Amy Dave

Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFNγ

Aims/hypothesisSubcutaneous immunisation with the 9–23 amino acid region of the insulin B chain (B:9-23) in incomplete Freund’s adjuvant (IFA) can protect the majority of 4- to 6-week-old prediabetic NOD mice and is currently in clinical trials. Here we analysed the effect of B:9-23/IFA immunisation at later stages of the disease and the underlying mechanisms.MethodsNOD mice were immunised once s.c. with B:9-23/IFA at 5 or 9 weeks of age, or when blood glucose reached 10 mmol/l or higher. Diabetes incidence was followed in addition to variables such as regulatory T cell (Treg) induction, cytokine production (analysed by Elispot) and emergence of pathogenic CD8+/NRP-V7+ cells.ResultsA single B:9-23/IFA immunisation protected the majority of NOD mice at advanced stages of insulitis, but not after blood glucose reached 13.9 mmol/l. It increased Treg numbers and lost its protective effect after IFNγ or IL-10 neutralisation, but not in the absence of IL-4. CD4+CD25+ and to a lesser extent IFNγ-producing cells from mice protected by B:9-23/IFA induced tolerance upon transfer into new NOD animals, indicating that a dominant Treg-mediated effect was operational. Reduced numbers of CD8+/NRP-V7+ memory T cells coincided with protection from the disease.Conclusions/interpretationProtection from diabetes after B:9-23/IFA immunisation cannot be achieved once diabetes is fully established, but can be achieved at most prediabetic stages of the disease. Protection is mediated by Tregs that require IFNγ and IL-10. These findings should provide important guidance for ongoing human trials, especially for the development of suitable T cell biomarkers.

Nasal cardiac myosin peptide treatment and OX40 blockade protect mice from acute and chronic virally-induced myocarditis

Myocarditis poses a severe health problem, can lead to dilated cardiomyopathy (DCM) and death, and is thought to be triggered by infections. Enteroviruses such as Coxsackie virus B3 (CVB3) have been implicated as a culprit, since they can cause acute and chronic heart disease in susceptible mice. CVB was detected in human cardiac myocytes in some cases, whereas acute CVB infection was thought to have caused death. Here we studied, whether nasal administration of cardiac myosin (CM) major histocompatibility class (MHC) II peptides CM₉₄₇-₉₆₀ and CM₇₃₅-₇₄₇ and OX40 blockade would be able to ameliorate immunopathology and heart disease in BALB/C mice infected with CVB3. We found that nasal CM-peptide prophylactic treatment significantly reduced myocarditis and mortality by enhancing Treg and IL-10 induction and that blockade of OX40 signaling could reduce heart inflammation when administered late during pathogenesis. Altogether, these results chart the way for novel prevention and intervention strategies for viral myocarditis.

Virtual Optimization of Nasal Insulin Therapy Predicts Immunization Frequency to Be Crucial for Diabetes Protection

OBJECTIVE Development of antigen-specific strategies to treat or prevent type 1 diabetes has been slow and difficult because of the lack of experimental tools and defined biomarkers that account for the underlying therapeutic mechanisms. RESEARCH DESIGN AND METHODS The type 1 diabetes PhysioLab platform, a large-scale mathematical model of disease pathogenesis in the nonobese diabetic (NOD) mouse, was used to investigate the possible mechanisms underlying the efficacy of nasal insulin B:9-23 peptide therapy. The experimental aim was to evaluate the impact of dose, frequency of administration, and age at treatment on Treg induction and optimal therapeutic outcome. RESULTS In virtual NOD mice, treatment efficacy was predicted to depend primarily on the immunization frequency and stage of the disease and to a lesser extent on the dose. Whereas low-frequency immunization protected from diabetes atrributed to Treg and interleukin (IL)-10 induction in the pancreas 1–2 weeks after treatment, high-frequency immunization failed. These predictions were confirmed with wet-lab approaches, where only low-frequency immunization started at an early disease stage in the NOD mouse resulted in significant protection from diabetes by inducing IL-10 and Treg. CONCLUSIONS Here, the advantage of applying computer modeling in optimizing the therapeutic efficacy of nasal insulin immunotherapy was confirmed. In silico modeling was able to streamline the experimental design and to identify the particular time frame at which biomarkers associated with protection in live NODs were induced. These results support the development and application of humanized platforms for the design of clinical trials (i.e., for the ongoing nasal insulin prevention studies).

