FasL-mediated death of activated intratumoral T cells drives secondary resistance to cancer immunotherapy

Cancer progression following an initial response to immunotherapy (secondary resistance; 2°R) is a major and poorly understood problem. We treated mice bearing B16 melanoma with a combination of a regulatory T cell (Treg) depleting, non-IL-2 blocking antibody (anti-CD25NIB) and an autologous cancer cell vaccine (GVAX). The regimen yielded initial tumor shrinkage followed by 2°R; lethal progression occurred in ~90% of partially responsive (PR) tumors by days 35-50. Cell lines derived from 2°R tumors retained treatment sensitivity upon re-implantation into naïve mice, suggesting resistance was related to a loss of immune control over time. Profiling PR and 2°R tumors by flow cytometry and single-cell RNA/TCR sequencing, we found that activated CD8⁺ T cells with tumor-reactive features declined in abundance, whereas non-activated T cells and Tregs increased. Activated CD8⁺ cells showed heightened TCR stimulation, clonal expansion, and expression of apoptotic signatures and death receptors, including Fas. Their loss was not explained by lymph node accumulation or differentiation to non-activated states. These findings were validated in a clinically relevant MC38 colon carcinoma model treated with anti-PD-L1 checkpoint blockade, confirming that depletion of activated, tumor-reactive clones is a shared mechanism of 2°R across therapeutic modalities. Fas ligand (FasL) blockade reversed this loss and prolonged survival. Longitudinal transcriptional and tissue staining data from human checkpoint blockade studies similarly indicated that activated T cells decline in abundance over time and at 2°R. These findings implicate death of activated T cells as a mechanism of 2°R and suggest Fas-FasL blockade may extend response durability.