Supplemental Figure 3 from Augmenting CAR T-cell Functions with LIGHT

(A) Flow cytometric analysis of LTβR expression of MIAPACA2 cells after CRISPR knockout (KO). MIAPACA2 LTβR KO cells were also transduced with non-signaling LTβR without intracellular signaling portion (tLTBR). (B) MIAPACA2 LTβR KO healthy human donor-derived mesothelin-targeted CAR T cells were cocultured with tumor cells expressing GFP and firefly luciferase at different effector to tumor ratios. Bioluminescence was measured 72 hours later and plotted as a percentage of the signal detected in a coculture of non-functional Meso-DEL-CAR T cells. Plots represent 3 independent experiments. Data errors were analyzed with mean ± SEM. (C) No killing advantage was observed with LIGHT CAR T cells co-cultured with the MIAPACA2 non-signaling, (truncated) tLTβR cancer cell line. (D) Cytotoxicity assay of MIAPACA2 and MIAPACA2 LTβR KO cell lines with non-functional CAR T cells (Meso-DEL and Meso-DEL-LIGHT). CAR T cells have been activated by CD3/CD28 Dynabeads for 1 day prior to adding cancer cells at 4 to 1 effector to tumor (E:T) ratio for 48 hours. Plots represent 3 independent experiments from 3 different donor T cells. Data errors were analyzed with mean ± SEM. (E) (F) Flow cytometric analysis of HVEM expression of CAR T cells before and after CRISPR KO. (red) represents isotype control, (blue) represents wild type CAR T cell expression, and (orange) represents the HVEM expression after CRISPR KO in CAR T cells. HVEM KO CAR T cells were cocultured with moderately high mesothelin expression AsPC1 tumor cells expressing GFP and firefly luciferase at different effector to tumor ratios. Bioluminescence was measured 48 hours later and plotted as a percentage of the signal detected in tumor alone. Plots represent 2 independent experiments from 2 healthy donor T cells. Data errors were analyzed with mean ± SEM. (G) HVEM KO CAR T cells were cocultured with low mesothelin expression MIAPACA2 tumor cells expressing GFP and firefly luciferase at different effector to tumor ratios. Bioluminescence was measured 48 hours later and plotted as a percentage of the signal detected in tumor alone. Plots represent 2 independent experiments from 2 healthy donor T cells. Data errors were analyzed with mean ± SEM. (H) Protein array analysis of 35 apoptosis-related proteins in MIAPACA2 cells treated with Meso-DEL, Meso-28z, and Meso28z-LIGHT. Representative array images indicating the protein levels of various apoptosis-related proteins in MIAPACA2 after 48 hours of coculture with CAR T cells at 4:1 E:T ratio. (I) (J) The relative fold change of the apoptosis-related proteins in MIAPACA2 treated with various conditions of CAR T cells was normalized to Meso-DEL (control). Most differentially expressed proteins in MIAPACA2 between Meso-28z and Meso-28z-LIGHT CAR T cells treated group.