Comparison of transcriptional changes after CD28/CD3z and 4-1BB/CD3z chimeric antigen receptor ligation

The adoptive transfer of chimeric antigen receptor- (CAR) modified T cells is revolutionizing the treatment of B cell malignancies and has the potential to be applied to other diseases. CARs redirect T cell specificity by linking an antigen recognition domain to T cell signaling modules comprised of CD3z to provide signal 1, and CD28 or 4-1BB to provide costimulation. CD28/CD3z and 4-1BB/CD3z CARs confer differences in effector function and cell fate that affect clinical efficacy and toxicity. These differences may result from activation of divergent transcriptional programs. To gain this insight, we analyzed changes in gene expression in stimulated and resting CD28/CD3z or 4-1BB/CD3z CAR T cells. CD28/CD3z CAR stimulation initiated more marked early transcriptional changes with greater fold increases in the expression of effector molecules including GZMB, IFNG, IL2, TNF, and IL6. Direct comparison of CD28/CD3z and 4-1BB/CD3z samples stimulated for 6 hours identified 1,673 differentially expressed genes. Of these, the memory T cell-associated genes KLF2, IL7R, and FAM65B were expressed at lower levels in CD28/CD3z CAR T cells. KLF2 and IL7R are FOXO transcription factor family targets and we found that FOXO4 expression was similarly reduced in CD28/CD3z CAR T cells. CD28/CD3z CAR stimulation induces an effector T cell-like transcriptional profile that may underlie the decreased persistence and increased risks of toxicities observed with CD28/CD3z CAR T cells in early clinical trials. Overall design: Purified CD28/CD3z and 4-1BB/CD3z CAR T cells were prepared from healthy donors and stimulated by incubation with anti-CAR beads, or left unstimulated by incubation with control beads. Total RNA was harvested 6 or 24 hours after treatment. Three biological replicates for each treatment condition were prepared, yielding 24 total samples for analysis. A42 and A44 denote 4-1BB/CD3z CARs, A43 and A45 denote CD28/CD3z CARs.

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Salter AI, Ivey RG, Kennedy JJ, Voillet V, Rajan A, Alderman EJ, Voytovich UJ, Lin C, Sommermeyer D, Liu L, Whiteaker JR, Gottardo R, Paulovich AG, Riddell SR