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. The emergence of CRISPR Cas9-based screen provides a superior and powerful tool in gene function profiling. The application of CRISPR Cas9 screen in primary immune cells and tumor cells such as diffuse large B cell lymphoma (DLBCL) cells will accelerate the identification of key regulators in tumor-immune interaction.

Methods CRIPSR screen using membrane protein-focused sgRNA library and genome-scale sgRNA library; primary T cell and tumor cell co-culture

Results First of all, we developed a CRISPR-Cas9 gene targeting method that can achieve efficient gene disruption in primary CD8+T cells isolated from mouse (~60% efficiency) or human (~70% efficiency). We have applied this method to a pooled CRISPR library screen for key modulators of T cell-induced cytotoxicity against cancer cells in vitro. This customized library contains sgRNAs targeting nearly all membrane proteins expressed in both murine and human T cells. For our in vitro screen, mouse colorectal cancer cell line MC38 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 dampened by tumor cells in an antigen-dependent way. On the other hand, we successfully developed a genome-scale CRISPR screen platform on the difficult-to-transduce DLBCL cells. The platform is currently deployed to validate modulators involved in bispecific antibody-mediated tumor cell killing by T cells.

Conclusions We have established CRISPR Cas9 pooled screen platforms for identification of modulators of tumor-immune interaction by either target primary T cells or difficult-to-transduce DLBCL cells.