IntroductionDespite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+T cells and monocytes. However, its specific role in the HCC TME remains unclear.MethodsIn this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings.ResultsFrom CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+TRMin TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+TRMfrom HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+CD8+T cells and CD38+PD-1+TRMwere significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease.ConclusionTaken together, the concurrent expression of CD38 with exhaustion markers on CD8+TRMunderpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.