Figure 5.

Highly functional CAR-T_RM cells appear to be transcriptionally and epigenetically exhausted

(A) Top transcription factor motifs enriched in CAR-T_RM cells.

(B) Heatmap showing expression of bZIP and IRF transcription factor genes (left) and genes associated with CAR T cell exhaustion in CD8⁺CAR T cells (right).

(C) GSEA showing enrichment of exhaustion signatures.

(D) The frequency of exhausted CAR T cells (PD1⁺TOX⁺ CD8⁺) is shown (two-way ANOVA, n = 3 biological replicates).

(E and F) M5 CAR T cells were challenged with AsPC-1 tumor cells at an E:T ratio of 2:1. Supernatants were collected at 18 h after coincubation and cytokine levels measured. (E) IL-2 production levels (two-way ANOVA, n = 4 biological replicates). (F) Heatmap showing effector cytokine and cytotoxic molecule production levels.

(G) Cytolytic activity of M5 CAR T cells was determined by coculturing the cells with Capan-2 cells at an effector to target ratio of 1:6 after three antigen stimulations. The normalized cell index at 31 h post co-culture was measured using the xCELLigence RTCA system. Results are shown as mean ± SEM of replicates (two-way ANOVA).

(H) Frequencies of resident memory CAR T cells (CD103⁺ or CD49a⁺ CD8⁺) are shown (two-way ANOVA, n = 3 biological replicates). ∗p < 0.05; ∗p < 0.01; ∗∗∗p < 0.001; ns., not significant.