Fig. 3: CD83-expressing tumor-infiltrating CAR-T cells display phenotypic and functional attributes of precursor exhausted T cells.

a–i NSG mice subcutaneously inoculated with the A375-mesothelin were infused with mesothelin-targeting CAR-T cells intratumorally and peripheral blood T cells from the same donor intravenously. T cells within the tumor and spleen were analyzed 5 days after infusion. b, c The expression of CD83 was analyzed in CCR7^+/− T cells within the tumor and spleen. Representative flow cytometry plots (b) and the frequency of CD83⁺ cells in the CCR7^+/−CD8⁺ T cell populations are shown (c), (n = 5 mice, repeated measures one-way ANOVA with multiple comparison test). d–f The expression of CCR7 and TIM3 was compared between CD83^+/−PD1⁺CD8⁺ CAR-T cells within the tumor. The data shown are representative flow cytometry plots (d), the frequency of CCR7⁺ cells in CD83^+/−PD1⁺ CAR-T cells in the tumor (e), (n = 5 mice, paired two-tailed t-test), and mean fluorescence intensity of TIM3 in the indicated T cell populations (f), (n = 5 mice, repeated measures one-way ANOVA with multiple comparison test). The data in c, e, and f are derived from the same mice. g–j Expression levels of TCF7 and granzyme B of intratumoral CAR-T cells were analyzed by intracellular flow cytometry. The data shown are representative flow cytometry plots (g, i) and the mean fluorescence intensity of TCF7 (h) and granzyme B (j) in the CD83⁺ and CD83⁻ CAR-T cell populations. (n = 6 or 5 mice, paired two-tailed t-test). k–o Mesothelin-targeting CAR-T cells were daily stimulated with K562-mesothelin for 7 days, and CCR7⁺CD83⁺, CCR7⁻CD83⁺, and CD83⁻ CD8⁺ CAR-T cells were purified by flow cytometry (l), (CD83-transduced cultured T cells were analyzed using the same panel to determine gating threshold). The isolated T cells were then restimulated by K562-mesothelin to analyze fold expansion (m), (n = 6 different samples) and the secretion of IL-2 (n), (n = 5 different samples; two samples from CD83⁺CCR7⁻ and four samples from CD83⁻ cells were under detection limit) and IFN-γ (o), (n = 5 different samples; one sample from CD83⁻ cells was under detection limit). In (m–o), statistical significance was tested by repeated measures one-way ANOVA with multiple comparison test. For (n) and (o), log-transformed values were used for calculation. Horizontal lines indicate mean values. NS not significant.