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^negPD-1^high CD8⁺ TILs, 4-1BB^posPD-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.