Claude Carnaud

Impairment of NK Cell Function by NKG2D Modulation in NOD Mice

Nonobese diabetic (NOD) mice, a model of insulin-dependent diabetes mellitus, have a defect in natural killer (NK) cell-mediated functions. Here we show impairment in an activating receptor, NKG2D, in NOD NK cells. While resting NK cells from C57BL/6 and NOD mice expressed equivalent levels of NKG2D, upon activation NOD NK cells but not C57BL/6 NK cells expressed NKG2D ligands, which resulted in downmodulation of the receptor. NKG2D-dependent cytotoxicity and cytokine production were decreased because of receptor modulation, accounting for the dysfunction. Modulation of NKG2D was mostly dependent on the YxxM motif of DAP10, the NKG2D-associated adaptor that activates phosphoinositide 3 kinase. These results suggest that NK cells may be desensitized by exposure to NKG2D ligands.

Prevention of diabetes in NOD mice treated with antibody to murine IFNγ

The NOD mouse is studied as an animal model of human insulin-dependent diabetes mellitus (IDDM). To evaluate the role of IFN gamma in the pathogenesis of the disease, we have studied the effect of anti-IFN gamma mAb on the expression of insulitis and clinical diabetes. Treatment of mice with anti-IFN gamma mAb prevented the induction of early IDDM by cyclophosphamide as well as the adoptive transfer of diabetes by spleen cells from diabetic NOD mice. The protection against induction of diabetes by cyclophosphamide was observed in animals treated with the anti-IFN gamma mAb within 24 h following the first cyclophosphamide injection but not in animals in which mAb treatment was started 7 days later. Transfer of disease was prevented both in adult irradiated and in newborn recipients. The absence of clinical signs in these mice was corroborated by a significant reduction of both the extent and severity of insulitis. Over-expression of Ia antigen on endothelial cells lining the islets was also considerably reduced in mice treated with mAb. These data strongly suggest a role for IFN gamma during the autoimmune process leading to beta cell destruction in diabetes and prompt further investigation of the use of such antibodies in the immunoprevention of IDDM.

Damage control, rather than unresponsiveness, effected by protective DX5 + T cells in autoimmune diabetes

The progression of autoimmune diabetes is regulated. We examined here the cellular controls exerted on disease that developed in the BDC2.5 T cell receptor–transgenic model. We found that all BDC2.5 mice with a monoclonal, β cell–reactive, T cell repertoire developed diabetes before 4 weeks of age; transfer of splenocytes from young standard NOD (nonobese diabetic) mice into perinatal monoclonal BDC2.5 animals protected them from diabetes. The protective activity was generated by CD4+ αβ T cells, which operated for a short time at disease initiation, could be partitioned according to DX5 cell surface marker expression and split into two components. Protection did not involve clonal deletion or anergy of the autoreactive BDC2.5 cells, permitting their full activation and attack of pancreatic islets; rather, it tempered the aggressiveness of the insulitic lesion and the extent of β cell destruction.

Treatment With Neutralizing Antibodies Specific for IL-1β Prevents Cyclophosphamide-Induced Diabetes in Nonobese Diabetic Mice

Interleukin-1 (IL-1) has been shown to be involved in the pathogenesis of IDDM, but it is not clear which form, IL-1α or IL-1β, is predominantly implicated. In this study, we have evaluated the contribution of IL-1β by treating diabetes-prone nonobese diabetic (NOD) mice with specific neutralizing antibodies. First, we assessed the neutralizing potential of these antibodies in C57BL/6 mice under acute septic shock by measuring IL-1β in sera 4 h after lipopolysaccharide injection. One milligram and 0.1 mg of anti–IL-1β antibodies (Abs) were capable of neutralizing the IL-1β produced, and the effect persisted for at least 5 days. Second, we evaluated the role of IL-1β in the cyclophosphamide (CY)-accelerated model of diabetes. Nondiabetic male NOD mice were injected with 200 mg/kg CY and treated twice weekly with anti–IL-1β Ab. The incidence of diabetes reached 76 and 100% in the control groups treated with 0.25 and 0.1 mg rabbit IgG, respectively. In contrast, only 34% of mice treated with 0.25 mg of anti–IL-1β Ab became diabetic. In the group treated with 0.1 mg of anti–IL-1β Ab, 89% of the mice became diabetic in the same period of time, demonstrating that the protective effect was dose dependent. Our results show that IL-1β is a critical effector molecule in this model of IDDM and that its specific inhibition could be an attractive target for therapeutic intervention.

