Abstract 3229: Investigating the transcriptomic signature of HHLA2-mediated suppression of NK cell activity

Abstract Determining how immune checkpoint inhibitors (ICI) alter functional characteristics of lymphocytes can improve our understanding of ICI. HERV-H LTR-associating 2 (HHLA2, B7-H7) is an immune checkpoint expressed by many tumors. Multiple HHLA2:KIR3DL3 blocking antibodies are currently in Phase I clinical trials and enrolling cancer patients (NCT05958199, NCT06240728), but little is known about how different subsets of NK cells are affected by HHLA2 expression. Our group previously found that in addition to an activating receptor, TMIGD2, HHLA2 has an inhibitory receptor, KIR3DL3. The KIR family of receptors is expressed in NK cells as well as T cells. Through co-culture experiments of CD56+ NK cells in the presence of K562 cells, we sought to investigate the mechanisms of NK cell engagement. Additionally, we generated transgenic K562 cell lines, over-expressing HHLA2, validated through flow cytometry, to maximize our ability to glean insight into HHLA2-KIR3DL3 mediated activation of NK cells. To this end, we generated a high quality scRNA-seq dataset of more than 100, 000 cells, comprising of more than 90, 000 NK cells, with approximately 30, 000 cells either cultured alone, co-cultured with K562 or K562 over-expressing HHLA2 tumor cells, allowing unprecedented granularity. In conjunction with published NK cell states and newly identified ones through this study, we recapitulate important known cytokines, growth factors and other regulators underlying NK cell maturation and substates that are differentially affected by treatment, in abundance, expression, and regulation. Furthermore, we shed some light on the complex interplay of positive and negative regulators, including CD48 - 2B4 and CD56 related interactions in CCL4 and XCL2 high NK cells, as well as TGFb related pathways. Uncovering NK cell activity modulators, such as amphiregulin, NKG7, FAM49B, and TXNIP, we then focused on NK cells in the context of KIR3DL or TMIGD2 expression. We add to previous efforts to understand immune checkpoint inhibition by the HHLA2-KIR3DL3 axis and in a concerted effort with blocking antibodies, provide evidence of enhanced NK cell efficacy. Better understanding of how HHLA2 pathway affects the function of different subsets of lymphocytes could determine which patients may best benefit from targeting this pathway. Citation Format: Nahuel Perrot, Nikolaos Kalavros, Deepthi Chowbene, Shoushuo Wang, Yered Pita-Juarez, Antonella Arruda de amaral, David McDermott, Gordon Freeman, Ioannis Vlachos, Kathleen Mahoney. Investigating the transcriptomic signature of HHLA2-mediated suppression of NK cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3229.