Abstract The immune checkpoint HERV-H LTR-associating 2 (HHLA2) is expressed in many tumors, including clear cell renal cell carcinoma (ccRCC). Multiple therapeutics directed against HHLA2 or its inhibitory receptor KIR3DL3 are about to enter clinical trials in metastatic RCC; however, little is known about the regulation of HHLA2 expression. Better understanding the regulation of HHLA2 in tumor cells and within the tumor microenvironment will facilitate translation of these therapeutic agents into the clinic by assisting in patient selection and development of rational drug combinations. Using flow cytometry, immunohistochemistry (IHC), and RNA analysis, HHLA2 expression was examined in monocytes, primary nephrectomy specimens and the established ccRCC cell lines, A498 and 786-0. A panel of cytokines was screened for the induction of HHLA2 in vitro. In vivo expression of HHLA2 in A498 and 786-0 human kidney cancer xenograft mouse models was examined. Tumor cells obtained from these xenograft models (either freshly isolated or cultured ex vivo) were characterized by RNASeq and flow cytometry. Analysis of HHLA2 expression in monocytes shows that among a wide range of cytokines tested, only IL-10 and BMP4, and to a lesser extent, IL-1b and IL-6, modestly enhanced HHLA2 mRNA and protein expression. These combinations of cytokines failed to induce HHLA2 in both A498 and 786-0. HHLA2 was frequently expressed in primary ccRCC tumors by IHC. Interestingly, analysis of HHLA2 by flow cytometry in primary RCC tumors showed that HHLA2 is progressively lost over 4 weeks when cells are cultured ex vivo. Hypoxia alone did not change HHLA2 expression. While HHLA2 was not expressed on either A498 or 786O cells in vitro, both A498 and 786-0 cells expressed HHLA2 when grown as subcutaneous xenografts in NSG immunodeficient mice. A498 xenografts express significantly more HHLA2 than 786-0 xenografts. HHLA2 expression is differentially regulated in monocytes and kidney cancer epithelial cells. High concentrations of cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in ccRCC; however, the tumor microenvironment in NSG mice significantly induced HHLA2 expression in both A498 and 786-0 xenografts. Complementary whole genome CRISPR knock out and gain-of-function screens are underway to assess by an unbiased means both nonimmune and immunologic regulators or repressors that may be responsible for inducing HHLA2 in kidney cancer in vivo. Furthermore, the A498 xenograft mouse model is an excellent humanized HHLA2 model for testing regulation of HHLA2 and HHLA2-directed therapeutics. Citation Format: Tomonari Shigemura, Nahuel Perrot, Zimo Huang, Aseman Bagheri Sheshdeh, Nourhan El Ahmar, David McDermott, Sabina Signoretti, Gordon Freeman, Kathleen Mahoney. Regulation of HHLA2 expression in renal cell carcinoma and myeloid cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A005.