Abstract 1340: The zinc finger 7 motif of TNFAIP3/A20 enforces a critical block in exhausted CD8 T cell degranulation and represents a potent target for enhancing adoptive cellular therapies for cancer.

Abstract Introduction: CD8 T cells eliminate tumors through effector cytokines (IFNgamma, TNF) and cytotoxic granule protein release (perforin, granzymes). The relative contribution of these mechanisms varies across tumor contexts, suggesting distinct modes of regulation. CD8 T cell exhaustion within the tumor microenvironment limits their cytotoxic potential and poses a major obstacle for cancer immunotherapy and adoptive cellular therapies (ACTs). While most studies of exhausted CD8 T cell (Tex) dysfunction have focused on impaired cytokine production, the regulation of perforin- and granzyme-mediated cytotoxicity, as well as the pathways governing CD8 T cell degranulation in general, remain poorly defined. Methods & Results: We performed a genome-wide CRISPR knockout screen in repetitively stimulated human T cells and identified the multifunctional ubiquitin-modifying enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3, or A20) as a key suppressor of T cell persistence and cytotoxicity in the face of exhaustive stress. Using a large language model, base editor tiling screen, and tumor models in hypomorphic A20 knock-in mice, we pinpointed the zinc finger (ZF7) motif of A20 as the crucial regulator of CD8 T cell cytotoxicity. We then incorporated base-editing into standard human ACT production workflow to introduce a single inactivating missense mutation in ZF7 that significantly improved ACT efficacy in preclinical leukemia and melanoma models. Although ZF7-edited T cells produced more IFNgamma and TNF, genetic ablation experiments revealed these cytokines were dispensable for improved tumor control. Instead, through in vivo and in vitro functional assays, we revealed an underappreciated block in perforin and granzyme degranulation — rather than expression — that was relieved by ZF7 ablation. Finally, genetic epistasis analyses uncovered a surprising role for receptor-interacting serine/threonine-protein kinase 3 (RIPK3) — a canonical activator of necroptosis without a defined role in CD8 T cell effector function — as the critical orchestrator of degranulation in ZF7-deficient cells. Conclusions: Our findings highlight an underrecognized yet critical post-translational block in Tex degranulation, regulated independently of cytokine production, and relieved upon targeted disruption of the ZF7 motif in a RIPK3-dependent manner. These results demonstrate that selective targeting of central suppressors such as A20 can not only reverse CD8 T cell exhaustion but also engage alternative signaling programs to unleash cytotoxicity. These insights address critical gaps in our understanding of the biological networks that restrain perforin-dependent cytotoxicity in Tex and provide a foundation for enhancing the potency and durability of ACTs. Citation Format: Adam Blaisdell, Stefanie Bachl, Luis R. Sandoval, Carter Ching, Manu Prabandham, Rommel Advincula, Nika Lenci, Alexander Marson, Chun Jimmie Ye, Barbara A. Malynn, Justin Eyquem, Julia Carnevale, Averil Ma, . The zinc finger 7 motif of TNFAIP3/A20 enforces a critical block in exhausted CD8 T cell degranulation and represents a potent target for enhancing adoptive cellular therapies for cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1340.