The immune profile of circulating autoreactive CD4 T cells is imprinted through tissue activation during autoimmune liver diseases (TTm)

Autoimmune liver diseases (AILD) are immune-mediated disorders in which CD4 T cells play a central role. However, the link between circulating self-antigen-specific CD4 T cells and the targeted tissue has not been extensively studied in AILD. We hypothesized that circulating autoreactive CD4 T cells were clonally and functionally related to dominant intra-hepatic pathogenic CD4 T cell clones. Single cell transcriptomic analysis of circulating self-antigen-specific CD4 T cells revealed a specific B-helper and immuno-exhausted transcriptional profile, which was conserved for different autoantigens, but distinct from several other types of foreign antigen specificities. In the blood, the dominant hepatic CD4 T cell clones had a similar transcriptomic signature and were enriched in the PD-1+ TIGIT+ HLA-DR+ CD4 T cell subset. In a mouse model, antigen-specific CD4 T cells acquired the immuno-exhausted transcriptional profile when they accumulated in the liver after local antigen reactivity. Locally, immune checkpoint molecules controlled the response of antigen-specific CD4 T cells responsible for liver damage. Our study reveals the origin and biology of liver-derived autoreactive CD4 T cells in the blood of AILD patients that are imprinted by the liver environment, and suggest a dysregulation of the immune checkpoint molecules pathways. Our study enables tracking and isolating circulating autoreactive CD4 T cells for future diagnostic and therapeutic purposes. Overall design: For tetramer staining, 30 million PBMCs in 200 µL of 5% human serum RPMI medium were stained with 20 µg/mL PE-labeled tetramers at room temperature for 100 minutes. Cells were washed and incubated with anti-PE magnetic beads (Miltenyi Biotec, Germany), and a one-tenth fraction was saved for analysis. The other fraction was passed through a magnetic column (Miltenyi Biotec). After enrichment, cells were stained with appropriate antibodies (Supplementary table 1). Sepsecs187-197 HLA-DRB1 03:01 tetramer was generated by the tetramer core at the Benaroya research institute (Seattle, USA). For scRNA-seq of tetramer-stained CD4 T cells, we used an updated version of FB5P-seq (Flash-FB5P-seq) modified to implement advances developed by Hahaut et al. in the Flash-seq protocol59.