Fig. 6

CardAP-EVs diminish anti-CD3 induced T cell proliferation only in the presence of CD14⁺ cells. CD3⁺ and CD14⁺ cells were isolated by MACS and cultured unstimulated for 48 h. Here, CD14⁺ cells were additionally treated with either unstimulated (EVs) or cytokine stimulated (EVs^(cyt)) EVs, PBS in equal volume of the EVs (PBS) or left untreated. Afterwards, CD14⁺ cells were co-cultured 1:5 with CD3⁺ cells and stimulated with anti-CD3. Additionally, a negative control was incorporated by treating anti-CD3 stimulated CD3⁺ cells with either CardAP-EV variant, PBS or left untreated. After 3 days, the cells were harvested and analysed by flow cytometry. T cell proliferation frequencies were normalized to the untreated control. a The general immune assay design is shown. Representative flow cytometric plots display the frequencies of proliferated CD4⁺ and CD8⁺ T cells in anti-CD3 stimulated monocultures of CD3⁺ cells (b, left) and anti-CD3 stimulated co-cultures of CD3⁺ cells with primed CD14⁺ cells (c, left). The normalized proliferation of CD4⁺ (upper graph) and CD8⁺ (lower graph) T cells in anti-CD3 stimulated monocultures of CD3⁺ cells (b, right) or in co-culture with primed CD14⁺ cells (c, right) is presented for the treatment with either CardAP-EV variant and PBS as median with interquartile range (monocultures n = 7; four different CardAP donors; five different PBMC donors) (co-cultures n = 5; four different CardAP donors; four different PBMC donors). Friedman Test with Dunn’s multiple comparison test: ***p < .001, **p < .01, *p < .05 or Wilcoxon matched-pairs signed rank test; ^###p < .001, ^##p < .01, ^#p < .05