GSM5502397: STIM_24h_CSP; Homo sapiens; OTHER
Eight anti-GPC2 and six anti-CD276 CAR T- cells were first thawed separately and recover for overnight. Then they were pooled the same way according to its counterpart experiment co-cultured with tumor cells using an E:T ratio of 1:1 for individual anti-GPC2 CAR T- cells expect G27; and 1:4 for each anti-CD276 and G27 CAR T- cells. Pooled CAR T-cells were evenly divided into seven fractions for multimodal single cell assay. One of the 7 fractions were taken as CTRL_0h and remaining 6 fractions were split into two groups: one group of 3 fractions cultured without target tumor cells for 24 hours (CTRL_24h) and other group of 3 fractions co-cultured with target tumor cells for 24 hours (STIM_24h). To improve the cell viability to >85%, dead cells were removed by magnetic negative selection (Dead Cell Removal Kit, Miltenyi Biotec, Cat# 130-090-101). For 2 groups of pooled CAR T-cells at 24 hours, Totalseq-C human 'hashtag' antibodies were used for each of CAR T-cell triplicates in 2 groups allowing identification of different samples in the analysis, and then triplicates in each group were combined. Next, three CAR T- cell pools (CTRL_0h, CTRL_24h, and STIM_24h) were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 31 surface proteins. After washed three times, CAR-T cells were resuspended in PBS, and counted before proceeding immediately to the single cell immune profiling using a 10X Genomics Chromium system (10x Genomics, Pleasanton, CA). Briefly, CAR T-cells were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5' Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry. Cell surface protein feature libraries were prepared as instructed by 10x Genomics user guides and sequenced on Illumina Nextseq500.