Project description:Background & Aims: In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies targeting specific self-antigen like the Soluble Liver Antigen (SLA or SepSecs) in autoimmune hepatitis (AIH). But the associated autoreactive CD4 T cells have not been characterized yet. Here we isolated and deeply characterized SLA-specific CD4 T cells in AIH patients. Methods: We used brief ex vivo re-stimulation with overlapping SLA-derived peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterize their transcriptome and T cell receptor (TCR) repertoire. Identified auto-reactive TCRs were reconstituted cloned and expressed in T cell hybridoma to identify dominant SLA-derived CD4 T cell epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing, to identify their phenotypic niche. We further characterized disease-associated peripheral blood T cells by high content flow cytometry in an additional cohort of n=42 AIH patients and n=17 non-alcoholic steatohepatitis (NASH) controls. Results: Frequency of autoreactive SLA-specific CD4 T cells was associated with anti-SLA autoantibodies and had a memory CD45RA-CD27+PD-1+CXCR5-CCR6- phenotype. ScRNA-seq revealed their pro-inflammatory/B-Helper profile (IL21, IFNG, TIGIT, CTLA4, NR3C1, CD109, KLRB1 and CLEC2D). SLA81-100 and SLA177-204 contain dominant T cell epitopes. Autoreactive TCR clonotypes were restricted to the memory PD-1+CXCR5- CD4 T cells which was significantly increased in the blood of AIH patients and supported B cell differentiation through IL-21. Finally, we identified a specific phenotype (CD45RA-PD-1+CD38+CXCR5-CD127-CD27+) of T cells linked to disease activity and IgG response during AIH. Conclusions: This work provides for the first time a deep characterization of rare circulating autoreactive CD4 T cells and the identification of their peripheral reservoir in AIH. We also propose a generic phenotype of pathogenic T cells related to AIH disease activity which will be essential to track, delineate and potentially target those pathogenic cells.

Project description:Background: During the past 25 years, selection of production traits highly increased pig production but diseases have emerged that may cause economic loss of extreme importance. Designing sustainable animal production that better balances productivity with resistance to disease is a major concern and challenge for the next decade. In order to address questions related to immunity and resistance to disease, it is necessary to increase knowledge on the pig immune system and to produce efficient tools dedicated to this species. In this context we produced a generic array enriched in immunity genes and validated its relevance by studying innate immune response of pigs by stimulating porcine mononuclear cells with lipopolysaccharide (LPS) or a mixture or phorboml myristate acetae (PMA) and ionomycin for 24 hours. Results: A long oligonucleotide-based chip was produced by combining a generic set of 13K probes targeting 8541 genes to a newly designed SLA-RI set that targets all genes and pseudogenes of the pig major histocompatibility complex region (SLA complex) in both orientations (906 probes) and immunity genes outside the SLA complex (2957 porbes). The porcine chip was referred to as SLA-RI/NRSP8-13K. Transcripotme analysis of PBMCs stimulated by LPS or PMA/ionomycin was carried out. Ten times more genes were up regulated after PMA/ionomycin stimulation by comparison to LPS stimulation. The LPS response was more related to the catalog Diseases and Disorders and the PMA/ionomycin response to the catalog Molecular and Cellular Function. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 genes. In addition, a very intense repression of THBS1 was observed, suggesting a predominant role of this matricellular glycoprotein during T/B cell stimulation by tumor inducers. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing and repression of MHC class II genes was observed after both stimulations. A significant reduction of antigen presentation to T cells was shared by the two types of responses that is likely to be due to separate mechanisms that need further elucidation. In addition, our results provided preliminary data suggesting a role of antisense transcripts mapping to the SLA complex during immune response. Conclusion: The SLA-RI/NRSP8-13K was highly relevant to. On the one hand the SLA-RI/NRSP8-13K was shown to be relevant and accurate to decipher two distinct innate immune responses by PBMCs indicating that this chip will constitute a valuable tool to further study immunity and resistance to disease in pig. On the other hand, the transcriptom analysis revealed specific and common features of the innate immune response according to stimulation that increase knowledge on pig immunity. Keywords: immune response activation in pig PBMCs Two-condition experiment, LPS stimulated PBMCs vs. mock-stimulated PBMCs, PMA_ionomycine stimulated PBMCs vs.mock-stimulated PBMCs. Biological replicates: 1 control, 1 LPS stimulated and 1 PMA_ionomycine stimulated from 7 animals independently grown and harvested. Dye-swap design. One replicate per array. 28 slides.

Project description:Background: During the past 25 years, selection of production traits highly increased pig production but diseases have emerged that may cause economic loss of extreme importance. Designing sustainable animal production that better balances productivity with resistance to disease is a major concern and challenge for the next decade. In order to address questions related to immunity and resistance to disease, it is necessary to increase knowledge on the pig immune system and to produce efficient tools dedicated to this species. In this context we produced a generic array enriched in immunity genes and validated its relevance by studying innate immune response of pigs by stimulating porcine mononuclear cells with lipopolysaccharide (LPS) or a mixture or phorboml myristate acetae (PMA) and ionomycin for 24 hours. Results: A long oligonucleotide-based chip was produced by combining a generic set of 13K probes targeting 8541 genes to a newly designed SLA-RI set that targets all genes and pseudogenes of the pig major histocompatibility complex region (SLA complex) in both orientations (906 probes) and immunity genes outside the SLA complex (2957 porbes). The porcine chip was referred to as SLA-RI/NRSP8-13K. Transcripotme analysis of PBMCs stimulated by LPS or PMA/ionomycin was carried out. Ten times more genes were up regulated after PMA/ionomycin stimulation by comparison to LPS stimulation. The LPS response was more related to the catalog Diseases and Disorders and the PMA/ionomycin response to the catalog Molecular and Cellular Function. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 genes. In addition, a very intense repression of THBS1 was observed, suggesting a predominant role of this matricellular glycoprotein during T/B cell stimulation by tumor inducers. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing and repression of MHC class II genes was observed after both stimulations. A significant reduction of antigen presentation to T cells was shared by the two types of responses that is likely to be due to separate mechanisms that need further elucidation. In addition, our results provided preliminary data suggesting a role of antisense transcripts mapping to the SLA complex during immune response. Conclusion: The SLA-RI/NRSP8-13K was highly relevant to. On the one hand the SLA-RI/NRSP8-13K was shown to be relevant and accurate to decipher two distinct innate immune responses by PBMCs indicating that this chip will constitute a valuable tool to further study immunity and resistance to disease in pig. On the other hand, the transcriptom analysis revealed specific and common features of the innate immune response according to stimulation that increase knowledge on pig immunity. Keywords: immune response activation in pig PBMCs

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