Joan Verdaguer

Progression of autoimmune diabetes driven by avidity maturation of a T-cell population

For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of inflammation of mononuclear cells in target tissues. Here we show that progression of pancreatic islet inflammation to overt diabetes in nonobese diabetic (NOD) mice is driven by the ‘avidity maturation’ of a prevailing, pancreatic beta-cell-specific T-lymphocyte population carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) in the context of H-2K d class I molecules of the major histocompatibility complex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8+ T lymphocytes contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest affinity and lowest dissociation rates for peptide–MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell populations may be the key event in the progression of benign inflammation to overt disease in autoimmunity.

CD40 Ligation Releases Immature Dendritic Cells from the Control of Regulatory CD4+CD25+ T Cells

We report that disruption of CD154 in nonobese diabetic (NOD) mice abrogates the helper function of CD4+CD25- T cells without impairing the regulatory activity of CD4+CD25+ T cells. Whereas CD4+ T cells from NOD mice enhanced a diabetogenic CD8+ T cell response in monoclonal TCR-transgenic NOD mice, CD4+ T cells from NOD.CD154(-/-) mice actively suppressed it. Suppression was mediated by regulatory CD4+CD25+ T cells capable of inhibiting CD8+ T cell responses induced by peptide-pulsed dendritic cells (DCs), but not peptide/MHC monomers. It involved inhibition of DC maturation, did not occur in the presence of CD154+ T-helper cells, and could be inhibited by activation of DCs with LPS, CpG DNA, or an agonistic anti-CD40 mAb. Thus, in at least some genetic backgrounds, CD154-CD40 interactions and innate stimuli release immature DCs from suppression by CD4+CD25+ T cells.

Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice.

Autoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic beta cells by T lymphocytes. It is believed that CD8(+) cytotoxic T lymphocytes (CTLs) effect the initial beta-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of beta cell-reactive CD8(+) T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8(+) CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2Kd complex on beta cells, to effect beta-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide-pulsed non-beta-cell targets via both perforin and Fas, but they killed NOD beta cells via Fas exclusively. Perforin-deficient 8.3-TCR-transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8(+) CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill beta cells in a Fas-dependent and perforin-independent manner.

IL-1α, IL-1β, and IFN-γ mark β cells for Fas-dependent destruction by diabetogenic CD4+ T lymphocytes

Cytokines such as IL-1alpha, IL-1beta, and IFN-gamma have long been implicated in the pathogenesis of autoimmune diabetes, but the mechanisms through which they promote diabetogenesis remain unclear. Here we show that CD4(+) T lymphocytes propagated from transgenic nonobese diabetic (NOD) mice expressing the highly diabetogenic, beta cell-specific 4.1-T-cell receptor (4.1-TCR) can kill IL-1alpha-, IL-1beta-, and IFN-gamma-treated beta cells from NOD mice. Untreated NOD beta cells and cytokine-treated beta cells from Fas-deficient NOD.lpr mice are not targeted by these T cells. Killing of islet cells in vitro was associated with cytokine-induced upregulation of Fas on islet cells and was independent of MHC class II expression. Abrogation of Fas expression in 4.1-TCR-transgenic NOD mice afforded nearly complete protection from diabetes and did not interfere with the development of the transgenic CD4(+) T cells or with their ability to cause insulitis. In contrast, abrogation of perforin expression did not affect beta cell-specific cytotoxicity or the diabetogenic potential of these T cells. These data demonstrate a novel mechanism of action of IL-1alpha, IL-1beta, and IFN-gamma in autoimmune diabetes, whereby these cytokines mark beta cells for Fas-dependent lysis by autoreactive CD4(+) T cells.

IFNβ Accelerates Autoimmune Type 1 Diabetes in Nonobese Diabetic Mice and Breaks the Tolerance to β Cells in Nondiabetes-Prone Mice

Genetic and environmental factors are decisive in the etiology of type 1 diabetes. Viruses have been proposed as a triggering environmental event and some evidences have been reported: type I IFNs exist in the pancreata of diabetic patients and transgenic mice expressing these cytokines in β cells develop diabetes. To determine the role of IFNβ in diabetes, we studied transgenic mice expressing human IFNβ in the β cells. Autoimmune features were found: MHC class I islet hyperexpression, T and B cells infiltrating the islets and transfer of the disease by lymphocytes. Moreover, the expression of β2-microglobulin, preproinsulin, and glucagon in the thymus was not altered by IFNβ, thus suggesting that the disease is caused by a local effect of IFNβ, strong enough to break the peripheral tolerance to β cells. This is the first report of the generation of NOD (a model of spontaneous autoimmune diabetes) and nonobese-resistant (its homologous resistant) transgenic mice expressing a type I IFN in the islets: transgenic NOD and nonobese-resistant mice developed accelerated autoimmune diabetes with a high incidence of the disease. These results indicate that the antiviral cytokine IFNβ breaks peripheral tolerance to β cells, influences the insulitis progression and contributes to autoimmunity in diabetes and nondiabetes- prone mice.

