GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes

Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion.     

B-Cell-Delivered Gene Therapy Induces Functional T Regulatory Cells and Leads to a Loss of Antigen-Specific Effector Cells

Previous reports have shown that B-cell-mediated gene therapy can induce tolerance in several animal models for autoimmune diseases and inhibitory antibody formation in hemophilia A mice. We know from our previous work that the induction of tolerance following B-cell therapy is dependent upon CD25⁺ regulatory T cells (Tregs). To extend these studies and identify the effects of this gene therapy protocol on the target CD4 T cells, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using carboxyfluorescein succinimidyl ester (CFSE) dilution as a measure of T-cell responsiveness to FVIII, we show that CD25⁺ Tregs from treated mice are more suppressive than those from control animals. To monitor the induction of antigen-specific Tregs, we repeated these studies in ovalbumin (OVA) peptide-specific DO11.10 T-cell receptor (TCR) transgenic mice. Tregs from DO11.10 mice treated with a tolerogenic OVA–Ig construct are better than polyclonal Tregs at suppressing the proliferation of responder cells stimulated with OVA peptide 323–339 (pOVA). Furthermore, we show that following B-cell therapy, there is an increase in antigen-specific FoxP3⁺ Tregs, and there is also a distinct decrease in antigen-specific CD4⁺ effector T cells. These changes in the lymphocyte population shift the balance away from effector function toward a tolerogenic phenotype.     

Nonviral Delivery of Small Interfering RNA Into Pancreas-associated Immune Cells Prevents Autoimmune Diabetes

The development of small interfering RNA (siRNA) for the treatment of human disorders has been often hampered by their low transfection efficiency in vivo. In order to overcome this major drawback, various in vivo siRNA transfection methods have been developed. However, their capacity to transfect immune or insulin-producing β-cells within the pancreas for the treatment of autoimmune diabetes remains undetermined. We found that lipid- or polyethylenimine-based delivery agents were efficient to address siRNA molecules within pancreas-associated antigen-presenting cells (APCs) (but not β-cells) and particularly a CD11b⁺ cell population comprising both CD11b⁺CD11c^neg macrophages and CD11b⁺CD11c⁺ dendritic cells. However, the route of administration and the carrier composition greatly affected the transfection efficacy. Therapeutically, we showed that early (starting at 6-week-old) short-course treatment with lipid/Alox15-specific siRNA complex promoted long-term protection from type 1 diabetes (T1D) in wild-type (WT) nonobese diabetic (NOD) mice. Alox15 downregulation in pancreas-associated CD11b⁺ cells significantly upregulated a variety of costimulatory molecules and particularly the programmed death 1 ligand 1 (PD-L1) pathway involved in tolerance induction. Concomitantly, we found that regulatory T cells were increased in the pancreas of lipid/Alox15 siRNA-treated NOD mice. Collectively, our data provide new insights into the development of siRNA-based therapeutics for T1D.     

CD4(+) T cells from glutamic acid decarboxylase (GAD)65-specific T cell receptor transgenic mice are not diabetogenic and can delay diabetes transfer

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286-300 (p286) of GAD65. These mice have GAD65-specific CD4(+) T cells, as shown by staining with an I-A(g7)(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon gamma, interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, and IL-10 when stimulated in vitro with GAD65 peptide 286-300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4(+) T cells, or p286-tetramer(+)CD4(+) Tcells, from GAD65 286-300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286-300-specific T cells have disease protective capacity and are not pathogenic.     

GAD65 autoantibody responses in Japanese latent autoimmune diabetes in adult patients

