Agnès Lehuen

NKT Cells Inhibit the Onset of Diabetes by Impairing the Development of Pathogenic T Cells Specific for Pancreatic β Cells

To determine the precise regulatory effect of NKT cells on CD4(+) T cells involved in autoimmune diabetes, we developed an in vivo model in which transferred naive transgenic T cells are stimulated by their antigen in the presence or absence of NKT cells or in the presence of another conventional transgenic alphabeta T cell. The presence of NKT cells did not block the initial activation and expansion of the CD4(+) T cells but did inhibit their IL-2 and IFN-gamma production and later proliferation, resulting in an anergic phenotype. These CD4(+) T cells did not induce significant insulitis and were unable to destroy the beta cells. Thus, NKT cells prevent alphabeta CD4 T cell differentiation into effector cells.

Tolerance to Islet Antigens and Prevention from Diabetes Induced by Limited Apoptosis of Pancreatic β Cells

Crosspresentation of self-antigens by antigen-presenting cells is critical for the induction of peripheral tolerance. As apoptosis facilitates the entry of antigens into the crosspresentation pathway, we sought to prevent the development of autoimmune diabetes by inducing pancreatic beta cell apoptosis before disease onset. Accordingly, young nonobese diabetic (NOD) mice injected with a single low dose of streptozotocin (SZ), a drug cytotoxic for beta cells, exhibited impaired T cell responses to islet antigens and were protected from spontaneous diabetes. Furthermore, beta cell apoptosis was necessary for protection since SZ did not protect RIP-CrmA transgenic NOD mice in which beta cells expressed the caspase inhibitor CrmA. Our results support a model in which apoptosis of pancreatic beta cells induces the development of regulatory cells leading to the tolerization of self-reactive T cells and protection from diabetes.

Phenotypic and Functional Differences Between NKT Cells Colonizing Splanchnic and Peripheral Lymph Nodes

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-γ. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vβ repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vβ8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-γ, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-γ than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.

Role of the Complementarity-Determining Region 3 (CDR3) of the TCR-β Chains Associated with the Vα14 Semi-Invariant TCR α-Chain in the Selection of CD4+ NK T Cells

The NK1.1+TCRαβint CD4+, or double negative T cells (NK T cells) consist of a mixture of CD1d-restricted and CD1d-unrestricted cells. The relationships between CD4+NK1.1+ T cells and conventional T cells are not understood. To compare their respective TCR repertoires, NK1.1+TCRαβint, CD4+ T cells have been sorted out of the thymus, liver, spleen, and bone marrow of C57BL/6 mice. Molecular analysis showed that thymus and liver used predominantly the Vα14-Jα281 and Vβ 2, 7, and 8 segments. These cells are CD1d restricted and obey the original definition of NK T cells. The complementarity-determining region 3 (CDR3) sequences of the TCR Vβ8.2-Jβ2.5 chain of liver and thymus CD4+ NK T cells were determined and compared with those of the same rearrangements of conventional CD4+ T cells. No amino acid sequence or usage characteristic of NK T cells could be evidenced: the Vβ8.2-Jβ2.5 diversity regions being primarily the same in NK T and in T cells. No clonal expansion of the β-chains was observed in thymus and liver CD1d-restricted CD4+NK T cells, suggesting the absence of acute or chronic Ag-driven stimulation. Molecular analysis of the TCR used by Vα14-Jα281 transgenic mice on a Cα−/− background showed that the α-chain can associate with β-chains using any Vβ segment, except in NK T cells in which it paired predominately with Vβ 2, 7, and 8+ β-chains. The structure of the TCR of NK T cells thus reflects the affinity for the CD1d molecule rather than a structural constraint leading to the association of the invariant α-chain with a distinctive subset of Vβ segment.

Immune complex-mediated glomerulopathy in experimental Chagas' disease

To investigate the development of glomerulopathy during the chronic phase of experimental Chagas disease, C3H-Hej mice were infected with Trypanosoma cruzi trypomastigotes. Deposits of IgG, IgM, and C3 in renal mesangium were observed by immunofluorescence (IF) to increase in size as a function of time after infection (4-6 months). T. cruzi antigens were codeposited in glomeruli with Ig and C3. Electron-dense deposits were visualized in mesangial and paramesangial areas by electron microscopy. Anti-T. cruzi and rheumatoid factor (RF) antibodies (of IgG isotypes) were detected both in serum and in renal eluates. In serum, the titers of both antibodies progressively decreased as a function of time after infection. In renal eluates, titers of anti-T. cruzi antibodies appeared to be stable during the three time periods after infection. By contrast, titers of RF antibodies in renal eluates were shown to increase progressively during these same time periods, paralleling the increase in size of mesangial Ig deposits observed by IF. Several T. cruzi proteins were immunoprecipitated from radiolabeled renal eluates by a control anti-T. cruzi antibody. In addition, antibodies from renal eluates specifically precipitated a 85-kDa protein from radiolabeled T. cruzi lysates, whereas serum antibodies precipitated a broad pattern of T. cruzi proteins. These results demonstrate that mice experimentally infected with T. cruzi can develop a mesangial glomerulopathy during the chronic phase of the disease, which appears to be mediated through immune complexes containing parasite antigens associated with secondary deposition of RF.

