Pooled screening for CAR function in glioblastoma

Human primary CD3+ T cells expressing IL13Ra2-targeted chimeric antigen receptors (CARs) that had been identified through high throughput pooled screening were rechallenged with an IL13Ra2+ human glioblastoma cell line , then stained with CITESEQ antibodies indicative of T cell memory and exhaustion state and prepared for single cell sequencing in order to determine their phenotypic responses to chronic tumor challenge relative to that of a clinical standard CAR. Overall design: CD3+ T cells from two donors expressing two novel CARs and one control CAR were subject to antigen stimulation with mitomycin treated IL13Ra2+ U87 cells four times in vitro. These were then stained with TotalSeq-C hashing, sorted to remove residual tumor cells, and pooled before encapsulation in two channels of the Chromium Single Cell 3' v3.1 platform (10X Genomics). Gene expression (GEX) libraries were constructed based on manufacturer' instructions, while hashing antibody libraries were constructed as reported previously. The resulting libraries were pooled at 12.5% antibody to 87.5% GEX before sequencing on an Illumina NextSeq500 to a depth of 34,380 reads per cell. Reads were aligned to the Genome Reference Consortium Human Build 38 (GRCh38), and a cell–gene matrix was generated using the CellRanger pipeline (10X Genomics; v4.0.0). Downstream analysis was performed using the Seurat package (v4.0.0).