Figure 4. Natural killer-derived extracellular vesicles (NK-EVs) promote CD4⁺ T cell activation and IL-2 release but not regulatory T cell (Treg) responses.

(A) Flow cytometry analysis of isolated CD4⁺ T cells incubated under non-cytokine-polarizing (upper panel) and Th1 cytokine-polarizing (with a mixture of IL-2 and IL-12, lower panel) conditions. Dot plots show the expression of CD4 and CD25 in gated single live cells ± SEM, after addition of NK-EVs. Plots show the quantification of n≥4 independent experiments. Significance was assessed with paired Student’s t-test; *p<0.05, **p<0.01. (B) ELISA quantification of soluble IL-2 in supernatants from CD4⁺ cultured T cells in the indicated conditions (unpolarized, upper panel; cytokine polarized, lower panel). The graph shows the mean concentration from n≥3 independent experiments. Significance was assessed by paired Student’s t-test; *p<0.05. (C,D) Quantitative real-time PCR showing FOXP3 (C, left); IL10 (D, right) and PTEN (D) mRNA levels at days 3 and 6 in CD4⁺ T cells after culture in the indicated conditions. Relative expression is shown, normalized to GAPDH and ACTB. Significance was assessed by paired Student’s t test; *p<0.05.

Figure 4—figure supplement 1. mRNA target modulation in CD4⁺ T cells mediated by natural killer-derived extracellular vesicles (NK-EVs).

(A–F) Quantitative real-time PCR at days 3 and 6, as indicated in either non-polarizing (upper panels) or cytokine-polarizing (lower panels) conditions, showing mRNA levels of IL6 (A), IL1B (B), SOCS1 (C), TGFB (D), TNFA (E), and INPP5D (F) respectively, normalized to GAPDH and ACTB. Significance was assessed with paired Student’s t test; *p<0.05.

Figure 4—figure supplement 2. T cell activation is specific of natural killer-derived extracellular vesicles (NK-EVs).

(A,B) Isolated CD4⁺ T cells were incubated with 80 µM of Dynasore prior to functional analyses. (A) Flow cytometry analysis showing CD4 and CD25. A representative experiment is shown. (B) Bar charts plots show the percentage of CD4⁺ CD25⁺ cells in gated live T cells (min to max and median values), normalizing NK-EV (obtained by ultracentrifugation, as previously described) to PBS-treated cells. Panel show the quantification of n≥4 independent experiments. Significance was assessed with paired Student’s t-test; *p<0.05. (C,D) NK-EVs from activated NK cells were isolated by size-exclusion chromatography (SEC). (C) Flow cytometry analysis showing CD4 and CD25 expression. A representative experiment is shown. (D) Bar charts show the percentage of CD4⁺ CD25⁺ T cells, normalizing NK-EV (obtained by SEC) to PBS-treated cells. Panel shows the quantification of n≥4 independent experiments. Significance was assessed with paired Student’s t-test; *p<0.05, **p<0.01, **p<0.001. (E,F) Small EVs were isolated by differential ultracentrifugation from Raji and HEK human cell lines and their effects on isolated CD4⁺ T cells were analysed. (E) Flow cytometry analyses showing CD4 and CD25 expression in CD4⁺ T cells treated with either PBS, HEK-EVs, or Raji-EVs, as indicated, either in the presence or absence of Th1-polarizing cyokines. A representative experiment is shown. (F) Charts show the quantification of the percentage of CD4⁺ CD25⁺ T cells in the different conditions from four independent experiments. Significance was assessed with paired Student’s t-test; **p<0.01.