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 1

Generation and functional characteristics of CAR T cells.

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Generation and functional characteristics of CAR T cells. (A) Scheme of ...
(A) Scheme of EphA2-CAR constructs. (B) Summary plot of %tCD19+ T cells (n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). (C) Summary plot of %F(ab′)2-positive T cells (n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). (D) CAR T cell production of Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines after 24-hour coculture at a 2:1 ratio against EphA2-positive (LM7, U373 WT) and EphA2-negative (BV173, U373 EphA2 KO) cell lines or in media alone (n = 5, mean ± SEM, 2-way ANOVA with Dunnett’s test for multiple comparisons, all statistical analysis is in comparison with NT cells). Dot colors: black, media; light gray, BV173 (EphA2 negative); dark gray, U373 EphA2 KO (EphA2 negative); dark blue: U373 (EphA2 positive); light blue, LM7 (EphA2 positive). (E) Summary plots of Th1 and Th2 cytokine production against EphA2-positive cell lines U373 and LM7 (n = 5, mean ± SEM, values were log transformed before 2-way ANOVA with Tukey’s test for multiple comparisons). (F) CAR T cells were incubated with increasing amounts of tumor cells for 24 hours, and the remaining live tumor cells were quantified with an MTS assay (2-way ANOVA with Tukey’s test for multiple comparisons, mean ± SEM, LM7: n = 4, U373 WT and U373 EphA2 KO: n = 9). For LM7 and U373, asterisks refer to statistical comparison of MC-CAR with NT and CD28-CAR with MC-CAR. For U373 EphA2 KO, asterisks refer to statistical comparison of 41BB-CAR with NT and CD28-CAR with NT. #P < 0.1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.