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. 2024 Nov 20;12(11):e009743. doi: 10.1136/jitc-2024-009743

Figure 5. Zip18R signaling alters the transcriptome of C.TNC-CAR T cells. (A) Schematic of experiment. C.TNC-CAR.Zip18R T cells were collected from culture in IL-7/IL-15 and frozen for analysis or freshly collected from a co-culture assay with LM7.GFP.ffLuc (LM7.GL) tumor cells in the presence of IL-15 at 12, 24, and 48 hours post tumor cell stimulation. All four populations are present in the same culture. (B) Percentages of C.TNC-CAR+Zip18R+, C.TNC-CAR+, Zip18R+, and NT T cells at 12, 24, and 48 hours post co-culture. (C,D) Gene Set Enrichment Analysis comparing C.TNC-CAR.Zip18R T cells to C.TNC-CAR T cells at (C) baseline or (D) 48 hours post stimulation with LM7.GFP.ffLuc cells. Top 10 activated or suppressed significant (p.adjust<0.1) KEGG pathways are shown. (E–I) 1×106 NT, Zip18R, C.TNC-CAR, and C.TNC-CAR.Zip18R T cells were cultured in media or against LM7 cells at a 2:1 effector to target ratio for 48 hours and then collected for flow cytometric analysis. (E) Transduction (TDX) percentage of NT, Zip18R, C.TNC-CAR, and C.TNC-CAR.Zip18R T cells alone and after 48 hours of stimulation. CD3+ expression is shown for NT samples. (n=3, mean+SEM), two-way ANOVA, ****p<0.0001. (F–I) Expression within pure isolated populations of T cells gated on CD3+ (NT), mClover+ (Zip18R+), (G4S)3+ (C.TNC-CAR+), and (G4S)3+ mClover+ (C.TNC-CAR+Zip18R+) for (F) CD69, (G) CD28, (H) CD39, and (I) TIM3 (n=3, mean+SEM), two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Only showing significance values for each cohort comparing Alone versus +LM7 and C.TNC-CAR versus C.TNC-CAR.Zip18R for both conditions. All p values are reported in online supplemental SFigure 16. ANOVA, analysis of variance; CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; ffLuc, firefly luciferase; GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; KEGG, Kyoto Encylopedia of Genes and Genomes; NT, non-transduced; TNF, tumor necrosis factor; Zip18R, interleukin-18 receptor-based leucine zipper receptor.

Figure 5