BackgroundCD8+ T cells differentiate into exhausted status within tumors, including hepatocellular carcinoma (HCC), which constitutes a solid barrier to effective anti-tumor immunity. A detailed characterization of exhausted T cells and their prognostic value in HCC is lacking.MethodsWe collected fresh tumor tissues with adjacent non-tumor liver tissues and blood specimens of 56 HCC patients, as well as archived samples from two independent cohorts of HCC patients (n = 358 and n = 254), who underwent surgical resection. Flow cytometry and multiplex immunostaining were used to characterize CD8+ T cells. Patient prognosis was evaluated by Kaplan-Meier analysis and Cox regression analysis.ResultsCD8+ T cells were classified into three distinct subpopulations: PD1Hi, PD1Int and PD1−. PD1Hi CD8+ T cells were significantly enriched in tumor compared to adjacent non-tumor liver tissues. PD1Hi CD8+ T cells highly expressed exhaustion-related inhibitory receptors (TIM3, CTLA-4, etc.) and transcription factors (Eomes, BATF, etc.). In addition, PD1Hi CD8+ T cells expressed low levels of cytotoxic molecules and displayed a compromised capacity to produce pro-inflammatory cytokines while the expression of anti-inflammatory IL-10 was up-regulated following mitotic stimulation. Furthermore, PD1Hi CD8+ T cells shared features with tissue resident memory T cells and were also characterized in an aberrantly activated status with an apoptosis-prone potential. In two independent cohorts of HCC patients (n = 358 and n = 254), we demonstrated that PD1Hi or TIM3+PD1Hi CD8+ T cells were significantly correlated with poor prognosis, and the latter was positioned in close proximity to PD-L1+ tumor associated macrophages.ConclusionThe current study unveils the unique features of PD1Hi CD8+ exhausted T cells in HCC, and also suggests that exhausted T cells could act as a biomarker to select the most care-demanding patients for tailored therapies.
The CD4 ؉ CD25 ؉ FOXP3 ؉ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-␥, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.
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