Study On The Mechanism Of Invasive CD8~+T Cell Depletion In Liver Cancer

Part 1 Genetic and phenotypic difference in CD8~+T cell exhaustion in liver cancerBackground&Aims:Recent studies published have suggested that T cell exhaustion exists both in chronic infection and cancer.However,to date,few studies have investigated their differences.Here we designed this study to explore the genetic and phenotypic difference in CD8~+T cell exhaustion between chronic hepatitis B(CHB)and hepatocellular carcinoma(HCC).Methods:In this study,we assayed the phenotypes and functional states of CD8~+T cells separating from human chronic hepatitis B(CHB)tissues and hepatocellular carcinoma(HCC)tissues,and re-analyze the single-cell sequencing data(GSE98638)of T cells from HBV-positive liver cancers.Results:CD8~+T cells from liver tissues of both CHB and HCC showed high levels of exhaustion markers(PD1,TIM3,LAG3 and CTLA4),decreased proliferation(Ki67)and cell activity(CD69),and reduced production of effector cytokines(IFN-γ,IL-2 and TNF-α).Comparing to CD8~+T cells from CHB tissues,those from HCC tissue showed higher expression levels of exhaustion markers,lower levels of proliferation,cell activity and the production of effector cytokines.Cluster analysis showed that exhaustion associated genes in CHB and liver cancerConclusion:CD8~+T cell exhaustion existed both in CHB and HCC,but the phenotypes,functional states and underlying mechanisms are somewhat different between the two.Part 2 TOX promotes the exhaustion of antitumor CD8+ T cells by preventing PD1 degradation in hepatocellular carcinomaBackground & Aims: T cell exhaustion drives compromised antitumor immunity.We investigated the key regulator of T cell exhaustion in hepatocellular carcinoma(HCC),aiming to find potential therapeutic targets to restore the antitumor function of exhausted CD8+ T cells.Methods: Fully functional(PD1-TIM3-),partially exhausted(PD1intTIM3+)and severely exhausted(PD1hiTIM3+)TIL-CD8+ T cells,were sorted by flow cytometry and subjected to transcriptome sequencing analysis.CD8+ T cells partially or completely deficient in Tox were transferred to Cd8-/-mice bearing HCC.TIL-CD8+ T cells with TOX knockdown or overexpression were transferred into patient-derived tumor xenografts(PDX)HCC mice in combination with anti-PD1 therapy.Furthermore,inhibitory receptors,transcription factors,cytokines,cell proliferation and apoptosis were assayed in TIL-CD8+ T cells.Transcriptome sequencing analysis was performed in TOX-overexpressing and control CD8+ T cells.The mechanism underlying the regulation of PD1 expression by TOX was studied in TIL-CD8+ T cells.Results: We found that TOX was upregulated in exhausted CD8+ T cells in HCC.The knockdown of TOX in CD8+ T cells inhibited tumor growth,increased the infiltration of CD8+ T cell in tumors,and alleviated CD8+ T cell exhaustion and improved the response of CD8+ T cells to anti-PD1 therapy in a PDX HCC model.TOX facilitated the endocytic recycling of PD1 in TIL-CD8+ T cells,thus maintaining abundant PD1 expression.High expression of TOX in peripheral CD8+ T cells was in correlation with larger tumor size,poorer differentiation and laterTNM stages.Conclusions: TOX promotes CD8+ T cell exhaustion in HCC by regulating endocytic recycling of PD1.Downregulating TOX expression in CD8+ T cells exerts synergistic effects with anti-PD1 therapy,highlighting a promising strategy for cancer immunotherapy.