T Cell Exhaustion in the Cervical Cancer Tumor Microenvironment: PD-1 Overexpression and Co-Expression with TIGIT, Tim-3, LAG-3, and NKG2A.

Objective: T cell exhaustion is a major mechanism of immune evasion in cancer, characterized by the sustained expression of multiple inhibitory receptors. This study aimed to evaluate the expression of immune checkpoints in peripheral and tumor-infiltrating CD8(+) T cells from cervical cancer patients. Methods: We enrolled 104 participants: 37 treatment-naïve patients, 36 treated patients, and 31 age-matched healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from all participants. Ten cervical biopsies were collected for tumor-infiltrating lymphocyte (TIL) isolation and paraffin fixation. Immune checkpoint expression was analyzed by multiparametric flow cytometry and immunohistochemistry. Results: In peripheral CD8(+) T cells, we found a significant upregulation of exhaustion-associated markers PD-1, TIGIT, Tim-3, and LAG-3. In the tumor infiltrating lymphocytes, these same molecules, with the addition of NKG2A, were notably upregulated further. While BTLA and NKG2A showed no systemic changes, NKG2A increased in TILs and BTLA decreased in TILs. The co-expression of PD-1 with TIGIT, Tim-3, LAG-3, and NKG2A was notably enriched between 2- and 6-fold in TILs compared with patient PBMCs. The tumor microenvironment was highly immunosuppressive, characterized by enrichment with PD-1, PD-L1, and TIGIT; TIGIT was notably upregulated in locally advanced versus early-stage tumors. Conclusions: Our findings highlight the strongly immunosuppressive environment of cervical tumors in treatment-naïve patients and the presence of elevated inhibitory checkpoint expression in peripheral blood of both pre- and post-treatment patients. These results underscore the importance of investigating immune regulation within the tumor site itself and suggest that immune checkpoint co-expression may serve as a biomarker of T cell exhaustion and therapeutic resistance. Understanding how treatment alters these pathways could guide rational combination immunotherapies to restore CD8(+) T cell function in cervical cancer.