Cross-Priming of Diabetogenic T Cells Dissociated from CTL-Induced Shedding of β Cell Autoantigens

Cross-presentation of self Ags by APCs is key to the initiation of organ-specific autoimmunity. As MHC class I molecules are essential for the initiation of diabetes in nonobese diabetic (NOD) mice, we sought to determine whether the initial insult that allows cross-presentation of β cell autoantigens in diabetes is caused by cognate interactions between naive CD8+ T cells and β cells. Naive splenic CD8+ T cells from transgenic NOD mice expressing a diabetogenic TCR killed peptide-pulsed targets in the absence of APCs. To ascertain the role of CD8+ T cell-induced β cell lysis in the initiation of diabetes, we expressed a rat insulin promoter (RIP)-driven adenovirus E19 transgene in NOD mice. RIP-E19 expression inhibited MHC class I transport exclusively in β cells and rendered these cells resistant to lysis by CD8+ (but not CD4+) T cells, both in vitro and in vivo. Surprisingly, RIP-E19 expression impaired the accumulation of CD8+ T cells in islets and delayed the onset of islet inflammation, without affecting the timing or magnitude of T cell cross-priming in the pancreatic lymph nodes, which is the earliest known event in diabetogenesis. These results suggest that access of β cell autoantigens to the cross-presentation pathway in diabetes is T cell independent, and reveal a previously unrecognized function of MHC class I molecules on target cells in autoimmunity: local retention of disease-initiating clonotypes.

CD154-Dependent Priming of Diabetogenic CD4+ T Cells Dissociated from Activation of Antigen-Presenting Cells

We followed the fate of K(d)- or I-A(g7)-restricted beta cell-autoreactive T cells in monoclonal TCR-transgenic NOD mice expressing or lacking CD154. 8.3-NOD.RAG-2(-/-)/CD154(-/-) mice, which bear autoreactive CD8(+) T cells, developed diabetes with the same incidence and tempo as 8.3-NOD.RAG-2(-/-)/CD154(+) mice. Recruitment of CD154(-/-) 8.3-CD8(+) CTL was accelerated by CD154(+)CD4(+) T cells, by expression of a B7.1 transgene in beta cells or by treatment of the mice with CpG-DNA or an agonistic anti-CD40 antibody. In contrast, the autoreactive CD4(+) T cells maturing in 4.1-NOD.RAG-2(-/-) mice lost their diabetogenic potential if they lacked CD154, even in the presence of CD154(+)CD4(+) T cells, B7.1 molecules on beta cells, CpG-DNA treatment, or systemic CD40 ligation. These results demonstrate the existence of a novel, CD154-dependent pathway of CD4(+) T cell activation that is independent of CD40-mediated activation of APCs.

Idd9.1 Locus Controls the Suppressive Activity of FoxP3+CD4+CD25+ Regulatory T-Cells

OBJECTIVE The ∼45-cM insulin-dependent diabetes 9 (Idd9) region on mouse chromosome 4 harbors several different type 1 diabetes–associated loci. Nonobese diabetic (NOD) mice congenic for the Idd9 region of C57BL/10 (B10) mice, carrying antidiabetogenic alleles in three different Idd9 subregions (Idd9.1, Idd9.2, and Idd9.3), are strongly resistant to type 1 diabetes. However, the mechanisms remain unclear. This study aimed to define mechanisms underlying the type 1 diabetes resistance afforded by B10 Idd9.1, Idd9.2, and/or Idd9.3. RESEARCH DESIGN AND METHODS We used a reductionist approach that involves comparing the fate of a type 1 diabetes–relevant autoreactive CD8+ T-cell population, specific for residues 206–214 of islet-specific glucose 6 phosphatase catalytic subunit–related protein (IGRP206–214), in noncongenic versus B10 Idd9–congenic (Idd9.1 + Idd9.2 + Idd9.3, Idd9.2 + Idd9.3, Idd9.1, Idd9.2, and Idd9.3) T-cell receptor (TCR)–transgenic (8.3) NOD mice. RESULTS Most of the protective effect of Idd9 against 8.3-CD8+ T-cell–enhanced type 1 diabetes was mediated by Idd9.1. Although Idd9.2 and Idd9.3 afforded some protection, the effects were small and did not enhance the greater protective effect of Idd9.1. B10 Idd9.1 afforded type 1 diabetes resistance without impairing the developmental biology or intrinsic diabetogenic potential of autoreactive CD8+ T-cells. Studies in T- and B-cell–deficient 8.3-NOD.B10 Idd9.1 mice revealed that this antidiabetogenic effect was mediated by endogenous, nontransgenic T-cells in a B-cell–independent manner. Consistent with this, B10 Idd9.1 increased the suppressive function and antidiabetogenic activity of the FoxP3+CD4+CD25+ T-cell subset in both TCR-transgenic and nontransgenic mice. CONCLUSIONS A gene(s) within Idd9.1 regulates the development and function of FoxP3+CD4+CD25+ regulatory T-cells and, in turn, the activation of CD8+ effector T-cells in the pancreatic draining lymph nodes, without affecting their development or intrinsic diabetogenic potential.

Phenotype and Functional Characteristics of Islet-Infiltrating B-Cells Suggest the Existence of Immune Regulatory Mechanisms in Islet Milieu

B-cells participate in the autoimmune response that precedes the onset of type 1 diabetes, but how these cells contribute to disease progression is unclear. In this study, we analyzed the phenotype and functional characteristics of islet-infiltrating B-cells in the diabetes-prone NOD mouse and in the insulitis-prone but diabetes-resistant (NOD×NOR)F1 mouse. The results indicate that B-cells accumulate in the islets of both mice influenced by sex traits. Phenotypically and functionally, these B-cells are highly affected by the islet inflammatory milieu, which may keep them in a silenced status. Moreover, although islet-infiltrating B-cells seem to be antigen experienced, they can only induce islet-infiltrating T-cell proliferation when they act as accessory cells. Thus, these results strongly suggest that islet-infiltrating B-cells do not activate islet-infiltrating T-cells in situ, although they may affect the progression of the disease otherwise.