scRNA-seq of human intra-hepatic CD4? T cells reveals unique Treg transcriptional identities and two steady-state tissue-adaptation programs across healthy and HCC liver samples

We employed a single-cell sequencing approach using the 10x Genomics platform, including scRNA-seq, CITE-seq, and paired TCR libraries, to investigate the molecular programs of human tissue-resident regulatory T cells (Tregs). By analyzing CD4? T cells from healthy liver and matched peripheral blood mononuclear cells (PBMCs), as well as hepatocellular carcinoma (HCC) tissue with paired non-tumoral liver samples, we characterized the transcriptional adaptations of intra-hepatic Tregs under steady-state conditions and their reprogramming within the HCC tumor microenvironment. Overall design: PBMCs were freshly isolated, and liver specimens were enzymatically digested immediately after surgical collection. The resulting single-cell suspensions were cryopreserved and stored until analysis. Upon thawing, CD4? T cells were enriched using magnetic bead–based negative selection, followed by staining with CITE-seq antibodies and additional markers for cell sorting. Sorted CD4? populations were then processed for scRNA-seq, including CITE-seq and TCR library preparations.