T cell repertoire in cholangiocarcinoma lesion and liver of a mouse affected with primary biliary cholangitis

We report in this study the expansion of T cell clonotypes inflitrating both ectopic cholangiocarcinoma (CCA) and liver tissues in a mouse suffering from primary biliary cholangitis (PBC) (Paillet et al, J Exp Med, 2021). We wondered whether the specificity of T cells found in PBC livers and tumors developing post-PBC might be shared to some extent, comparing them to circulating T cells from the blood (Paillet et al, J Exp Med, 2021). Single-cell TCR sequencing coupled to single-cell RNA sequencing (scTCR/RNA-seq) led to the identification of 8112 distinct clonotypes with a hierarchy of clonotypes enrichment that was significantly higher in PBC livers than in peripheral blood, as well as in CCA compared to blood and liver. Indeed, 12 distinct clonotypes accounted for ~20% of the tumor T cell infiltrate. The overlap of clonotypes between liver and tumor was superior to the overlap between blood and liver, or blood and tumor. Among the 128 clonotypes enriched (i.e. accounting for > 0.1% of total T cells infiltrating the tissue) in the tumor, 25 were also detected and enriched in the liver. Two clonotypes (#2 and #13) were particularly expanded, each representing over 0.5% of the total T cell population in both liver and tumor post-PBC (Paillet et al, J Exp Med, 2021). Bioinformatic analysis of the scRNA-seq data of total T cells isolated from the liver and tumor of a PBC mouse revealed a net distinction between the transcriptome of T lymphocytes located in the two organs. Only one phenotypic cluster (h) was observed in both liver and tumor. The 25 liver/tumor dual-enriched (>0.1%) clonotypes were spread across few clusters (mostly c, j, k in the tumor; b and i in the liver). Among these clusters, three corresponded to CD8+ T cells (c, i, k) and two corresponded to CD4+ T cells (b, j). They exhibited a preponderant Tc1/Th1 polarization as illustrated by the expression of the enhancers Tbx21 and/or Eomes, and of effector molecules, namely IFNγ for Th1 (b, j), or the cytotoxic markers granzymes B and K for Tc1 (c, i, k) (Paillet et al, J Exp Med, 2021). Interestingly, the two clusters containing most of the enriched CD4+ T cell clonotypes presented a multifunctional signature characterized by the co-expression of IFNγ along with either IL4 (cluster b in the liver) or GzmB and KLRG1 (cluster j in the tumor). Clearly, the two most enriched clonotypes shared between CCA tumor and PBC liver belonged to the category of CD8+ cytotoxic T lymphocytes (clusters c + k in tumor, and cluster i in the liver). Moreover, they showed signs of prolonged activation/exhaustion with the expression of Pdcd1 (encoding PD-1), Lag3, Harvc2/Tim3 and Tigit, yet in a lesser extent than another cluster of CD8+ T cells (tumor cluster f) that contains only little shared clonotypes (Paillet et al, J Exp Med, 2021). Autoimmune primary biliary cholangitis (PBC) was induced in a C57Bl/6 mice as described before: Chang et al., 2014; Wakabayashi et al., 2008. Briefly, mice were immunized with 100 µg of 2-octynoic acid BSA conjugate (2OA-BSA) intraperitoneally (i.p.) in a mix of PBS and complete Freund’s adjuvant (CFA, Sigma-Aldrich) at day 0 and were reboosted at days 14 and 28 with 2OA-BSA and incomplete Freund’s Adjuvant (IFA, Sigma Aldrich). Concomitantly with the two first injections of 2OA-BSA, mice were also injected intravenously (i.v.) with 2 μg of α-galactosylceramide (α-GalCer, Abcam) in PBS. Thirty-five days after the beginning of cholangitis induction, 3x10^6 SB1-JP4 cells (murine cholangiocarcinoma cells) were resuspended in 100 µL of PBS and injected subcutaneously, under anesthesia with 2.5% isoflurane, in the right flank of C57Bl/6 mice. For sinlge cell (sc)-RNA-seq and sc-TCR (T cell receptor)-seq, blood was drawn 7 days before and 34 days after PBC induction. One hundred five days after cholangitis induction (equivalent to 70 days after the engraftment of the CCA cells), blood, liver and the CCA tumor were collected from a PBC mouse (Paillet at al, J Exp Med, 2021).