FasL-mediated death of activated intratumoral T cells drives secondary resistance to cancer immunotherapy
Cancer progression following an initial response to immunotherapy (secondary resistance; 2oR) 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 2oR; lethal progression occurred in ~90% of partially responsive (PR) tumors by days 35-50. Cell lines derived from 2oR tumors retained treatment sensitivity upon re-implantation into naive mice, suggesting resistance was related to a loss of immune control over time. Profiling PR and 2oR 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 2oR 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 2oR. These findings implicate death of activated T cells as a mechanism of 2oR and suggest Fas-FasL blockade may extend response durability.