Abstract 3966: NK receptors enable TCR-mediated CTL recognition and killing of cancer cells expressing non-mutated tumor-associated antigens

Abstract Background: Responsiveness or ignorance of antigens presented to naïve T cells by dendritic cells (DC) involves the CD28-driven “signal 2”, but it is unclear how activated CTLs respond to the same non-mutated tumor-associated antigens (TAAs) presented by cancer versus healthy cells to avoid autoimmunity. Since CD8+ T cells express activating NK receptors (NKRs) and NKR ligands are commonly expressed by cancer cells but not healthy tissues, we evaluated the crosstalk between NKRs and TCR during recognition of cancer cells by DC-primed TAA-specific CTLs. Methods: Associations between DNAM-1, NKG2D, CTL markers, and patients’ survival was analyzed using TCGA. In vitro sensitization was used to induce human TAA-specific CTLs by TAA-loaded DCs. The roles of DNAM-1 and NKG2D in CTL recognition and killing were tested using IFN-γ ELISpot, conjugate formation, degranulation, and cytotoxicity assays. The mechanisms and molecular effects of NKR-mediated costimulation were analyzed using IsoPlexis, calcium flux, western blot, and RNA sequencing. Double-transduction of T cells with TCR and NKR constructs or pre-treatment of cancer cells by chemotherapy was used to manipulate the levels of NKRs on T cells or their ligands on cancer cells. Results: TCGA analysis revealed that DNAM-1 and NKG2D are strongly associated with intratumoral CD8+ T cells rather than NK cells, being critical for the long-term survival of melanoma patients. Human DC-primed CTLs significantly upregulated DNAM-1 and NKG2D compared to naïve CD8+ T cells, but retained strict dependence on TCR in cancer cell recognition. Unexpectedly, blockade of DNAM-1 and NKG2D prevented the TCR-mediated CTL recognition and killing of cancer cells expressing low-TAA levels, but were redundant in the recognition of high-TAA-expressing cancer cells. DNAM-1, and to a lesser extent NKG2D, lowered TCR activation threshold and enhanced proximal TCR signaling and CTL polyfunctionality. NKR overexpression in TCR-transgenic T cells or chemotherapy-driven elevation of NKR ligands on cancer cells allowed effective recognition and killing of weakly immunogenic cancer cells. Conclusions: Reduced TCR activation threshold in the presence of NKR costimulation enables CTL activation by low levels of cognate MHC I-peptide complexes on cancer cells. Our data helps explain the ability of non-mutated “self” antigens to mediate tumor rejection in the absence of autoimmunity, and provides new tools to enhance the effectiveness of cancer therapies. Citation Format: Bowen Dong, Nataša Obermajer, Takemasa Tsuji, Junko Matsuzaki, Cindy Bonura, Henry Withers, Mark Long, Colin Chavel, Scott H. Olejniczak, Hans Minderman, Robert P. Edwards, Walter J. Storkus, Pedro Romero, Pawel Kalinski. NK receptors enable TCR-mediated CTL recognition and killing of cancer cells expressing non-mutated tumor-associated antigens abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3966.