TOX promotes the exhaustion of antitumor CD8+ T cells by preventing PD1 degradation in hepatocellular carcinoma

Abstract Background & Aims The thymocyte selection-associated high mobility group box protein (TOX) plays a vital role in T cell development and differentiation, however, its role in T cell exhaustion was unexplored. Here, we aim to investigate the role of TOX in regulating antitumor effect of CD8 + T cells in hepatocellular carcinoma (HCC). Methods PD1 - TIM3 - /PD1 int TIM3 + /PD1 hi TIM3 + TIL-CD8 + T cells, were sorted by flow cytometer and subjected to transcriptome sequencing analysis. Upregulated TOX expression was validated in human TIL-CD8 + T cells and in mouse infection and tumor models by flow cytometer. The antitumor function of CD8 + T cells with TOX downregulation or overexpression was studied in mouse HCC model and HCC patient derived xenograft mouse model. Transcriptome sequencing analysis was performed in TOX-overexpressing and control CD8 + T cells. The mechanism underlying the TOX-mediated regulation of PD1 expression was studied in TIL-CD8 + T cells through laser confocal detection, immune co-precipitation and flow cytometer. Results TOX was upregulated in exhausted CD8 + T cells in HCC. TOX downregulation in CD8 + T cells inhibited tumor growth, increased of CD8 + T cell infiltration, alleviated CD8 + T cell exhaustion and improved anti-PD1 response of CD8 + T cells. In mechanism, the binding of TOX to PD1 in the cytoplasm facilitated the endocytic recycling of PD1, thus maintaining abundant PD1 expression at cell surface. High expression of TOX in peripheral CD8 + T cells was in correlation with poorer anti-PD1 response and prognosis. 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. Lay summary Abundant TOX expression in CD8 + T cells impairs their antitumor function of in hepatocellular carcinoma. Mechanically, TOX reduces PD1 degradation and promote PD1 translocate to cell surface in CD8 + T cells, thus maintaining highly PD1 expression at cell surface. Downregulating TOX expression improves the antitumor function of CD8 + T cells, which shows synergetic role with anti-PD1 therapy, highlighting a promising strategy for enhancement of cancer immunotherapy.