journal contribution
posted on 2023-04-04, 01:00 authored by Dinali Wijewarnasuriya, Christina Bebernitz, Andrea V. Lopez, Sarwish Rafiq, Renier J. Brentjens (A) Media from in vitro co-culture of CAR T cells with EL4mCD19 tumor cells was assessed for IL12p70 on day 7 after CAR T cell treatment. Data shown is mean +/- s.e.m. of two independent experiments. (B) EL4mCD19 tumor bearing C57BL/6 mice, pretreated with 250mg/kg per mouse of cyclophosphamide day -3, treated i.v. with CAR T cells day 0. Significance determined bylong-rank Mantel-Cox Test, with 95% confidence interval (n=3 per group). *p=0.0224 m1928� compared to m1928� + cyclophosphamide and *p=0.0295 m19� compared to m19� + cyclophosphamide (C) Flow cytometry demonstrating CD25 expression on CAR T cells prior to injection in terms of percentage and representative flow cytometry plot (ns by ordinary one-way ANOVA). Data shown is mean +/- s.e.m. of three independent experiments (n=3 per group). (D) CAR T cells were co-cultured with EL4mCD19 tumor cells and assessed for cytotoxicity 4 hours later, using a luciferase killing assay. m1928�, m19�IL-12, and m1928�IL-12 CAR T cells showed significantly increased cytotoxicity compared to m19� CAR T cells at E:T ratios of 2:1, 1:1, and 0.5:1. There was no difference in tumor lysis between 19�12 and m1928�IL-12 CAR T cells (*p=<0.001 by two-way ANOVA). Data shown is mean +/- s.e.m. of three independent experiments. Serum was obtained through retro-orbital bleeds and assessed for IL12p70 through luminex on day 7 after CAR T cell treatment. Data shown is mean +/- s.e.m. of three independent
Funding
US National Institutes of Health
Annual Terry Fox Run for Cancer Research
Carson Family Charitable Trust
Commonwealth Foundation for Cancer Research
Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center
History
ARTICLE ABSTRACT
Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation.See related article by Drakes et al., p. 743