OBJECTIVE—To determine whether development of insulin requirement in patients with latent autoimmune diabetes in adults (LADA) is accompanied with the emergence of a type 1 diabetes–like autoimmune response.RESEARCH DESIGN AND METHODS—We correlated β-cell–specific autoimmunity reflected in autoantibodies to the 65-kDa isoform of GAD (GAD65) with insulin requirement. We determined GAD65Ab epitope specificities in type 1 diabetic patients, LADA patients without insulin requirement (nonprogressed), and LADA patients that had developed insulin requirement (progressed).RESULTS—Recognition of a type 1 diabetes–specific GAD65Ab epitope was more pronounced in type 1 diabetic patients than in nonprogressed (P < 0.001) or progressed (P < 0.01) LADA patients, with no significant differences between the two LADA cohorts. These differences were particularly pronounced in samples with GAD65Ab titers <1,000 units/ml, with no differences in epitope specificities in samples with higher GAD65Ab titers. Disease duration (initial diabetes diagnosis until sample collection or development of insulin requirement) in nonprogressed and progressed LADA patients, respectively, was not correlated with epitope specificity, suggesting lack of epitope maturation. This was supported by epitope analyses of longitudinal samples from LADA patients during progression to insulin requirement.CONCLUSIONS—First, the GAD65Ab-specific autoimmune reaction in type 1 diabetic patients with low and moderate GAD65Ab titers differs from that in LADA patients, irrespective of insulin requirement. Second, the GAD65Ab-specific autoimmune response in LADA patients does not change after their initial diabetes diagnosis. Finally, LADA patients with high GAD65Ab titers resemble type 1 diabetic patients in their GAD65Ab epitope specificity.Latent autoimmune diabetes in adults (LADA) consists of a subgroup (∼10%) of adult patients initially diagnosed with type 2 diabetes, who show signs of β-cell autoimmunity and eventually develop insulin requirement (1,2). Signs of β-cell autoimmunity, such as the well-characterized insulin autoantibodies, glutamate decarboxylase (GAD65), and the tyrosine phosphatase–like protein insulinoma-associated protein-2, indicate significant damage of the β-cells and subsequent development of insulin requirement in these patients (1). While autoantibodies to insulin and insulinoma-associated protein-2 antibody (Ab) are inversely correlated with age at onset, GAD65Ab shows no, and in some studies even a positive, correlation with age at onset and is therefore a particularly attractive marker for autoimmune diabetes in the adult population (3,4). Moreover, GAD65Ab can be detected years after the clinical onset of the disease, indicating that these autoantibodies may be permanent markers for the autoimmune response (5,6).Notably, not all LADA patients progress to insulin requirement, raising the possibility that the autoimmune response in these patients resembles that in autoantibody-positive healthy individuals, with no significant risk for development of insulin requirement (7,8). A better understanding of the autoimmune response is necessary to predict insulin requirement in LADA patients, which is important to prevent escalation of blood glucose levels and subsequent complications.In previous studies, we have investigated the humoral immune response toward GAD65 as a reflection of islet cell destruction (9). It remains unclear whether the autoimmune response in LADA patients and type 1 diabetic patients differs or whether only the duration of the prodomal period distinguishes between the two groups (10). Therefore, we compared the GAD65-specific humoral autoimmune response in type 1 diabetic patients with that in LADA patients who had or had not progressed to insulin requirement.     

Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes

We previously demonstrated that a spontaneous Th1 response against glutamate decarboxylase (GAD65) arises in NOD mice at four weeks in age and subsequently T cell autoimmunity spreads both intramolecularly and intermolecularly. Induction of passive tolerance to GAD65, through inactivation of reactive T cells before the onset of autoimmunity, prevented determinant spreading and the development of insulin- dependent diabetes mellitus (IDDM). Here, we examined whether an alternative strategy, designed to induce active tolerance via the engagement of Th2 immune responses to GAD65, before the spontaneous onset of autoimmunity, could inhibit the cascade of Th1 responses that lead to IDDM. We observed that a single intranasal administration of GAD65 peptides to 2-3-wk-old NOD mice induced high levels of IgG1 antibodies to GAD65. GAD65 peptide treated mice displayed greatly reduced IFN gamma responses and increased IL-5 responses to GAD65, confirming the diversion of the spontaneous GAD65 Th1 response toward a Th2 phenotype. Consistent with the induction of an active tolerance mechanism, splenic CD4+ (but not CD8+) T cells from GAD65 peptide- treated mice, inhibited the adoptive transfer of IDDM to NOD-scid/scid mice. This active mechanism not only inhibited the development of proliferative T cell responses to GAD65, it also limited the expansion of autoreactive T cell responses to other beta cell antigens (i.e., determinant spreading). Finally, GAD65 peptide treatment reduced insulitis and long-term IDDM incidence. Collectively, these data suggest that the nasal administration of GAD65 peptides induces a Th2 cell response that inhibits the spontaneous development of autoreactive Th1 responses and the progression of beta cell autoimmunity in NOD mice.     

Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR⁺γδ T cells were expanded on CD19⁺ artificial antigen-presenting cells (aAPC), which resulted in >10⁹ CAR⁺γδ T cells from <10⁶ total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR⁺γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19⁺ tumor cell lines compared with CAR^negγδ T cells. CD19⁺ leukemia xenografts in mice were reduced with CAR⁺γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.     

