Huub T. C. Kreuwel

Memory CD8+ T Cells Undergo Peripheral Tolerance

Memory T cells differ from naive T cells in that they respond more rapidly and in greater numbers. In addition, memory T cells are generally believed to be less susceptible to tolerance induction than naive T cells. In this study, we show that this is not the case. Using two different methods of tolerance induction, peptide-induced tolerance and crosstolerance, we present evidence that memory CD8(+) T cells are as susceptible to tolerance as naive cells. These results have a direct impact on manipulating T cell responses to self-antigens in order to improve immunotherapy of cancer and autoimmune diseases.

Defective CD8+ T Cell Peripheral Tolerance in Nonobese Diabetic Mice

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet β cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (KdHA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.

CD8+ T Cell Tolerance in Nonobese Diabetic Mice Is Restored by Insulin-Dependent Diabetes Resistance Alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.

The Apoptotic Pathway Contributing to the Deletion of Naive CD8 T Cells during the Induction of Peripheral Tolerance to a Cross-Presented Self-Antigen

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet β-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim−/− clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.

Self-tolerance and the composition of T cell repertoire.

T cell recognition of self-major histocompatibility complexpeptide complexes dictates the composition of the T cell receptor repertoire. Research projects in our laboratory deal with the mechanisms that regulate the composition of the repertoire specific for self-antigens and the defects that can result in autoimmunity. Two different types of disease models are under investigation: juvenile (type 1) diabetes and cancer. Both of these diseases are impacted by the presence of anti-self CD8 cells, yet in opposite ways. By understanding the mechanisms of peripheral tolerance and the reasons they fail in autoimmunity, we may learn how to prevent undersirable autoimmunity and how to encourage an autoimmune response when it is needed to eliminate tumor cells.

The Role of Fas–FasL in CD8+ T-Cell-Mediated Insulin-Dependent Diabetes Mellitus (IDDM)

In the past few years a number of studies have evaluated the contributions of different cytolytic pathways in the autoimmune destruction of pancreatic β cells, which results in insulin-dependent (type I) diabetes mellitus. Conflicting results continue to emerge regarding the role of Fas-mediated apoptosis in β-cell destruction. This is likely to reflect differences inherent to the model systems under investigation, as well as the pleiotropic nature of the genes that are involved in cytotoxicity. Despite these complications, it may be possible to reconcile some of these apparently conflicting results by considering that T-cell-mediated cytotoxicity can occur simultaneously by several mechanisms and that variables such as the cytokine milieu and the strength of the signal to the T cell received through the T-cell receptor complex may alter the relative contribution of each cytolytic pathway to β-cell destruction.

The virtual NOD mouse: applying predictive biosimulation to research in type 1 diabetes.

Abstract:  Type 1 diabetes is a complex, multifactorial disease characterized by T cell–mediated autoimmune destruction of insulin‐secreting pancreatic β cells. To facilitate research in type 1 diabetes, a large‐scale dynamic mathematical model of the female non‐obese diabetic (NOD) mouse was developed. In this model, termed the Entelos® Type 1 Diabetes PhysioLab® platform, virtual NOD mice are constructed by mathematically representing components of the immune system and islet β cell physiology important for the pathogenesis of type 1 diabetes. This report describes the scope of the platform and illustrates some of its capabilities. Specifically, using two virtual NOD mice with either average or early diabetes‐onset times, we demonstrate the reproducibility of experimentally observed dynamics involved in diabetes progression, therapeutic responses to exogenous IL‐10, and heterogeneity in disease onset. Additionally, we use the Type 1 Diabetes PhysioLab platform to investigate the impact of disease heterogeneity on the effectiveness of exogenous IL‐10 therapy to prevent diabetes onset. Results indicate that the inability of a previously published IL‐10 therapy protocol to protect NOD mice who exhibit early diabetes onset is due to high levels of pancreatic lymph node (PLN) inflammation, islet infiltration, and β cell destruction at the time of treatment initiation. Further, simulation indicates that earlier administration of the treatment protocol can prevent NOD mice from developing diabetes by initiating treatment during the period when the disease is still sensitive to IL‐10s protective function.

Dosing and Timing Effects of Anti-CD40L Therapy

Abstract:  Several publications describing the use of anti‐CD40L monoclonal antibodies (anti‐CD40L) for the treatment of type 1 diabetes in non‐obese diabetic (NOD) mice have reported different treatment responses to similar protocols. The Entelos® Type 1 Diabetes PhysioLab® platform, a dynamic large‐scale mathematical model of the pathogenesis of type 1 diabetes, was used to study the effects of anti‐CD40L therapy in silico. An examination of the impact of pharmacokinetic variability and the heterogeneity of disease progression rate on therapeutic outcome provided insights that could reconcile the apparently conflicting data. Optimal treatment protocols were identified by exploring the dynamics of key pathophysiological pathways.

Mechanisms mediating anti-CD3 antibody efficacy: insights from a mathematical model of type 1 diabetes.

Abstract:  Anti‐CD3 antibody therapy, a promising clinical approach for the treatment of type 1 diabetes (T1D), was investigated using a mathematical model of T1D in the female nonobese diabetic (NOD) mouse. Analyses of model simulation results indicate that, in addition to the known direct effects of anti‐CD3 antibody on T lymphocytes, two additional mechanisms are required for sustained disease remission: (a) rapid regrowth of healthy β cells following clearance of islet inflammation and (b) enhanced regulatory T cell activity and/or phenotypic changes in antigen presenting cells (APCs) that promote a stable regulatory environment in the pancreas.