Targeting SNX9 rescues CD28-mediated T cell exhaustion for cancer immunotherapy - Targeted Pooled CRISPR Screen for T cell Exhaustion

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. To explore new pathways for reinvigoration of anti-tumor immune functions, we developed a human ex vivo exhaustion model by repetitive antigenic stimulation of primary CD8 T cells. This results in T cells that resemble patient-derived T cells in tumors on a phenotypic and transcriptional level. A pooled targeted CRISPR screen was performed on ex vivo exhausted T cells. The readout was elevated CD107a degranulation after repetitive stimulation (CD107a+ vs CD107- cells after 4h co-culture with peptide loaded tumor cells). CD8+ T cells from five healthy donor PBMCs were used in two batches to generate NY-ESO-1 specific T cells by lentiviral transduction and co-transduced with a lentiviral gRNA-Cas9 library. The library targeted 32 genes selected by analysis of mRNA expression of ex vivo exhausted T cells and published exhaustion gene sets. Additionally 20 guides against regions without known gene functions (intergenic) were used as controls. After repetitive stimulation for 12 days (4 rounds of stimulations of 3 days each), the cells were co-cultured with NY-ESO-1 peptide loaded T2 tumor cells in presence of anti-CD107a antibody. After 4h co-culture the cells were sorted for CD107a+ or CD107a- cells (both CD8+ DAPI-). The cells were lysed and genomic DNA extracted. gRNAs were amplified from the genomic DNA and sequenced to measure gRNA enrichment in one of the cell fractions.