Immunoregulatory mechanisms triggered by viral infections protect from type 1 diabetes in mice

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing β cells. Viral infections induce immune responses that can damage β cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on β cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death–1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8⁺ T cells expressing programmed cell death–1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4⁺CD25⁺ Tregs, which produced TGF-β and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4⁺CD25⁺ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.     

Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20–40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4⁺ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4⁺CD25⁺ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.     

GAD-reactive CD4+ Th1 cells induce diabetes in NOD/SCID mice.

Although glutamic acid decarboxylase (GAD) has been implicated in IDDM, there is no direct evidence showing GAD-reactive T cells are diabetogenic in vivo. To address this issue, 3-wk-old NOD mice received two injections of purified rat brain GAD; one mouse rapidly developed diabetes 3 wk later. Splenocytes from this mouse showed a proliferative response to purified GAD, and were used to generate a CD4+ T cell line, designated 5A, that expresses TCRs encoding Vbeta2 and Vbeta12. 5A T cells exhibit a MHC restricted proliferative response to purified GAD, as well as GAD65 peptide 524-543. After antigen-specific stimulation, 5A T cells secrete IFNgamma and TNFalpha/beta, but not IL-4. They are also cytotoxic against NOD-derived hybridoma cells (expressing I-Ag7) that were transfected with rat GAD65, but not nontransfected hybridoma cells. Adoptive transfer of 5A cells into NOD/SCID mice produced insulitis in all mice. Diabetes occurred in 83% of the mice. We conclude that GAD injection in young NOD mice may, in some cases, provoke diabetes due to the activation of diabetogenic T cells reactive to GAD65 peptides. Our data provide direct evidence that GAD65 autoimmunity may be a critical event in the pathogenesis of IDDM.     

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.     

Distinct Effects of IL-18 on the Engraftment and Function of Human Effector CD8+ T Cells and Regulatory T Cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8⁺ effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Rα was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.     

α/β–T Cell Receptor (TCR)+CD4−CD8− (NKT) Thymocytes Prevent Insulin-dependent Diabetes Mellitus in Nonobese Diabetic (NOD)/Lt Mice by the Influence of Interleukin (IL)-4 and/or IL-10

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in α/β-T cell receptor (TCR)⁺CD4⁻CD8⁻ NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 10⁶ CD4^−/lowCD8⁻ or CD4⁻CD8⁻ thymocytes from female (BALB/c × NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of “allogenic tolerance” or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When α/β-TCR⁺ and α/β-TCR⁻ subsets of CD4⁻CD8⁻ thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the α/β-TCR⁺ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti–IL-4 and anti–IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.     

T Helper 2 (Th2) T Cells Induce Acute Pancreatitis and Diabetes in Immune-compromised Nonobese Diabetic (NOD) Mice

Autoimmune diabetes is caused by the CD4⁺, T helper 1 (Th1) cell-mediated apoptosis of insulin-producing β cells. We have previously shown that Th2 T cells bearing the same T cell receptor (TCR) as the diabetogenic Th1 T cells invade islets in neonatal nonobese diabetic (NOD) mice but fail to cause disease. Moreover, when mixed in excess and cotransferred with Th1 T cells, Th2 T cells could not protect NOD neonates from Th1-mediated diabetes. We have now found, to our great surprise, the same Th2 T cells that produced a harmless insulitis in neonatal NOD mice produced intense and generalized pancreatitis and insulitis associated with islet cell necrosis, abscess formation, and subsequent diabetes when transferred into immunocompromised NOD.scid mice. These lesions resembled allergic inflamation and contained a large eosinophilic infiltrate. Moreover, the Th2-mediated destruction of islet cells was mediated by local interleukin-10 (IL-10) production but not by IL-4. These findings indicate that under certain conditions Th2 T cells may not produce a benign or protective insulitis but rather acute pathology and disease. Additionally, these results lead us to question the feasibility of Th2-based therapy in type I diabetes, especially in immunosuppressed recipients of islet cell transplants.     

