Fig. 4

Antibody-mediated blocking experiments showing NK cell activating and inhibiting receptors mediating pMSC lysis. NK cells were pre-incubated with antibodies specific to indicated NK cell activating and inhibiting receptors; NK cells were then co-cultured with pMSCs at 25NK to 1pMSC ratio in a cytolytic experiment. pMSC lysis by NK cells was evaluated using microscopic examination within 24 h. Representative phase contrasts microscopic images showing complete lysis of pMSCs as no sign of intact adherent pMSCs and cells were also ruptured as cellular debris were evident in suspension (star pointing at adherent pMSCs) by untreated NK cells (NK cells were initially activated with 100 U/mL IL-2 for 72 h) by NKp30, by NKp44, NKp46, by DNAM, and by NKG2D and substantial inhibition of lysis of pMSC (arrow pointing at adherent pMSCs) by CD69. Blocking CD94 receptor did not increase the cytolytic activity of NK cells. Experiments were carried out in triplicate and repeated for ten times using NK cells and pMSCs prepared from the peripheral blood of ten different healthy donors and ten different normal human term placentae, respectively. Scale bars, 50 μm