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. 2020 May 4;130(6):3087–3097. doi: 10.1172/JCI133215

Figure 5. Mutation of the YMFM motif in the ICOS signaling domain to YMNM (CD28 motif) impairs antitumor effect and T cell persistence.

Figure 5

(A) A real-time cytotoxicity assay (xCELLigence) was used to evaluate the lysis of Capan-2 tumor cells when treated with CAR-T cells (E/T = 3:1) over a 120-hour period. (B) CAR-T cells were cocultured with mesothelin+ Capan-2 cells. Supernatants were obtained 24 hours later, and cytokine production was analyzed by ELISA. Fold-change cytokine production in second-generation versus first-generation (z) CAR-T cells was analyzed in 3 to 4 healthy donors. P values were calculated by 1-way ANOVA with Tukey’s post hoc test. (C–E) NSG mice bearing s.c. Capan-2 tumors were treated 30 days after tumor implantation with 2 doses of CAR-T cells (n = 6–9). (C) Tumor volume was analyzed at indicated time points. Statistical analysis was performed by 2-way ANOVA followed by Dunnett’s multiple-comparisons test. (D) Box plots of the change in tumor volume on day 35 versus baseline (n = 6–9). Box plots show median (line) and 25th to 75th percentile (box). The end of the whiskers represents the minimum and the maximum of all of the data. P values were calculated with 2-tailed Mann-Whitney tests. (E) The concentration of CD4+ and CD8+ T cells was determined in the blood of treated animals 36 days after T cell injection. P values were calculated by Kruskal-Wallis multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.