Chimeric Antigen Receptors Combining 4-1BB and CD28 Signaling Domains Augment PI3kinase/AKT/Bcl-XL Activation and CD8+ T Cell�Cmediated Tumor Eradication

To enhance the strength of activation afforded by tumor antigen-specific receptors, we investigated the effect of adding combined CD28 and 4-1BB costimulatory signaling domains to a chimeric antigen receptor (CAR) specific for prostate-specific membrane antigen (PSMA). Having transferred receptors encompassing the CD28, 4-1BB, and/or CD3ζ cytoplasmic domains in primary human CD8⁺ T cells, we find that the P28BBz receptor, which includes all three signaling domains, is superior to receptors that only include one or two of these domains in promoting cytokine release, in vivo T-cell survival and tumor elimination following intravenous T-cell administration to tumor-bearing severe combined immunodeficient (SCID)/beige mice. Upon in vitro exposure to PSMA, the P28BBZ receptor-induced the strongest PI₃Kinase/Akt activation and Bcl-X_L expression, and the least apoptosis in transduced peripheral blood CD8⁺ T cells. These findings further support the concept of integrating optimized costimulatory properties into recombinant antigen receptors to augment the survival and function of genetically targeted T cells within the tumor microenvironment.     

Targeted Coating With Antigenic Peptide Renders Tumor Cells Susceptible to CD8+ T Cell-mediated Killing

The potency of immunotherapies targeting endogenous tumor antigens is hindered by immune tolerance. We created a therapeutic agent comprised of a tumor-homing module fused to a functional domain capable of selectively rendering tumor cells sensitive to foreign antigen-specific CD8⁺ T cell-mediated immune attack, and thereby, circumventing concerns for immune tolerance. The tumor-homing module is comprised of a single-chain variable fragment (scFv) that specifically binds to mesothelin (Meso), which is commonly overexpressed in human cancers, including ovarian tumors. The functional domain is comprised of the Fc portion of IgG2a protein and foreign immunogenic CD8⁺ T cell epitope flanked by furin cleavage sites (R), which can be recognized and cleaved by furin that is highly expressed in the tumor microenvironment. We show that our therapeutic protein specifically loaded antigenic epitope onto the surface of mesothelin-expressing tumor cells, rendering tumors susceptible to antigen-specific cytotoxic CD8⁺ T lymphocytes (CTL)-mediated killing in vitro and in vivo. Our findings have important implications for bypassing immune tolerance to enhance cancer immunotherapy.     

Overexpression of Natural Killer T Cells Protects Vα14-Jα281 Transgenic Nonobese Diabetic Mice against Diabetes

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of α/β double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) α chain Vα14-Jα281 characteristic of NK T cells. Precise identification of NK1.1⁺ T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1⁺ T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Vα14-Jα281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.     

Recombinant Vesicular Stomatitis Virus Transduction of Dendritic Cells Enhances Their Ability to Prime Innate and Adaptive Antitumor Immunity

Dendritic cell (DC)–based vaccines are a promising strategy for tumor immunotherapy due to their ability to activate both antigen-specific T-cell immunity and innate immune effector components, including natural killer (NK) cells. However, the optimal mode of antigen delivery and DC activation remains to be determined. Using M protein mutant vesicular stomatitis virus (ΔM51-VSV) as a gene-delivery vector, we demonstrate that a high level of transgene expression could be achieved in ~70% of DCs without affecting cell viability. Furthermore, ΔM51-VSV infection activated DCs to produce proinflammatory cytokines (interleukin-12, tumor necrosis factor-α, and interferon (IFN)α/β), and to display a mature phenotype (CD40^highCD86^high major histocompatibility complex (MHC II)^high). When delivered to mice bearing 10-day-old lung metastatic tumors, DCs infected with ΔM51-VSV encoding a tumor-associated antigen mediated significant control of tumor growth by engaging both NK and CD8⁺ T cells. Importantly, depletion of NK cells completely abrogated tumor destruction, indicating that NK cells play a critical role for this DC vaccine-induced therapeutic outcome. Our findings identify ΔM51-VSV as both an efficient gene-delivery vector and a maturation agent allowing DC vaccines to overcome immunosuppression in the tumor-bearing host.     

Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4⁺ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4⁺ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP⁺ LV-IL-10–transduced human CD4⁺ T (CD4^LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4^LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4^LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4⁺ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.     

IL-28B/IFN-��3 Drives Granzyme B Loading and Significantly Increases CTL Killing Activity in Macaques

Type III/λ interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-λ3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-λ3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-λ3 has significant influence on antigen-specific CD8⁺ T-cell function, especially in regards to cytotoxicity. Peripheral CD8⁺ T cells from animals that were administered IFN-λ3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8⁺ T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-λ3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-λ3 is a potent effector of the immune system with special emphasis on CD8⁺ T-cell killing functions which warrants further study as a possible immunoadjuvant.