Transcriptional signature of T cells expressing ICOS-based CARs. Redirected TH17 cells from 3 different human normal donors were stimulated with immobilized recombinant mesothelin. Gene expression levels were determined previous to stimulation (day 0) and 4, 8, 24, and 96 hours on antigen recognition. (A) MDS analysis of 886 differentially expressed genes between the 3 sets of CAR T cells at 4 hours on activation show 3 distinct clusters. The number of differentially expressed genes (FDR < 0.05, FC > 2) between a pair of CARs is indicated with pair-connecting arrows. (B) Relative log2 expression of Il17a at indicated time points on antigen recognition. #FDR < 0.05, FC > 2 compared with both 28ζ and BBζ. The cytokine levels of IL-17A were validated by ELISA. Error bars represent SEM (3 different normal donors). *P < .05, **P < .01. (C) Expression of genes that were found significantly upregulated in ICOSζ T cells compared with both 28ζ and BBζ T cells was validated in at least 1 of the 3 healthy donors by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at indicated time points and shown as ratio vs β-actin. (D) The significant regulatory inputs for Ncs1 identified in the TH17 differentiation network model are displayed. Blue and orange nodes depict the extent of TH17 upregulation and downregulation, respectively (compared with a TH0 control). Edges connecting 2 genes are green for activation and red for repression. Edges emitting from IRF4, STAT3, BATF, MAF, and Fosl2 are supported by both TF ChIP-seq and TF knockout RNA-seq and thus should be considered as validated. Edges emitted from other TFs were inferred from transcriptomic analysis of a large compendium of mouse immune cells and thus should be considered as likely but not validated.
