On the other hand, CD16 expression was only minimally reduced C59 wnt on NK cells exposed to hypoxia (Fig. 2A). The inhibitory effect of hypoxia on triggering receptors expression was observed after 48 h and was even more pronounced after 96 h of culture. Statistical analysis of data obtained in NK cells derived from different healthy individuals (Fig. 2A, lower panels) confirmed that hypoxia could significantly decrease the expression of NCRs and NKG2D, while it had only marginal effect on CD16 expression. Analysis of Killer Ig-like Receptors (KIRs) by specific monoclonal

Ab (mAbs) indicated that in PB NK cells, the size of the various subsets (identified by the expression of different KIR patterns) was not substantially modified by hypoxia conditions (data not shown), thus suggesting that hypoxia did not affect the balance among NK-cell subsets. We tested whether the inhibitory effect of hypoxia on the surface receptor expression

could be exerted also in NK cells activated by cytokines other than IL-2, including IL-15, IL-12, and IL-21. In all cases (Fig. 2B and Supporting Information Table 1), cells cultured under hypoxia displayed a reduced expression of NCRs and, to a minor extent, of NKG2D and CD16. The only exception was represented by the lack of effect on NKG2D click here expression in IL-12-cultured cells. On the other hand, IL-12 induced only a small increment of NKG2D expression under normoxic conditions (Supporting Information Table 1). We also analyzed the effect of hypoxia on the expression of perforins, and granzymes A and

B. As shown in Figure 2C, a trend toward a reduction of granule expression is observed in NK cells cultured under hypoxic conditions. We next investigated whether the impaired expression of activating receptors had any effect on the NK-cell ability to recognize and SPTLC1 kill susceptible targets. To this end, NK cells cultured with IL-2 under either hypoxic or normoxic conditions for 96 h were mixed with target cells and assessed for surface expression of CD107a in order to measure degranulation. As shown in Figure 3A, NK cells cultured under normoxia showed high CD107a expression upon exposure to the FO-1 melanoma cell line (a highly NK-susceptible target), while NK cells cultured under hypoxia expressed significantly lower levels of this marker (60 versus 17%). These data indicate that the ability of NK cells to degranulate was sharply reduced by hypoxia. The degranulation assay was also performed by stimulating NK cells with the FcγR+ P815 target cells coated with anti-NK-receptor-specific mAbs. As shown in Figure 3B, NKp46, NKp30, NKp44, and NKG2D induced lower CD107a expression in NK cells cultured under hypoxia as compared with that of “normoxic” NK cells. On the other hand, CD16, only minimally altered by hypoxic conditions (see Fig.