MyD88/CD40 signaling retains CAR T cells in a less differentiated state
Brooke Prinzing, … , Giedre Krenciute, Stephen Gottschalk
Brooke Prinzing, … , Giedre Krenciute, Stephen Gottschalk
Published November 5, 2020
Citation Information: JCI Insight. 2020;5(21):e136093. https://doi.org/10.1172/jci.insight.136093.
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Research Article Immunology Therapeutics

MyD88/CD40 signaling retains CAR T cells in a less differentiated state

Abstract

Chimeric antigen receptor (CAR) T cell therapy for solid tumors has shown limited efficacy in early-phase clinical studies. The majority of CARs encode CD28 and/or 41BB costimulatory endodomains, and we explored whether MyD88 and CD40 (MC) costimulatory endodomains in CARs could improve their antitumor activity. We generated CD28-, 41BB-, and MC-CAR T cells and demonstrated that MC-CAR T cells have greater proliferative capacity and antitumor activity in repeat stimulation assays and in tumor models in vivo. Transcriptomic analysis revealed that MC-CAR T cells expressed higher levels of MYB and FOXM1, key cell cycle regulators, and were activated at baseline. After stimulation, MC-CAR T cells remained in a less differentiated state than CD28- and 41BB-CAR T cells as judged by low levels of transcription factor TBET and B lymphocyte induced maturation protein 1 expression and lower cytolytic activity in comparison with CD28- and 41BB-CAR T cells. Thus, including MyD88 and CD40 signaling domains in CARs may improve current CAR T cell therapy approaches for solid tumors.

Authors

Brooke Prinzing, Patrick Schreiner, Matthew Bell, Yiping Fan, Giedre Krenciute, Stephen Gottschalk

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Figure 6

MC-CAR T cells retain a less differentiated phenotype postactivation.

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MC-CAR T cells retain a less differentiated phenotype postactivation. (A...
(A) CAR T cells from 3 healthy donors were taken directly from culture or first stimulated with LM7 tumor cells for 24 hours before being sorted into CD4+CAR+ and CD8+CAR+ subsets. RNA was extracted from sorted cells and sequenced on an Illumina NovaSeq platform. Expression of PRDM1 (encodes BLIMP1) and TBX21 (encodes TBET) at baseline and after 24-hour stimulation with LM7 in CD4+ (left) and CD8+ (right) T cells (n = 3, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). FPKM, fragments per kilobase of transcript per million mapped reads. (B) CAR T cells were stimulated with rhEphA2 protein for 24 hours before intracellular staining for the transcription factors TBET and BLIMP1. Representative flow plots and summary of expression for all donors are shown (TBET: n = 7, BLIMP1: n = 4, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). (C) T cells were incubated with LM7 tumor cells at a 1:2 E/T ratio for 5 hours in the presence of GolgiStop and stained for CD107a, a surrogate marker for degranulation. Representative flow plots and summary expression for all donors are shown (T cells only: n = 13, T cells vs. LM7: n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). All statistical tests were performed in comparison with MC-CAR T cells (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Enlarged versions of A and B are provided in Supplemental Figures 22 and 23.