FIGURE 4.

NKG2D mediates the uptake of MICA*008⁺ EVs. (a) NKL cells were pre‐treated with αNKG2D and αDNAM‐1 blocking mAbs and then incubated for 3 h with PKH26‐labelled sEVs (30 μg/ml) or mEVs (20 μg/ml). Data reported represent the mean of three independent experiments. Values represent the percentage of PKH26⁺ cells. (b and c) NKL cells were treated overnight with MICA*008⁺ EVs to induce NKG2D downmodulation (NKG2D^low) or left untreated (NKG2D^high). (b) A representative experiment showing the different levels of NKG2D expression is shown. (c) The mean of three independent experiments is shown. (d) NKG2D^low and NKG2D^high NKL cells were incubated with PKH26‐labelled EVs for 3 h as described in panel (a). Values represent the percentage of PKH26⁺ cells. The mean of three independent experiments is shown. (e–g) NKL cells were infected with the pLKO lentiviral vector containing a scrambled sequence or shRNA for silencing NKG2D. NKG2D expression was evaluated by (e) FACS analysis and (f) real‐time PCR. (g) NKL/pLKO and NKL/shNKG2D were incubated with PKH26‐labelled EVs for 3 h as described in panel (a). Values represent the percentage of PKH26⁺ cells. The mean of at least four independent experiments is shown. (h) Ba/F3 or Ba/F3 stably expressing the NKG2D/DAP10 complex were stained with cIg or anti‐NKG2D mAb and analyzed by Immunofluorescence and FACS analysis. (i) Ba/F3 or Ba/F3‐NKG2D/DAP10 were plated at 5 × 10⁵ cells/ml and incubated for 1 h with PKH26‐labelled mEVs as described in panel (a). Values represent the percentage of PKH26⁺ cells. The mean of three (for sEVs) or four (for mEVs) independent experiments is shown