Abstract A180: HHLA2 is a novel tumor-expressed member of the B7 immune checkpoint family

Abstract

The purpose of this study was to assess the function and expression-regulation of the new B7 family member, HHLA2, in the tumor microenvironment. HHLA2 is a newly identified B7 family member that has been reported to both inhibit and stimulate T-cell function in vitro. Expression of HHLA2 has been noted on a wide variety of cancers and relatively few normal tissues. The function of HHLA2 in the tumor microenvironment remains elusive and the mechanisms regulating its expression are currently unknown. Challenges to addressing these questions include the lack of a functional HHLA2 gene in mice and, consequently, the lack of in vivo tumor models in which HHLA2 can be studied. Here we assess factors that drive expression of HHLA2 in vivo and present the first humanized mouse model to study the function of HHLA2 expression in the tumor microenvironment. Human cancer cell lines derived from lung, prostate, pancreas, kidney, and colon malignancies were identified with varying levels of HHLA2 expression. HHLA2 surface expression was monitored as these cell lines were grown in vivo as tumors in NSG mice and in vitro under conditions including cytokine stimulation, 3D culture, and hypoxia. Expression was assessed by both flow cytometry and qPCR. HHLA2-knockout cancer cell lines and controls were generated using CRISPR and grown as tumors in NSG mice with human immune cells. These tumors were dissociated and the immune infiltrates were phenotyped by flow cytometry. HHLA2 was upregulated in numerous human cancer cell lines in vivo and expression was reduced or lost in vitro under standard cell culture conditions. Conditions mimicking the tumor microenvironment including 3D culture, hypoxia, and cytokine stimulation all contributed to HHLA2 upregulation. We successfully generated HHLA2-knockout human cancer cell lines and developed a humanized tumor immunotherapy model. HHLA2-expressing tumors contained significantly less infiltrating CD8 and CD4 T-cells than the HHLA2-knockout tumors. The T-cells were also phenotypically less active in the HHLA2-expressing tumors. Expression of HHLA2 in human cancers is driven by environmental conditions that are specific to the tumor microenvironment and this expression appears to have a suppressive effect on infiltrating T-cells. HHLA2 is the newest B7 family member and a promising candidate for targeted immunotherapy against a wide range of cancers. Citation Format: Jordan Manek Chinai, Hao Wang, Xudong Tang, Murali Janakiram, Haiying Cheng, Xingxing Zang. HHLA2 is a novel tumor-expressed member of the B7 immune checkpoint family [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A180.