Identifying gene combinations for targeting innate immune cells to enhance T cells activation [CITE-seq]

Genetic engineering of hematopoietic cells ex-vivo opens new opportunities for programming immune cells and improving their functionality in response to illness and infections. Molecular methods for genetic manipulation are constantly improving, but it is challenging to reveal which genes are the most beneficial to target and whether multiple genes tackling can further improve the desired outcome. Current screening methods are limited by scale, the number of perturbations, and low content readouts. We developed a hybrid platform called “high multiplicity of perturbations and cellular indexing of transcriptomes and epitopes by sequencing (HMPCITE-seq)” that allows for finding combinations of genes to target and improves desired cellular functions. We used antigen-presenting cells and performed two genome-wide CRISPR screens to enhance the activation and co-stimulation programs and restrict the suppressive programs. Five genes were found in both screens including Cebpb gene. We showed that targeting Cebpb in antigen-presenting cells enhances the expression of costimulatory molecules, prevents attenuate cell migration and phagocytosis, improves T cell proliferation, and restricts tumor growth. To find the genetic interactions between validated targeted genes, and identify gene combinations for targeting, we performed high-order perturbations of validated genes from the screens with single-cell RNA-seq readouts. We found that targeting both Cebpb and Med12 genes shows a better phenotype compared to the single KOs. Thus, the HMPCITE-seq platform, starting from a genome-wide screen, allows the finding of gene combinations to target as demonstrated by functional experiments in-vitro and in-vivo. Overall design: The goal of this experiments was to find the effect of targeting Cebmb and Med12 on DCs, T cells, and tumor growth. We therefore generated perturb BMDCs from bone marrow or spleen incubate them with CD8 T cells or inject to tumors as detailed bellow.