Beyond Canonical Immune Checkpoints: Overexpression of TNFRSF Members 4‐1BB and OX‐40 Marks T Cells Exhibiting Phenotypic Features of Exhaustion in Cervical Carcinoma

ABSTRACT T cells are pivotal in combating cancer; however, they can become exhausted during tumour progression, losing their cytotoxic capacity and upregulating inhibitory receptors including PD‐1 and TIGIT. While checkpoint blockade has emerged as a potent treatment option for numerous cancers, patient selection, long‐term efficacy, and adverse effects still remain an issue. For these reasons, it is important to investigate other pathways that might lead to selective reactivation of the immune system. Co‐stimulatory TNFRSF receptors, including 4‐1BB and OX‐40, have emerged as promising targets for reactivating exhausted T cells. However, their expression on exhausted peripheral and tumour‐infiltrating lymphocytes (TILs) is not well characterised, particularly in cervical cancer (CC), which remains the leading cause of gynaecological cancer mortality in low‐ and middle‐income countries. To investigate the expression of these receptors, PBMCs were collected from CC patients and healthy donors, along with TILs from tumour biopsies, and analysed using multiparametric flow cytometry. Our findings revealed an increased population of phenotypically exhausted (PD‐1 + TIGIT + ) CD4 + and CD8 + T cells in TILs, and, to a lesser extent, in peripheral blood and from CC patients. These exhausted T cell subsets exhibited selective overexpression of 4‐1BB and OX‐40 compared to phenotypically non‐exhausted cells (PD‐1 − TIGIT − ). In TILs, 4‐1BB was overexpressed 12.7‐fold in CD8 cells with the exhausted phenotype, OX‐40 was overexpressed 3.3‐fold; in CD4 cells with the exhausted phenotype, the overexpression was 7.8× and 3.8× for 4‐1BB and OX‐40, respectively. CD8 and CD4 T cells that were PD‐1 + TIGIT+ 4‐1BB+ were 7.3× and 16× more likely to be found in the tumour versus peripheral blood. Additionally, subpopulations of PD‐1 high T cells were significantly elevated in the tumour‐infiltrating T cells and TIGIT expression was positively associated with PD‐1 levels in peripheral patient CD8 + and CD4 + T cells, potentially indicating an advanced state of exhaustion. These findings suggest that TNFRSF members, especially 4‐1BB, may serve as potential immunotherapeutic targets for reinvigorating exhausted T cells in CC.