Mapping Immune Exhaustion Markers in CD8⁺ T Cells within Syngeneic Tumor Microenvironments

Abstract The functional state of CD8⁺ T cells within the tumor microenvironment (TME) critically determines antitumor immunity and immunotherapy outcome. However, how tumor location, tumor cell intrinsic features and progression kinetics shape CD8⁺ T cell exhaustion remains poorly defined. We performed a spatial and temporal characterization of immune infiltration and CD8⁺ T cell phenotypes across multiple syngeneic murine tumor models implanted at subcutaneous (SC), orthotopic, and metastatic sites. We examined tumor-infiltrating immune cell dynamics, immune checkpoint (IC) acquisition, and antigen-specific T cell responses at baseline and under anti–PD-L1 treatment. The immune composition of the TME varied with tumor site, developmental stage, and tumor-intrinsic features. Orthotopic and metastatic tumors displayed distinct lymphoid and myeloid infiltration profiles compared to SC tumors. Among head and neck (HN) cancer models, HPV − MOC1 tumors were sensitive to PD-L1 blockade, with expansion of antigen-specific CD8⁺ T cells whereas HPV + TC1 tumors remained refractory despite harboring E7-specific CD8⁺ T cells. Across models, CD8⁺ T cells acquired ICs in a temporally ordered and tumor-dependent manner. TIM-3 emerged as an early marker of exhaustion preceding PD-1 expression, and advanced exhausted populations accumulated preferentially in slower-growing tumors. Taking all models into account, CD8⁺ T cell exhaustion appeared as a dynamic and context-dependent process predominantly governed by tumor antigenic profile and growth kinetics relative to anatomical location. These findings establish a mechanistic framework for understanding T cell differentiation across tumor types and emphasize the need for spatially and temporally faithful preclinical models to guide immunotherapy development.