4‐1BB Delineates Distinct Activation Status of Exhausted Tumor‐Infiltrating CD8+ T cells in Hepatocellular Carcinoma

BACKGROUND AND AIMS: Targeting costimulatory receptors with agonistic antibodies is a promising cancer immunotherapy option. We aimed to investigate costimulatory receptor expression, particularly 4-1BB (CD137 or tumor necrosis factor receptor superfamily member 9), on tumor-infiltrating CD8(+) T cells (CD8(+) tumor-infiltrating lymphocytes [TILs]) and its association with distinct T-cell activation features among exhausted CD8(+) TILs in hepatocellular carcinoma (HCC). APPROACH AND RESULTS: Tumor tissues, adjacent nontumor tissues, and peripheral blood were collected from HCC patients undergoing surgical resection (n = 79). Lymphocytes were isolated and used for multicolor flow cytometry, RNA-sequencing, and in vitro functional restoration assays. Among the examined costimulatory receptors, 4-1BB was most prominently expressed on CD8(+) TILs. 4-1BB expression was almost exclusively detected on CD8(+) T cells in the tumor-especially on programmed death 1 (PD-1)(high) cells and not PD-1(int) and PD-1(neg) cells. Compared to PD-1(int) and 4-1BB(neg) PD-1(high) CD8(+) TILs, 4-1BB(pos) PD-1(high) CD8(+) TILs exhibited higher levels of tumor reactivity and T-cell activation markers and significant enrichment for T-cell activation gene signatures. Per-patient analysis revealed positive correlations between percentages of 4-1BB(pos) cells among CD8(+) TILs and levels of parameters of tumor reactivity and T-cell activation. Among highly exhausted PD-1(high) CD8(+) TILs, 4-1BB(pos) cells harbored higher proportions of cells with proliferative and reinvigoration potential. Our 4-1BB-related gene signature predicted survival outcomes of HCC patients in the The Cancer Genome Atlas cohort. 4-1BB agonistic antibodies enhanced the function of CD8(+) TILs and further enhanced the anti-PD-1-mediated reinvigoration of CD8(+) TILs, especially in cases showing high levels of T-cell activation. CONCLUSION: 4-1BB expression on CD8(+) TILs represents a distinct activation state among highly exhausted CD8(+) T cells in HCC. 4-1BB costimulation with agonistic antibodies may be a promising strategy for treating HCCs exhibiting prominent T-cell activation.