Increased Memory Conversion of Naïve CD8 T Cells Activated during Late Phases of Acute Virus Infection Due to Decreased Cumulative Antigen Exposure

Background Memory CD8 T cells form an essential part of protective immunity against viral infections. Antigenic load, costimulation, CD4-help, cytokines and chemokines fluctuate during the course of an antiviral immune response thus affecting CD8 T cell activation and memory conversion. Methodology/Principal Findings In the present study, naïve TCR transgenic LCMV-specific P14 CD8 T cells engaged at a late stage during the acute antiviral LCMV response showed reduced expansion kinetics but greater memory conversion in the spleen. Such late activated cells displayed a memory precursor effector phenotype already at the peak of the systemic antiviral response, suggesting that the environment determined their fate during antigen encounter. In the spleen, the majority of late transferred cells exhibited a central memory phenotype compared to the effector memory displayed by the early transferred cells. Increasing the inflammatory response by exogenous administration of IFNγ, PolyI:C or CpG did not affect memory conversion in the late transferred group, suggesting that the diverging antigen load early versus later during acute infection had determined their fate. In agreement, reduction in the LCMV antigenic load after ribavirin treatment enhanced the contribution of early transferred cells to the long lasting memory pool. Conclusions/Significance Our results show that naïve CD8 cells, exposed to reduced duration or concentration of antigen during viral infection convert into memory more efficiently, an observation that could have significant implications for vaccine design.

Following the Fate of One Insulin-Reactive CD4 T cell Conversion Into Teffs and Tregs in the Periphery Controls Diabetes in NOD Mice

In diabetic patients and susceptible mice, insulin is a targeted autoantigen. Insulin B chain 9-23 (B:9-23) autoreactive CD4 T cells are key for initiating autoimmune diabetes in NOD mice; however, little is known regarding their origin and function. To this end, B:9-23–specific, BDC12-4.1 T-cell receptor (TCR) transgenic (Tg) mice were studied, of which, despite expressing a single TCR on the recombination activating gene–deficient background, only a fraction develops diabetes in an asynchronous manner. BDC12-4.1 CD4 T cells convert into effector (Teff) and Foxp3+-expressing adaptive regulatory T cells (aTregs) soon after leaving the thymus as a result of antigen recognition and homeostatic proliferation. The generation of aTreg causes the heterogeneous diabetes onset, since crossing onto the scurfy (Foxp3) mutation, BDC12-4.1 TCR Tg mice develop accelerated and fully penetrant diabetes. Similarly, adoptive transfer and bone marrow transplantation experiments showed differential diabetes kinetics based on Foxp3+ aTreg’s presence in the BDC12-4.1 donors. A single-specificity, insulin-reactive TCR escapes thymic deletion and simultaneously converts into aTreg and Teff, establishing an equilibrium that determines diabetes penetrance. These results are of particular importance for understanding disease pathogenesis. They suggest that once central tolerance is bypassed, autoreactive cells arriving in the periphery do not by default follow solely a pathogenic fate upon activation.

Regulatory T cells control diabetes without compromising acute anti-viral defense

While previous reports have demonstrated the efficacy of regulatory T cell therapy in the prevention of diabetes, systemic immunocompromise and Treg instability remain key safety concerns. Here we examined the influence of induced Treg (iTreg) cell therapy on anti-viral host defense and autoimmune T cell responses during acute viral infection in a murine model of autoimmune diabetes. Protective transfers of iTregs maintained IL-10 expression, expanded in vivo and controlled diabetes, despite losing FoxP3 expression. Adoptive transfer of iTregs affected neither the primary anti-viral CD8 T cell response nor viral clearance, although a significant and sustained suppression of CD4 T cell responses was observed. Following acute viral clearance, iTregs transferred early suppressed both CD4 and CD8 T cell responses, which resulted in the reversion of diabetes. These observations indicate that iTregs suppress local autoimmune processes while preserving the immunocompetent hosts ability to combat acute viral infection.

