Abdelaziz Amrani

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.

Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide

Antigen therapy may hold great promise for the prevention of autoimmunity; however, most clinical trials have failed, suggesting that the principles guiding the choice of treatment remain ill defined. Here, we examine the antidiabetogenic properties of altered peptide ligands of CD8+ T cells recognizing an epitope of islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP206–214), a prevalent population of autoreactive T cells in autoimmune diabetes. We show that islet-associated CD8+ T cells in nonobese diabetic mice recognize numerous IGRP epitopes, and that these cells have a role in the outcome of protocols designed to induce IGRP206–214-specific tolerance. Ligands targeting IGRP206–214-reactive T cells prevented disease, but only at doses that spared low-avidity clonotypes. Notably, near complete depletion of the IGRP206–214-reactive T-cell pool enhanced the recruitment of subdominant specificities and did not blunt diabetogenesis. Thus, peptide therapy in autoimmunity is most effective under conditions that foster occupation of the target organ lymphocyte niche by nonpathogenic, low-avidity clonotypes.

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.

Granulocyte-Macrophage Colony-Stimulating Factor Prevents Diabetes Development in NOD Mice by Inducing Tolerogenic Dendritic Cells that Sustain the Suppressive Function of CD4+CD25+ Regulatory T Cells

Autoimmune diabetes results from a breakdown of self-tolerance that leads to T cell-mediated β-cell destruction. Abnormal maturation and other defects of dendritic cells (DCs) have been associated with the development of diabetes. Evidence is accumulating that self-tolerance can be restored and maintained by semimature DCs induced by GM-CSF. We have investigated whether GM-CSF is a valuable strategy to induce semimature DCs, thereby restoring and sustaining tolerance in NOD mice. We found that treatment of prediabetic NOD mice with GM-CSF provided protection against diabetes. The protection was associated with a marked increase in the number of tolerogenic immature splenic DCs and in the number of Foxp3+CD4+CD25+ regulatory T cells (Tregs). Activated DCs from GM-CSF-protected mice expressed lower levels of MHC class II and CD80/CD86 molecules, produced more IL-10 and were less effective in stimulating diabetogenic CD8+ T cells than DCs of PBS-treated NOD mice. Adoptive transfer experiments showed that splenocytes of GM-CSF-protected mice did not transfer diabetes into NOD.SCID recipients. Depletion of CD11c+ DCs before transfer released diabetogenic T cells from the suppressive effect of CD4+CD25+ Tregs, thereby promoting the development of diabetes. These results indicated that semimature DCs were required for the sustained suppressive function of CD4+CD25+ Tregs that were responsible for maintaining tolerance of diabetogenic T cells in NOD mice.

Inflammatory Cytokine Production by Human Neutrophils Involves C/EBP Transcription Factors

A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPγ are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPα, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPβ and C/EBPε. However, nuclear C/EBPβ is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-κB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1α, and Mip-1β in this cellular model, whereas TNF-α generation was mostly unaffected. Finally, overexpression of a C/EBPβ mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.

Thymic stromal lymphopoietin and thymic stromal lymphopoietin-conditioned dendritic cells induce regulatory T-cell differentiation and protection of NOD mice against diabetes.

OBJECTIVE—Autoimmune diabetes in the nonobese diabetic (NOD) mouse model results from a breakdown of T-cell tolerance caused by impaired tolerogenic dendritic cell development and regulatory T-cell (Treg) differentiation. Re-establishment of the Treg pool has been shown to confer T-cell tolerance and protection against diabetes. Here, we have investigated whether murine thymic stromal lymphopoietin (TSLP) re-established tolerogenic function of dendritic cells and induced differentiation and/or expansion of Tregs in NOD mice and protection against diabetes. RESEARCH DESIGN AND METHODS—We examined the phenotype of TSLP-conditioned bone marrow dendritic cells (TSLP-DCs) of NOD mice and their functions to induce noninflammatory Th2 response and differentiation of Tregs. The functional relevance of TSLP and TSLP-DCs to development of diabetes was also tested. RESULTS—Our results showed that bone marrow dendritic cells of NOD mice cultured in the presence of TSLP acquired signatures of tolerogenic dendritic cells, such as an absence of production of pro-inflammatory cytokines and a decreased expression of dendritic cell costimulatory molecules (CD80, CD86, and major histocompatibility complex class II) compared with LPS-treated dendritic cells. Furthermore, TSLP-DCs promoted noninflammatory Th2 response and induced the conversion of naïve T-cells into functional CD4+CD25+Foxp3+ Tregs. We further showed that subcutaneous injections of TSLP for 6 days or a single intravenous injection of TSLP-DCs protected NOD mice against diabetes. CONCLUSIONS—Our study demonstrates that TSLP re-established a tolerogenic immune response in NOD mice and protects from diabetes, suggesting that TSLP may have a therapeutic potential for the treatment of type 1 diabetes.

