P-01.09 Fas-L mediated death of activated intratumoral T cells drives secondary resistance to Treg depletion in cancer

<h3>Background</h3> Secondary resistance (2°R) to immunotherapy is a poorly understood phenomenon, where cancer progresses after an initial positive response to treatment. Understanding the mechanisms behind this 2°R is crucial for improving the durability of immunotherapy responses. In this study, we explore the evolution of 2°R using a combination therapy model in a murine model. <h3>Materials and Methods</h3> We modelled 2°R by treating mice bearing B16 melanoma tumours with a novel non-IL-2 blocking antibody (anti-CD25^NIB) designed to deplete regulatory T cells (Tregs), combined with an autologous cancer cell vaccine (GVAX). This regimen induced initial tumour shrinkage (partial response), followed in almost all cases by tumor progression, recapitulating 2°R. To investigate evolution of the immune microenvironment prior to clinical progression, we carried out high dimensional flow cytometry and single cell whole transcriptome and T cell receptor targeted RNA sequencing (scRNAseq) on partially responsive and 2°R tumours. We performed FasL antibody blockade to assess its impact on activated CD8+ T cell and tumor progression. Longitudinal RNA-seq data from human checkpoint immunotherapy studies were analyzed to compare to the murine findings. <h3>Results</h3> During the period of partial response, there was a steady decrease in the abundance of activated T cells and an increase in non-activated T cells and Treg cells. Repeated depletion of re-accumulating Tregs had no impact on tumour growth suggesting the importance of activated CD8 loss. These populations had features of tumor reactivity including TCR stimulation, clonal expansion and expression of TCRs with sequence similarity to known antigen reactive clonotypes. There was no clonal relationship between the lost activated populations and the non-activated cells that became more abundant at relapse, suggesting death of activated cells. Transcriptional and protein expression data including Fas expression, were consistent with T cell death. Consolidation therapy with FasL blockade significantly increased the abundance of activated CD8+ T cells and improved the survival of tumor-bearing mice. Analysis of longitudinal scRNAseq data from patients undergoing cancer therapy with immune checkpoint blockade supported our finding that activated T cells are prone to death and undergo a reduction in abundance over time. <h3>Conclusions</h3> We provide evidence that death of activated CD8 T cells underlies acquisition of secondary resistance to immunotherapy. Interference with T cell death signaling pathways could be therapeutically relevant to enhance long-term immunotherapy outcomes, warranting further clinical investigation. <b>C. Qing:</b> None. <b>E. Ghorani:</b> None. <b>K. Foster:</b> None. <b>I. Uddin:</b> None. <b>G. Beattie:</b> None. <b>F. Galvez-Cancino:</b> None. <b>H. Walczak:</b> None. <b>M. Amann:</b> A. Employment (full or part-time); Significant; Roche. <b>K.S. Peggs:</b> None. <b>S.A. Quezada:</b> None.