Immunological Tolerance to a Pancreatic Antigen as a Result of Local Expression of TNFα by Islet β Cells

Recent experiments have suggested that tumor necrosis factor alpha (TNFalpha) can down-regulate islet-specific T cells and prevent the development of autoimmune diabetes. Here we demonstrate that transgenic mice expressing both TNFalpha and the Leishmania major LACK antigen in the pancreas (RIP-TNFalpha/RIP-LACK) exhibit an impaired ability to mount a CD4+ T cell response against LACK. In addition, peripheral CD4+ T cells from TCR transgenic mice (TCR-LACK/RIP-TNFalpha/RIP-LACK) produced reduced interleukin-2 but elevated levels of T helper 2 cytokines in response to LACK peptide in vitro. Taken together, our data suggest that TNFalpha may act in vivo to modulate a potentially damaging self-reactive T cell response by inducing tolerance to pancreatic antigens.

Adult thymectomy promotes the manifestation of autoreactive lymphocytes.

Abstract Spleen cells from adult thymectomized mice (ATX) were assayed in a syngeneic graft vs host (GVH) model based upon enlargement of the draining popliteal lymph node following syngeneic cell inoculation into the hind footpad. Spleen cells from ATX mice have been found to induce a significantly higher increase in the weight of the regional lymph node than that induced by the injection of normal spleen cells. Irradiated spleen cells from ATX donors did not cause a similar increase, suggesting either that proliferation of the transferred cells was required at some stage of the reaction or that autoreactive cells are radiosensitive. Autoreactive cells were found in the spleen of mice 2 to 3 months after the thymectomy but were never found in the lymph nodes of such animals or in the thymus of intact mice. They are not phagocytic adherent cells and are not retained on nylon wool columns, which suggests that they belong to the T-cell lineage. Autoreactivity is lost when spleen cells from ATX donors are depleted of autologous rosette-forming cells (A-RFC) by centrifugation on a Ficoll-Hypaque gradient after rosette formation. Autoreactive spleen lymphocytes might belong to the population of A-RFC previously characterized as a population of immature T cells.

Studies on mouse autoreactive cells. I. Role of h-2 antigens in mouse autologous rosette formation.

Abstract A minority (1–2%) of normal mouse lymphoid cells bind autologous erythrocytes and form rosettes. In this study we examined the antigenic specificity involved in the formation of such rosettes. A significant difference in the incidence of rosettes formed, respectively, with autologous and allogeneic mouse erythrocytes is found. Moreover, preincubation of lymphoid cells with low concentrations of syngeneic erythrocytic ghosts causes significant competitive inhibition of subsequent rosette formation. Allogeneic ghosts obtained from nonrelated or from congenic resistant strains of mice do not display this inhibitory effect under the same conditions. It is thus suggested that mouse autologous rosette-forming cells bear receptors for syngeneic H-2 antigens that are involved in the binding of autologous erythrocytes. More precisely, compatibility between lymphocyte and erythrocyte restricted to K or D only is sufficient to ensure a level of rosettes similar to that obtained when complete identity occurs for K, I, and D regions.

The suppressive effect of a supernate from concanavalin A-activated human lymphocytes: effects of concanavalin A-activated lymphocytes and their supernates on cytotoxic and mixed lymphocyte reactions.

