International audienceExopolysaccharides (EPS) from Lactobacillus rhamnosus RW- 9595M have been prepared from bacterial cultures, isolated, concentrated, fractionated and tested in vitro for their possible modulating properties on mouse splenocytes from the C57Bl/6 and BALB/c strains, on the murine RAW 264.7 macrophage-like cell line and on human Peripheral Blood Mononuclear Cells (PBMC) from a total of 14 healthy donors. A first step of EPS fractionation was attempted, using membranes with different molecular weight cut-off. Fractions were as follows: F1: $> $1000 kg$\cdot$mol$^{-1}$; F2: 1000-100 kg$\cdot$mol$^{-1}$; F3: 100-10 kg$\cdot$mol$^{-1}$; F4: $< $10 kg$\cdot$mol$^{-1}$. Total EPS, as well as F1, appeared slightly mitogenic in both mouse splenocytes and human PBMC in 2-3 d cultures, and F3 also exhibited such a property on human PBMC. Unfractionated concentrated ("total" ) EPS, as well as F1, elicited TNF, IL-6 and IL-12 p40 both in the mouse and human cells, in 6 h and 24 h cultures, with important variability depending on the cell source. In 24 h cultures, total EPS or F1 elicited bio-active IFN-$\gamma$ in both C57Bl/6 and BALB/c splenocytes, and this IFN-$\gamma$ secretion was sustained until at least 3 d of culture. In human PBMC, no IFN-$\gamma$ production was observed despite high IL-12p40 secretion. These results suggest the possibility of enhancing the immune system through EPS from lactic acid bacteria, in individuals responsive to such a stimulus.Productions de TNF, IL-6, IL-12 et IFN-g chez des cellules immunocompétentes traitées avec des exopolysaccharides du Lactobacillus rhamnosus RW-9595M. Différence entre les réponses de cellules de sang périphérique humain et de splénocytes de souris. Les exopolysaccharides (EPS) du Lactobacillus rhamnosus RW- 9595M ont été préparés à partir de cultures bactériennes, isolés, concentrés, fractionnés et testés in vitro quant à leur potentiel immunomodulateur sur des splénocytes de souris C57Bl/6 et BALB/c, sur la lignée macrophagique murine RAW.264.7 et sur des cellules mononuclées du sang humain provenant de 14 donneurs. Nous avons fractionné ces EPS en fonction des tailles moléculaires, avec des membranes filtrantes de différentes porosités. Les fractions correspondent aux tailles suivantes : F1 : $> $ 1000 kg$\cdot$mol$^{-1}$, F2 : 1000-100 kg$\cdot$mol$^{-1}$, F3 : 100-10 kg$\cdot$mol$^{-1}$ et F4 : $< $ 10 kg$\cdot$mol$^{-1}$. Les EPS concentrés, non fractionnés, ainsi que F1, se sont avérés légèrement mitogènes sur des splénocytes de la souris, ou des cellules sanguines humaines, cultivées 2-3 jours. Dans le dernier cas, F3 manifeste également cette propriété. Les EPS non fractionnés, ainsi que F1, sont capables de provoquer la production de TNF, IL-6, IL-12p40 après 6 ou 24 h de culture, l'origine des cellules étant importante pour l'intensité de la réponse. Après 24, 48 et 72 h de culture, les EPS non fractionnés, ainsi que F1, ont provoqué la production d'interféron gamma par des splénocytes de souris C57Bl/6 et BALB/c. Au contraire, dans le ca...

CD8+ T-cell immune response to liver antigens is often functionally diminished or absent. This may occur via deletion of these autoaggressive T cells, through the acquisition of an anergic phenotype, or via active suppression mediated by other cell populations. We generated a double transgenic model in which mice express CD8+ T cells specific for the lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) and LCMV-NP as a hepatic neo-autoantigen, to study the immunological response of potentially liver antigen autoagressive CD8+ T cells. Autoreactive transgenic CD8+ T cells were analyzed for functionality and cytotoxic effector status. Despite severe peripheral deletion of liver-specific CD8+ T cells, a fraction of autoreactive NP-specific CD8+ T cells accumulate in liver, resulting in hepatocyte injury and production of autoantibodies in both male and female mice. NP-specific intrahepatic T cells showed capacity to proliferate, produce cytokines and up-regulate activation markers. These data provide in vivo evidence that autoreactive CD8+ T cells are activated in the liver and developed an inflammatory process, but require additional factors to cause severe autoimmune destruction of hepatocytes. Our new model will provide a valuable tool for further exploration of the immunological response involved in inflammatory liver diseases, including autoimmune hepatitis.

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Identifying the type of diabetogenic CD8 T cells that initiate autoimmune diabetes (AID) is a critical step in designing appropriate strategies for the early detection of beta cell-directed autoimmunity and its progression to diabetes. We generated a novel double transgenic (Tg) mouse model on the naturally diabetes resistant C57Bl/6 background, co-expressing two transgenes including a specific TCR anti-lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) carried by CD8 T cells and LCMV-NP (as neo-self antigen) expressed by pancreatic beta cells. The resulting double Tg mouse showed partial thymic deletion of the NP-specific CD8 T cells. The escaping autoreactive NP-specific CD8 T cells joining the periphery were activated and gained effector functions. Both male and female mice mounted anti-NP antibodies, but only one-fourth adult males spontaneously developed AID. Significant upregulation of the CD44 and CD122 markers as compared to healthy male and female mice characterized the phenotype of diabetogenic CD8 T cells in diabetic male mice. We also show that only 10% of these CD8T cells expressed programmed death 1 receptor (PD-1). Together, these results suggest that in our double Tg mouse model, Ag-specific effector CD44CD122PD-1CD8 T cell subpopulation is associated with the pathogenesis of AID.