Abstract 3264: CRISPR-Cas9 library screen in primary T cells to identify cancer immunotherapeutic targets

Abstract

Abstract Background: Immunotherapy, especially checkpoint blockers targeting programmed cell death protein 1 (PD-1) pathways, has transformed cancer treatment. Current checkpoint blockers are limited by low response rate, side effect and treatment relapse. An efficient and high-throughput screen platform in primary immune cells will accelerate the identification of key regulators in the immune response to tumor. New targets identified from the screen will be validated in in vitro and in vivo models that recapitulate key components of tumor-immune interaction. Results: We developed a CRISPR-Cas9 genome targeting system that can achieve efficient gene disruption in primary CD8+T cells isolated from mouse (~60%) or human (~70%). We have applied this method to a pooled CRISPR library screen for key modulators of T cell-induced cytotoxicity to cancer cells in vitro and in vivo. This library contains sgRNAs targeting nearly all membrane proteins expressed in both murine and human T cells. In our in vitro model, cancer cells expressing chicken ovalbumin (OVA) were co-cultured with OVA-specific CD8+T cells isolated from OT-I transgenic mice. The proliferation and function of CD8+ T cell were damped by tumor cells in an antigen dependent way. In our in vivo model, OVA-specific CD8+T cells were transplanted into C57BL/6 mice implanted with OVA-expressing tumor. The accumulation, proliferation and function dynamics of transplanted CD8+T cells were monitored in tumors. They infiltrated into tumor in 3 days, substantially decreased in number in the following 4 days, and then continuously increased. Importantly, most CD8+T cells (~70%) in tumor were CD107a negative, which means they were non-functional. Summary: We have established a high-throughput platform for identification of membrane proteins in primary CD8+ T cells to regulate T cell proliferation and function in tumor microenvironment. Citation Format: Yangbo Yue, Qian Li. CRISPR-Cas9 library screen in primary T cells to identify cancer immunotherapeutic targets [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 3264.