Single-cell transcriptome and TCR repertoire sequencing of cholinergic CD4+ T cells in liver cancer

ACh was originally isolated from spleen back in 1929, however, its contribution to immune regulation has only recently been appreciated. Subsets of both T and B lymphocytes have been found to express choline acetyltransferase (Chat) and produce ACh. To date, Chat-expressing T cells have been described as relaying neural signals in the spleen to modulate immune responses; regulating blood pressure; and promoting anti-viral immune responses. In a murine multi-hit model of hepatocellular carcinoma, we observed activation of the adaptive immune response and induction of Chat-expressing CD4+ T cells. These cholinergic T cells curtail the development of liver cancer by supporting anti-tumor immune responses. We used single-cell RNA sequencing to investigate the transcriptome and TCR repertoire of the Chat-expressing CD4+ T cells in healthy and HCC-bearing livers. Overall design: HCC was induced in Chat-GFP mice by Myc overexpression and combined Trp53 and Pten ablation. HCC livers (from two male and two female mice) and control livers (from sex-matched littermates) were collected for hepatic mononuclear cell (MNC) isolation. Hepatic MNCs were stained with antibodies for CD4, CD8, CD19, TCR-ß, NK1.1, CD45 and CD1d tetramer, as well as with barcode antibodies (TotalSeq-C0304, C0305, C0306, or C0307) to hashtag cells from individual mice of the HCC or control group. CD45+DAPI-NK1.1-CD1dTetramer-CD19-TCR-ß+CD4+CD8-GFP+ (Chat-GFP+ CD4 T cells) and CD45+DAPI-NK1.1-CD1dTetramer-CD19-TCR-ß+CD4+CD8-GFP- (Chat-GFP- CD4 T cells) populations were sorted. The two CD4 T cell compartments were individually sorted from each mouse. The same CD4 T compartments from mice under the same treatment were pooled into 4 samples: control Chat-GFP+, control Chat-GFP-, HCC Chat-GFP+ and HCC Chat-GFP-. After sorting and pooling, the samples were immediately submitted to the Princess Margaret Genomics Centre for downstream processing.