Inducible nitric oxide synthase (iNOS) in endotoxemia: chimeric mice reveal different cellular sources in various tissues

The aim of these experiments was to determine the contribution of leukocyte‐derived iNOS to total iNOS expression induced by lipopolysaccharide (LPS). By transferring bone marrow between iNOS+/+ and iNOS–/– mice, we created chimeric mice in which iNOS expression was limited to either circulating leukocytes (leukocyte‐iNOS mice) or parenchymal cells (parenchyma‐iNOS mice). Analysis of congenic markers demonstrated that >95% of thymocytes in chimeric mice were of donor origin. Also, following LPS treatment, iNOS mRNA was detectable in blood from leukocyte‐iNOS mice but not parenchyma‐iNOS mice. Together these findings indicated that the host marrow had been replaced entirely by donor cells. In the lung, at least 50% of the LPS‐induced iNOS mRNA was derived from leukocytes, and immunohistochemical analysis indicated that leukocytes were the main source of iNOS protein. In contrast in the liver, colon, and muscle, iNOS expression was derived predominantly from parenchymal cells. This divergence is potentially explained by the high level of leukocyte recruitment to the lung, relative to the other tissues. Plasma levels of NOS byproducts indicated that parenchymal iNOS was the dominant source of systemic iNOS activity. These findings indicate that in tissues other than the lung, parenchymal cells are the principal source of iNOS during endotoxemia.

RAG-dependent peripheral T cell receptor diversification in CD8+ T lymphocytes

Rearrangement of T cell receptor (TCR) genes is driven by transient expression of V(D)J recombination-activating genes (RAGs) during lymphocyte development. Immunological dogma holds that T cells irreversibly terminate RAG expression before exiting the thymus, and that all of the progeny arising from mature T cells express the parental TCRs. When single pancreatic islet-derived, NRP-A7 peptide-reactive CD8+ T cells from nonobese diabetic (NOD) mice were repeatedly stimulated with peptide-pulsed dendritic cells, daughter T cells reexpressed RAGs, lost their ability to bind to NRP-A7/Kd tetramers, ceased to transcribe tetramer-specific TCR genes, and, instead, expressed a vast array of other TCR rearrangements. Pancreatic lymph node (PLN) CD8+ T cells from animals expressing a transgenic NRP-A7-reactive TCR transcribed and translated RAGs in vivo and displayed endogenous TCRs on their surface. RAG reexpression also occurred in the PLN CD8+ T cells of wild-type NOD mice and could be induced in the peripheral CD8+ T cells of nondiabetes-prone TCR-transgenic B10.H2g7 mice by stimulation with peptide-pulsed dendritic cells. In contrast, reexpression of RAGs could not be induced in the CD8+ T cells of B6 mice expressing an ovalbumin-specific, Kb-restricted TCR, or in the CD8+ T cells of NOD mice expressing a lymphocytic choriomeningitis virus-specific, Db-restricted TCR. Extra-thymic reexpression of the V(D)J recombination machinery in certain CD8+ T cell subpopulations, therefore, enables further diversification of the peripheral T cell repertoire.

Deregulation of Protein Phosphatase 2A and Hyperphosphorylation of τ Protein Following Onset of Diabetes in NOD Mice

The histopathological hallmarks of Alzheimer disease (AD) include intraneuronal neurofibrillary tangles composed of abnormally hyperphosphorylated τ protein. Insulin dysfunction might influence AD pathology, as population-based and cohort studies have detected higher AD incidence rates in diabetic patients. But how diabetes affects τ pathology is not fully understood. In this study, we investigated the impact of insulin dysfunction on τ phosphorylation in a genetic model of spontaneous type 1 diabetes: the nonobese diabetic (NOD) mouse. Brains of young and adult female NOD mice were examined, but young NOD mice did not display τ hyperphosphorylation. τ phosphorylation at τ-1 and pS422 epitopes was slightly increased in nondiabetic adult NOD mice. At the onset of diabetes, τ was hyperphosphorylated at the τ-1, AT8, CP13, pS262, and pS422. A subpopulation of diabetic NOD mice became hypothermic, and τ hyperphosphorylation further extended to paired helical filament-1 and TG3 epitopes. Furthermore, elevated τ phosphorylation correlated with an inhibition of protein phosphatase 2A (PP2A) activity. Our data indicate that insulin dysfunction in NOD mice leads to AD-like τ hyperphosphorylation in the brain, with molecular mechanisms likely involving a deregulation of PP2A. This model may be a useful tool to address further mechanistic association between insulin dysfunction and AD pathology.