Abstract The effects of Concanavalin A-treated human peripheral blood lymphocytes and their supernatants were evaluated on the MLC reaction and on the generation of cytotoxic lymphocytes assessed by cell-mediated lympholysis (CML). Experiments were performed with both allogeneic and xenogenic sensitization. It was found that Con A-activated cells suppressed the MLC and CML reactions in allogeneic and xenogeneic systems. On the other hand, the SIRS-like supernate was able to suppress the MLC reaction and blastogenesis, but had no suppressive effect on the generation of cytotoxic lymphocytes. We found no difference in the magnitude of suppression, whether or not Con A-activated lymphocytes were syngeneic to the responder cells. This finding suggests that there is no requirement for allogeneic restriction in the interaction between suppressor and suppressed cells, and demonstrates a soluble human suppressor substance capable of suppressing some cell-mediated reactions.

Acquired allo-tolerance to major or minor histocompatibility antigens indifferently contributes to preventing diabetes development in non-obese diabetic (NOD) mice

Diabetes in NOD mice represents the end stage of a genetically-programmed autoimmune process mediated by T lymphocytes and directed against insulin-producing beta cells. We have shown in a previous study that the course of the disease is significantly inhibited in NOD mice which have been made tolerant at birth to foreign histocompatibility antigens. This early T cell manipulation results in a significant delay of disease onset, reduced overall incidence and less severe alterations of islet cells. In order to characterize better the nature of the foreign tolerogenic determinants responsible for this protection, we have now examined separately the contribution of MHC and non-MHC antigens. Two lines of congenic mice were used as donors of tolerogenic cells, NOD.H-2b, which differ from NOD by the MHC-encoded antigens only, and B10.H-2g7, which differ by all the minor histocompatibility antigens encoded by the B10 background, but which share with NOD mice the same MHC haplotype. Our results show that NOD recipients of F1 semi-compatible cells become specifically tolerant to the set of alloantigens to which they were neonatally exposed. Unresponsiveness, assessed by lack of CTL generation, is profound and specific. Yet, despite the fact that distinct sets of alloreactive T cell precursors are silenced, mice made tolerant indifferently to major or minor histocompatibility antigens are significantly protected against overt diabetes. These results could mean that each set of MHC and non-MHC encoded determinants can independently cross-tolerize a sufficient proportion of the autoreactive repertoire to slow the natural course of the disease. Alternatively, neonatally-acquired tolerance might induce polyclonal activation of the immune system resulting in the suppression or the immunodeviation of potentially harmful, autoreactive T cell clones.

Cellular basis of T-cell autoreactivity in autoimmune diseases

There is no doubt that T cells play a key role in the pathogenesis of autoimmune diseases (AD) both as effector and regulatory cells. Despite spectacular progress in the understanding of natural tolerance to self, owing particularly to transgenic technology, important questions remain open regarding the pathogenesis of AD, the conditions favoring the transition from benign or ‘physiological’ autoimmunity to deleterious autoimmunity, and the precise effector mechanisms. This review on the cellular basis of T-cell-mediated AD begins with an enumeration of the main arguments in favor of direct T-cell involvement, special emphasis being given to two animal models which have been most extensively investigated: experimental allergic encephalomyelitis, and the nonobese diabetic mouse. The question as to whether pathogenic T cells use a restricted repertoire of Vβ genes is examined in the context of these two models. From here we proceed to an evaluation of the mechanisms of onset of AD, discussing both extrinsic and intrinsic factors responsible for the breakdown in T-cell tolerance and reviewing the arguments in favor of suppressor T cells being actively involved in the prevention of autoimmunity. The last two sections are devoted to the effector mechanisms responsible for tissue injury in organ-specific AD and to T-cell-directed therapeutic interventions, respectively. We discuss the two main pathogenic hypotheses based on direct intervention of cytotoxic T cells or indirect involvement of inflammatory cytokines and macrophages, and evaluate the importance of ecotaxis in leading autoreactive T cells to the site of injury. We conclude on a brief and nonexhaustive list of strategies aimed at selectively neutralizing potentially harmuful T cells.