Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

Abstract

Abstract Background & Aims In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-Soluble Liver Antigen (SLA or SepSecs) autoantibodies is associated with significantly reduced overall survival, 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 restimulation 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 TCR repertoire in n=5 AIH patients. 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=46 AIH patients and n=18 non-alcoholic steatohepatitis (NASH) controls. Results Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1 + CXCR5 − CCR6 − CD27 + phenotype. ScRNA-seq revealed their pro-inflammatory/B-Helper profile ( IL21, IFNG, TIGIT , CTLA4, NR3C1, CD109, KLRB1 and CLEC2D) . Autoreactive TCR clonotypes were restricted to the memory PD-1 + CXCR5 − CD4 T cells. This subset was significantly increased in the blood of AIH patients and supported B cell differentiation through IL-21. Finally, we identified a specific phenotype (PD-1 + CD38 + CD27 + CD127 − CXCR5 − ) of CD4 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 CD4 T cells related to AIH disease activity.