Figure 4. Zip18R bolsters C.TNC-CAR T-cell effector function in vitro. (A) Transduction efficiency of primary T cells was determined via flow cytometry on day 7 post-transduction (n=4, mean+SEM). Determined via F(ab’)₂ staining and mClover expression. (B) Repeat stimulation assay schematic. T cells were stimulated with LM7.green fluorescent protein.firefly luciferase at an effector to target ratio of 2:1 in the presence of IL-15 every 4 days. (C) Expansion of T cells in repeat stimulation assay. Each graph represents one donor. (D) Fold change of C.TNC-CAR and C.TNC.CAR.Zip18R T cells after stimulations 1 through 10, paired t-test, ****p<0.0001. (E–G) Quantification of (E) type 1 and (F) type 2/type 17 cytokines, and (G) chemokines 48 hours post first stimulation of the repeat stimulation assay (n=3, mean+SEM), data was log-transformed before statistical analysis, two-way analysis of variance, *p<0.05, **p<0.01, ***:p<0.001, ****p<0.0001. (H–J) Comparison of the sum of (H) type 1 cytokines, (I) type 2/type 17 cytokines, and (J) chemokines produced post first stimulation and fourth stimulation by C.TNC-CAR T cells and C.TNC-CAR.Zip18R T cells from repeat stimulation assay (n=3), unpaired t-test, *p<0.05, **p<0.01. CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; NT, non-transduced; TNF, tumor necrosis factor; Zip18R, interleukin-18 receptor-based leucine zipper receptor.