Figure 1.
Phenotypic and functional characterization of CAR-TRM cells
CAR T cells were cultured in T cell medium with or without 2 ng/mL TGF-β. (A) Representative flow cytometry plot showing CD39 and CD103 expression on CD8+ CAR T cells after manufacturing.
(B–D) Expression markers associated with (B) residency (CD39, CD103, CD49a), (C) exhaustion (PD1, LAG3, TIM3, TOX), and (D) memory/stemness (TCF1, IL7R, CCR7) in CD8+ CAR T cells.
(E) Anti-mesothelin M5 CAR T cells were co-cultured with AsPC-1 tumor targets at an effector to target (E:T) ratio of 1:2. Cytolytic activity of CAR-TCONV and CAR-TRM cells was measured in real time using the xCELLigence RTCA system. Cytotoxic capacity at 7 h post co-culture is shown.
(F and G) M5 CAR T cells were challenged with AsPC-1 targets at an E:T ratio of 2:1. Supernatant was collected at 18 h after coincubation, and cytokine levels were measured. (F) Granzyme A; (G) effector cytokines (IFN-γ, IL-2, TNFα, IL-6).
(H) CAR T cell expansion levels were measured during ex vivo manufacturing. Data represent results from four independent CAR T cell manufacturing experiments (paired t test). All in vitro experiments were performed using CAR T cells derived from various healthy subjects as biological replicates (n = 4–6). (A–G) Representative results from one donor. ∗p < 0.05; ∗p < 0.01; ∗∗∗p < 0.001; ns, not significant (Mann-Whitney U test).