CTLA-4 tail fusion enhances CAR-T anti-tumor immunity [single-cell RNA-seq]

To systematically investigate the effect of CCT engineering on CAR-T cell phenotypes in vivo, we used single-cell RNA sequencing (scRNA-seq) to characterize the full spectrum of engineered CAR-T cells and their transcriptomic profiles Overall design: NSG mice were inoculated with 0.5 million NALM6GL cells intravenously (i.v.) on day 0 and treated with 1 million CAR-T cells (i.v.) on day 4. Relapse was modeled by re-challenging all mice with 0.5 million NALM6GL cells(i.v.) on day 12. To better model the clinical response to CAR-T cells in NSG mice45, 2.5µg human recombinant IL2 (Peprotech) was administered subcutaneously (s.c.) everyday starting on from day 4 to day 14. All mice were sacrificed on day 15. Spleen and bone marrow tissues were collected and processed into single cell suspension. CAR-T cells were sorted using Aria II cell sorter (BD) based on mScarlet expression. For all four groups (CAR, CAR-1CCT, CAR-2CCT and CAR-3CCT), samples from 3 individual mice within the same group were pooled together to minimize sampling bias.