Figure 4

CD19⁺EVs accelerate exhaustion and impair CD19-CAR T cells’ killing activity. A Experimental timeline. B Changes in viability and surface phenotype of CAR T cells cultured in presence of CD19⁺, CD19⁻ or CD19⁺PD-L1⁺ EVs and analyzed daily for 7 days. Data were normalized against T cells treated with corresponding EVs and presented as heat map of the mean of two experimental replicates from two independent experiments. C Representative histograms show the difference in proportion of CAR T cells expressing markers of functional exhaustion: CD57 (left), PD-1 (center), TIGIT (right). CAR T cells stained for corresponding markers were analyzed by flow cytometry on days 4–5. D Analysis of differentiation state of CAR-positive cells cultivated for 5 days in the presence of EVs. CD19-CAR T cells were stained with anti-CD62L and anti-CD45RA mAbs and CD19-FITC conjugate, analyzed by flow cytometry, and plotted as two-dimensional dot plots. CAR T cells incubated for 5 days in the absence of EVs served as negative control. E In vitro killing activity at different time points indicated in Fig. 3A of CD19-CAR T-cells cultured for 20 h with Nalm-6 cells at 1:16 E:T ratio in the presence of CD19⁺, CD19⁻, CD19⁺PDL-1⁺EVs or without EVs. Data are presented as the mean ± SD of four experimental replicates from at least two independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test.