Frontline: Self‐peptides that bind with low affinity to the diabetes‐associated I‐Ag7 molecule readily induce T cell tolerance in non‐obese diabetic mice

Although non‐obese diabetic (NOD) mice spontaneously develop Tu2004cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self‐Ag. Furthermore, as autoimmunity has been postulated to result from Tu2004cell responses directed toward self‐peptides that bind with low affinity to NOD I‐Ag7 MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic β cells. We identified LACK peptides that were the targets of Tu2004cells in LACK‐immunized NOD mice while binding to I‐Ag7 with low affinity. While CD4+ Tu2004cells from NOD mice secreted IFN‐γ, IL‐4, IL‐5 and IL‐10 in response to LACK, those from LACK‐expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK‐expressing Tg mice exhibited self‐reactive CD4+ Tu2004cells with impaired proliferation capabilities. Hence, even self‐peptides that bind to I‐Ag7 with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that Tu2004cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.

Natural thymocytotoxic autoantibodies in non-obese diabetic (NOD) mice: characterization and fine specificity.

The NOD mouse is a model of human juvenile type I diabetes mellitus. As in humans and in the BB rat model, the development of diabetes in NOD mice is accompanied by evident manifestations of cell‐mediated and humoral autoimmunity. Beside autoantibodies directed at putative islet cell antigens, NOD sera contain antibodies with specificity for lymphocyte cell‐surface determinants. Here we demonstrate that these anti‐lymphocyte antibodies have the same characteristics of target cell specificity, of isotype, and of temperature reactivity, as do natural thymocytotoxic autoantibodies (NTA) from lupic NZB mice, or from mice undergoing polyclonal B cell activation. We also demonstrate that the thymocytotoxic activity of NOD sera is not due to cross‐reactive anti‐insulin antibodies. Biochemical characterization of the determinants recognized by these anti‐lymphocyte antibodies reveals two membrane‐associated proteins of 28 and 33 kD, partially similar to the two peptides recognized by NTA from NZB mice (30 and 33 kD). Altogether, these results suggest that NOD mice develop manifestations of polyclonal B cell activation similar to those observed in lupus‐prone mice. The relationship of these anomalies with the organ‐specific pancreatic disease remains to be properly evaluated.

Definition of a differentiation antigen on the surface of phagocytic cells of thymic reticulum which is down-regulated by interferon γ☆

Monoclonal antibodies (MoAb) were raised against phagocytic cells of thymic reticulum (P-TR) grown in vitro. Each of the two MoAb (TR-1N, TR-3N) defined two polypeptides of 46-57 kDa on P-TR membrane. TR-1N and TR-3N recognize respectively 48 and 81% of P-TR, but do not recognize any cells in spleen, lymph node, thymic lymphocytes, or bone marrow. They bind to part of peritoneal macrophages and to macrophage cell lines J 774 and P 388 D1. Cell binding of TR-1N and TR-3N was compared by immunofluorescence to that of anti-CR3 antibody (Mac-1) which recognizes P-TR, a small number of cells in bone marrow and spleen, and a much higher percentage of peritoneal macrophages. The polypeptides recognized by TR-1N/TR-3N may be defined as differentiation antigens on accessory cells as they appear on bone marrow cells during maturation in vitro in the presence of L-cell supernatant which contains colony stimulating factor (CSF-1). Interferon gamma is able to down-regulate the expression of TR-1N/TR-3N antigen on P-TR membrane while that of Mac-1 is unchanged and that of Ia is up-regulated.

Characterization of two molecules at 30,33 kDa as major target antigens of natural thymocytotoxic autoantibodies

We have attempted to characterize, by immunoblotting, the cell surface determinants which are recognized by natural thymocytotoxic autoantibodies (NTA). NTA-positive sera from spontaneously autoimmune mice and from mice rendered autoreactive by neonatal induction of tolerance were used as probes on blots of thymocytes. Fifty percent of the sera screened under these conditions reacted with a doublet of 30,33 kDa molecular weight present on membranes, but not detectable on blots of total cell extracts. Additional bands of various molecular weights were detected by NTA-positive sera at a much lower frequency than the 30,33 kDa determinants. Direct evidence of identity between the antibodies inducing thymocytotoxicity and those detecting the doublet in immunoblotting was provided by immunoadsorption tests. Among a panel of immunoadsorbents prepared with thymocyte membrane proteins of various molecular weights, only the one containing the 30,33 kDa molecules efficiently adsorbed cytotoxic antibodies whereas the other preparations containing some of the bands occasionally detected in immunoblotting had practically no effect. In addition we showed that the 30,33 kDa doublet is also detected on membrane preparations of T and B lymphocytes but not on preparations of a nonlymphoid cell line, and is composed of two independent molecules not covalently linked, in their native configuration, by disulfide bonds. Altogether these results strongly suggest that the 30,33 kDa molecules represent major cell surface determinants for thymocytotoxic autoantibodies.