CD103 is dispensable for anti-viral immunity and autoimmunity in a mouse model of virally-induced autoimmune diabetes

Recent studies suggest a beneficial role for blocking CD103 signaling in preventing islet allograft rejection and thus Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, antibody blockade approaches generally raise anti-microbial safety issues, necessitating additional studies to address the possible adverse effects of antibody therapy. Here we report that CD103 had no significant impact on the development of primary and memory CD8(+) or CD4(+) responses after acute lymphocytic choriomeningitis virus (LCMV) infection. In addition, CD103 was found to be dispensable for T1D progression in a rapid, CD8-mediated virally-induced T1D model (the rat insulin promoter [RIP]-LCMV), suggesting that its previous efficacy in the NOD mouse model may not be related to its effect on the generation, memory conversion and/or effector function of CD8(+) or CD4(+) T cells. While the data does not preclude a role for CD103 in T1D in its entirety, the current study does provide much evidence to suggest that CD103 blockade may prove to be a safe intervention for autoimmunity and allo-transplantation. While in cases of rapid microbial (CD8)-driven T1D CD103 antibody blockade may not limit disease progression or severity, in mucosally-driven cases of T1D anti-CD103 antibody treatment may provide a new and safe therapeutic avenue.

Beta-cell-specific production of IL6 in conjunction with a mainly intracellular but not mainly surface viral protein causes diabetes

Inflammatory mechanisms play a key role in the pathogenesis of type 1 and type 2 diabetes. IL6, a pleiotropic cytokine with impact on immune and non-immune cell types, has been proposed to be involved in the events causing both forms of diabetes and to play a key role in experimental insulin-dependent diabetes development. The aim of this study was to investigate how beta-cell specific overexpression of IL-6 influences diabetes development. We developed two lines of rat insulin promoter (RIP)-lymphocytic choriomeningitis virus (LCMV) mice that also co-express IL6 in their beta-cells. Expression of the viral nucleoprotein (NP), which has a predominantly intracellular localization, together with IL6 led to hyperglycemia, which was associated with a loss of GLUT-2 expression in the pancreatic beta-cells and infiltration of CD11b(+) cells, but not T cells, in the pancreas. In contrast, overexpression of the LCMV glycoprotein (GP), which can localize to the surface, with IL-6 did not lead to spontaneous diabetes, but accelerated virus-induced diabetes by increasing autoantigen-specific CD8(+) T cell responses and reducing the regulatory T cell fraction, leading to increased pancreatic infiltration by CD4(+) and CD8(+) T cells as well as CD11b(+) and CD11c(+) cells. The production of IL-6 in beta-cells acts prodiabetic, underscoring the potential benefit of targeting IL6 in diabetes.

BDC12-4.1 T-cell receptor transgenic insulin-specific CD4 T cells are resistant to in vitro differentiation into functional Foxp3+ T regulatory cells.

The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4+ T cells of the BDC12-4.1 clone to convert into Foxp3+ iTreg cells. We found that in vitro polarization toward Foxp3+ iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3+ BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4+ T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3+ cell phenotype and its associated in vivo regulatory potential.

Minimal Effect of CD103 Expression on the Control of a Chronic Antiviral Immune Response

Impaired antiviral CD8 and CD4 T-cell responses are often associated with chronic viral infections. Cell-intrinsic as well as cell-extrinsic mechanisms are thought to dampen such responses, for example programmed death 1 receptor (PD-1) expression on T cells, and interleukin (IL)-10 production primarily by dendritic cells (DCs), have been shown to support viral persistence by suppressing immune responses. Here we demonstrate that CD103, an alpha E integrin necessary for T-cell homing and retention in the gut and other epithelia expressed by the majority of naïve CD8(+), and CD4(+)CD25(+) T cells and some DC subsets, is unnecessary for controlling T-cell responses during chronic lymphocytic choriomeningitis virus clone 13 (LCMV cl13) infection. T-cell analysis following viral infection showed that the primary as well as the memory CD8(+) and CD4(+) T-cell responses among CD103-sufficient and CD103-deficient mice were identical. In addition, no rescue of cytokine production by virus-specific T cells or alterations in viral titers in the absence of intrinsic CD103 expression was observed. Interestingly, CD103 levels on the effector CD8(+) T cells became reduced soon after virus infection, with a small proportion of cells co-expressing PD-1 and CD103. In contrast, although no substantial differences in the frequency and number of the CD4(+)CD25(+) cell population were seen, CD103 expression increased significantly over time in this population, correlating with viral persistence. Thus, a lack of CD103 expression does not affect functional impairment of effector T-cell responses during chronic viral infection.