In vivo genome-wide CRISPR screen identifies key factors modulating antigen-specific CD8+ T cells responses to recombinant MVA viral immunotherapy

Summary: Abstract Body: Viral-based cancer immunotherapy represents a novel and versatile platform to modulate the tumor microenvironment (TME) by activating innate and adaptive immune responses. We have previously reported that intratumoral delivery of recombinant MVA (rMVA) deleting cGAS inhibitor E5R and expressing membrane-anchored Flt3L and OX40L generates potent antitumor effects by activating the cGAS/STING DNA-sensing pathway, depleting OX40hi Tregs, and activating CD8+ T cells. In this study, we show that local modulation of the TME by immunogenic viruses is critical for anti-tumor immunity, independent of recruiting antigen-specific T cells from secondary lymphoid organs. Using FTY-720, an inhibitor of T-cell egress, we show that anti-tumor activity induced by intratumoral (IT) rMVA remains intact. Integrated single-cell RNA and TCR sequencing revealed significant reprogramming of CD8+ T cells within injected tumors. Stem-like TCF7-positive cells exhibited the lowest clonality, while greater clonal expansion was observed in effector and proliferating populations. Following rMVA injection, stem-like T cells decreased, while clone proliferation increased in effector and proliferating cells. Interestingly, with FTY-720, T-cell clone proliferation further increased in these populations. These findings underscore the critical role of local TME modulation by rMVA in driving T-cell reprogramming and clonal expansion, independent of T-cell recruitment. An in vivo CRISPR screen using antigen-specific OT1 CD8+ T cells expressing Cas9 identified Socs1 and Zc3h12a as top negative regulators and Gpn2 and Adra2A as top positive regulators in response to rMVA therapy. These findings highlight the critical role of local TME modulation by viral immunotherapy and provide insights into mechanisms driving T-cell activation and clonal expansion. Ning Yang¹, Yueqi Wang^{1, 2}, Yi Wang¹, Shanza Baseer Tariq¹, Charles M. Rice², Liang Deng^{1, 2} 1. Memorial Sloan Kettering Cancer Center, New York, NY, United States. 2. The Rockefeller University, New York, NY, United States. Pigmentation, Melanoma, and Melanoma Immune Surveillance