Synergistic function screening of multiple checkpoint perturbation in TIL

A synergistic function screening system was established, in which a multiplexed CRIPSR knockout library targeting different checkpoint combinations was constructed and transduced into CD8+T cells to block multiple checkpoints in single cell. Based on this system, we performed a in vivo synergistic function screening of several checkpoints in tumor infiltrating CD8+T cells and find the best combined checkpoint blockade strategy to boost the CD8+T cell-mediated tumor elimination in vivo. In the library designing, a total of 6 checkpoints PDCD1, CTLA4, TIGIT, BTLA, TIM3 and A2AR were selected to make a total of 56 combinations (Table 1). Meanwhile, two distinct biological control groups were created. One group contained 30 combinations covering 5 co-stimulatory molecules for T cell activation, another one contained 15 combinations covering 4 critical molecules in TCR signaling. 6 groups of sgRNA were designed against individual combination. In each combination that contained less than 4 targeting sgRNAs, the non-targeting sgRNAs were included as the patch. At last, a total of 84 non-targeting 4-sgRNA combinations were included as the negative control.