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

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. For mini-bulk RNA-seq of in vitro stimulated CD4 T cells, we used a slightly modified version of the Flash-FB5P-seq protocol described above. First, CD4 T cell subsets were sorted on BD FACSAriaII. 500 to 2000 cells per well were stimulated in presence of 2.5µg/ml anti-CD3 and 1µg/ml anti-CD28. After 24h, cells were washed gently in PBS and resuspended in 4µL of specific lysis buffer with the composition described above except for ERCC spike-in mix which we used 10-time more concentrated (0.125 pg/µl). We performed RT-PCR with 16 µl of the RT-PCR mix described above, using 16 cycles of PCR for cDNA amplification (instead of 22 cycles for single-cell assay). Sequencing libraries were prepared and sequenced as described above for Flash-FB5P-seq.