Abstract 3781: A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity

Abstract

Abstract Introduction: Dendritic cells (DCs) are professional antigen-presenting cells with two major subsets, the classical DC type 1 and 2 (cDC1 and cDC2). The cDC1, also known as the CD8α+ DC, is especially equipped for the process of cross-presentation in which dead cell antigen is presented on MHC-I to prime cytotoxic T cells. This process is relevant to anti-cancer immunity, as mice deficient in the transcription factor BATF3, which specifically lack the cDC1, succumb to highly immunogenic tumors that are normally rejected and cleared in wild-type mice. The mechanism of cross-presentation by the cDC1 is poorly understood. In this study, we developed a focused CRISPR/Cas9 screen to identify novel genes that endow the cDC1 with the ability to cross-prime cytotoxic T cells. Methods: A curated list of target cDC1 genes was constructed using the Immunological Genome Project Database as the primary resource (www.immgen.org). 120 genes specifically expressed by the cDC1 in comparison to other murine immune cells were selected. Guide RNAs (gRNAs) were designed to early exon sequences using the MIT CRISPR Design Tool to minimize off-target effects (www.crispr.mit.edu), and 2-3 different gRNAs were evaluated in tandem for each candidate. These gRNAs were transduced via retroviral vector into lineage depleted c-Kithi bone marrow cells isolated from C57BL/6J Cas9 knock-in mice. These cells were differentiated in vitro for 8 days in the presence of the cytokine Flt3L. Transduced DCs were sorted by FACS and co-cultured in the presence of a source of dead cell antigen (heat-killed L. monocytogenes expressing ovalbumin, or HKLM-OVA) as well as CFSE-labelled transgenic CD8+ T cells with an ovalbumin peptide-specific T cell receptor (OT-1). The proliferation of OT-1 cells was measured by CFSE dilution. Results: To validate our CRISPR/Cas9-based screening approach, we targeted the first exon of the beta-2 microglobulin gene (B2M), which is required for cell surface expression of MHC-I. B2M knockout DCs were co-cultured in the presence of HKLM-OVA and CSFE-labelled OT-1 cells for 3 days. Controls included DCs transduced with a scramble gRNA with or without antigen. As expected, targeting B2M in DCs led to near-complete reduction in OT-1 proliferation compared to scramble control (3.7% vs. 20.6%, p=0.003), similar to the absence of antigen (3.7% vs. 0.96%, p=0.99). We then expanded our assay to our curated list. Promising candidate genes have been discovered and are currently being validated in murine knockout models. Conclusions: The CRISPR/Cas9 system can be adapted to screen the cDC1 for novel genes involved in cross-priming cytotoxic T cells. Validation of candidates in vivo, including tumor assays, will be presented at the meeting. These findings may have important implications for adaptive immunity against cancer. Citation Format: Jesse T. Davidson, Derek J. Theisen, Carlos G. Briseño, Vivek Durai, William E. Gillanders, Theresa L. Murphy, Kenneth M. Murphy. A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3781.