Abstract A014: Blocking the HHLA2 immune checkpoint alters transcriptomic signature in specific subsets of NK cells

Abstract Background: Determining how immune checkpoint inhibitors (ICI) alter functional characteristics of lymphocytes can improve our understanding of these treatments. HERV-H LTR-associating 2 (HHLA2, B7-H7) is an immune checkpoint expressed by many tumors. HHLA2 has an activating receptor, TMIDG2, and an inhibitory receptor, KIR3DL3. The KIR family of receptors is expressed in NK cells as well as T cells. We developed a series of monoclonal antibodies that block the inhibitory HHLA2:KIR3DL3 interaction. HHLA2:KIR3DL3 blocking antibodies in Phase I clinical trials are currently enrolling cancer patients (NCT05958199, NCT06240728). Method : We performed single-cell RNA sequencing on co-cultures of NK cells isolated from peripheral blood cells with or without the presence of K562 cells expressing HHLA2, which are NK cells target, to assess the effects of HHLA2 antibodies blocking HHLA2:KIR3LD3. Co-cultured cells were treated with different HHLA2 blocking antibodies or isotype control. Results : We found different subsets of NK cells in the cocultures delineated by different marker sets, including the XCL2 and CCL4 chemokines, further differentiated TXNIP positive NK cells and BCL2 positive subsets. Additionally, we observed differential abundance between the interaction blocking conditions as compared to isotypes controls. Furthermore, differential gene expression analysis after non-selective HHLA2 blockade was more pronounced in the XCL2+ subset, followed by the CCL4+ subset, and included genes implicated in phagocytosis, regulation of macrophages and chemotaxis, signaling detection, cytokine secretion, and the humoral immune response respectively for each subset. Finally, by iteratively sub-clustering our subsets based on known NK cell markers, we have discovered clusters reflecting a milieu of NK cell states and activity profiles. We will explore whether these subsets of NK cells, specifically XCL2 and+ CCL4+ subsets, are expressed in tumors and characterize the interaction in greater depth. Conclusion : Better understanding of how blocking the HHLA2:KIR3DL3 pathway affects lymphocyte function 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. Blocking the HHLA2 immune checkpoint alters transcriptomic signature in specific subsets of NK cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A014.