Fig. 6: C-JUN re-activates ERK and upregulates co-stimulatory molecules and cytokines.

A, B GSEA results from RNAseq of U937 co-incubated- control versus C-JUN CAR T (A). Nominal P values, FDR q values, and NES were calculated using GSEA software (Broad Institute). Heat maps (B) indicating the expression of genes enriched in GSEA from (A) and the known related genes not in the GSEA gene set. The genes in heatmaps meet: fold change ≥ 1.5-fold in each of the two biological replicates. Each RNA sample was pooled from three technical replicates with T cells from one donor, and we conducted experiment with two different donors, n = 2. C Differentially accessible regions of indicated genes from ATAC-seq analysis. D Intensity of intracellular calcium in CAR T cells, n = 3. Expression of p-ZAP70 (E), p-ERK1/2 (F), p-C-JUN (G), and p-JNK (H) in CAR T cells pre-incubated with U937 and re-stimulated with U937, n = 3. I Percentage of 4-1BB, CD28, CD86, and IL-21 in CAR T cells cocultured with U937, n = 3. J Cytolytic activity of C-JUN CAR T cells against U937 with 50 µM U0126 or 50 µM SP600125, n = 3. Percentage of 4-1BB, CD28, CD86 (K), and IL-2, IL-21, IFN-γ (L) in T cells from (J), n = 3. (M) Percentage of p-ERK1/2 in U937 pre-incubated-control CAR T, C-JUN CAR T, and C-JUN CAR T with 1, 5, 10 µM U0126, n = 3. Percentage of 4-1BB, CD28, and CD86 (N) and IL-2, IL-21, and IFN-γ (O) in U937-co-incubated control CAR T, C-JUN CAR T, and C-JUN CAR T with 5 µM U0126, n = 4 in CD28 and CD86 expression, n = 3 otherwise. For all bar plots, data are shown as mean ± SD. Assays were performed on day 10 after T-cell initial activation. Two-sided unpaired t-tests or multiple two-sided unpaired t tests were used to assess significance in (D–I). One-way ANOVA was used in (J–O). All numbers defined by “n” indicate the number of biological replicates with different human donors. Data are representative of two independent experiments. NS not significant. Source data are provided in the Source Data file.