Abstract 926: Binding of the novel inhibitory receptor KIR-X to its ligand HHLA2 negatively regulates T cell activation
Authors: Klee Alyssa N. , Mahoney Kathleen M. , Arulanandam Antonio R. , Bhatt Rupal , Zhu Baogong , Freeman Gordon J. 
Published: September 2020

Abstract
Cancer immunotherapies blocking immuno-inhibitory pathways have become a successful treatment method in the last decade. HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) is a B7 family member and ligand that is often expressed on cancer cells and antigen presenting cells. HHLA2 has the ability to contribute to both T cell stimulation and inhibition. Previous results have shown that HHLA2 binding to TMIGD2 induces T cell activation, however, the inhibitory receptor was unknown. We identified KIR-X as an additional receptor for HHLA2. In order to test the functional consequence of HHLA2 binding to KIR-X, we set up a KIR-X receptor transfected Jurkat T cell model. Jurkat cells transfected with KIR-X or TMIGD2 and luciferase under the control of an NFAT-response element or IL-2 promoter were stimulated with CHO cells expressing scFv anti-human CD3 +/- HHLA2. The bioluminescence produced by the firefly luciferase reporter reflects NFAT or IL2 transcriptional activation. We confirmed that HHLA2 binding to TMIGD2 stimulates T cells in this Jurkat T cell model as we saw an increase in NFAT mediated transcription of luciferase. The goal of this project was to first test the immunological effect of HHLA2 binding to KIR-X on T cells, and second, test a panel of candidate therapeutic antibodies directed against KIR-X or HHLA2 for their ability to reverse effects observed in Jurkat cells. We found that HHLA2 binding to KIR-X inhibited a T cell antigen receptor/CD28 signal. Next, the antibodies were tested for their capacity to reverse HHLA2 mediated effects on T cell activation. I tested HHLA2 antibodies which block binding to KIR-X, TMIGD2 or both as well as KIR-X antibodies that block binding to HHLA2. I also tested antibodies known to not block binding to ligand. My results show that KIR-X antibodies 2F11 and 1G7 and the HHLA2 antibodies 2G2, 1C8, 6F10, and 2C4 enhanced activation of KIR-X Jurkat cells, consistent with the blockade of a KIR-X mediated inhibitory signal. The HHLA2 antibody 6F10 also reduced TMIGD2 Jurkat T cell activation while the 2C4 antibody did not, consistent with their ability or inability to block TMIGD2 binding, respectively. We found that antibodies that did not block the KIR-X/HHLA2 interaction did not have a functional consequence in the Jurkat T cell luciferase assay. Therefore, these assays showed that there was good correlation between blockade of receptor/ligand interactions and effects on T cell activation and that the binding sites on HHLA2 for KIR-X and TMIGD2 were close but distinguishable. We believe that blocking the KIR-X inhibitory receptor will be valuable for increasing T cell activation in cancer, while blocking TMIGD2 has potential to alleviate autoimmune disease.This work was supported by the Dana-Farber/Harvard Cancer Center Kidney Cancer SPORE P50CA101942, (GJF and KMM), Department of Defense Early Investigator Idea Development Award W81XWH1810500 (KMM), Kidney Cancer Association Advanced Discovery Award (KMM, GJF), and P01AI056299 (GJF).
RSB was supported by NIH R01 CA196996.
Citation Format: Alyssa N. Klee, Kathleen M. Mahoney, Antonio R. Arulanandam, Rupal Bhatt, Baogong Zhu, Gordon J. Freeman. Binding of the novel inhibitory receptor KIR-X to its ligand HHLA2 negatively regulates T cell activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 926.