogenous T-cells and thus to define the requirement for transferred T-cell subsets as a function of underlying diabetes development in the NOD donor. Total T-cells and T-cell subsets prepared from either prediabetic or diabetic NOD.NON-Thy-1a donors were adoptively transferred into 6-wk-old NOD-scid/scid recipients that were monitored for diabetes development. Both flow cytometric and histological analysis of recipient spleen and pancreas after adoptive transfer showed lymphocytes of donor (Thy1.1+) origin exclusively. Total T-cell and enriched CD4+ T-cell preparations from both diabetic and young prediabetic donors transferred diabetes to NOD-scid/scid recipients. However, the mean time to diabetes onset was doubled when CD4+ lymphocytes were isolated from prediabetic versus diabetic donors, and these transfers were complicated by the generation of small but significant numbers of CD8+ cells over time. Enriched CD8+ populations alone were unable to transfer disease. More rigorous exclusion of CD8+ cells by means of anti-CD8 MoAb treatment in vivo of the recipients of enriched CD4+ cells demonstrated a significant difference in the diabetogenic potency of CD4+ lymphocytes from diabetic versus nondiabetic donors. Diabetes was adoptively transferred to 58% of the recipients of enriched CD4+ lymphocytes from diabetic donors. In contrast, none of the recipients of enriched CD4+ lymphocytes from young prediabetic donors developed diabetes after MoAb treatment in vivo. The ability of a T-cell population to produce severe insulitis and sialitis in NOD-scid/scid recipients of T-cells closely paralleled its ability to induce diabetes. In an effort to suppress insulitis by suppression of macrophage migration to the islets, NOD-scid/scid mice were treated with silica in conjunction with adoptive transfer of T-cells from diabetic donors. Chronic silica treatment failed to deplete tissue macrophages and did not prevent diabetes development after transfer of unfractionated T-cells. Evidence is discussed indicating that the age-associated differences in ability of CD4+ T-cells to adoptively transfer diabetes in the absence of the CD8+ T-cells subset is a function of prior, chronic exposure of the CD4+ lymphocytes to beta-cell antigens in the donor.(ABSTRACT TRUNCATED AT 400 WORDS)

Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors

Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors

Staphylococcal Enterotoxin-Activated Spleen Cells Passively Transfer Diabetes in BB/Wor Rat

BB/Wor rats develop spontaneous autoimmune diabetes similar to human insulin-dependent diabetes mellitus. A T-cell-mediated pathogenesis for BB/Wor diabetes is indicated because disease is prevented by neonatal or adult thymectomy and treatment of diabetes-prone rats with monoclonal antibodies directed against CD5 or CD8 T-cell surface markers. Disease can be adoptively transferred with injections of concanavalin A-activated spleen cells from either acutely diabetic or RT6.1 T-cell-depleted diabetes-resistant BB/Wor rats. We used microbial superantigens to stimulate spleen cells from RT6.1 T-cell-depleted diabetes-resistant rats and demonstrated that such cells activated with staphylococcal enterotoxins (SEs) can also transfer diabetes. The diabetogenic effector T cells are readily activated by SEA, SEC3, and SEE, whereas SEB- and SEC2-activated cells are far less effective in the adoptive transfer of diabetes. These results demonstrate that microbial superantigens are capable of activating self-reactive and diabetes-inducing T cells in vitro in the BB/Wor rat. Ubiquitous microorganisms may be the environmental trigger for autoimmunity in susceptible individuals.