Neutrophils are essential for NK cell function at the steady state. (A) Representative FACS profiles of spleen cells from Rat IgG2b- or RB6-8C5–treated mice (top) or, Rat IgG2a-, or 1A8-treated mice (bottom), stained with anti-Ly6C and anti-CD11b mAb. 1A8 is specific for Ly6G, whereas RB6-8C5 recognizes Ly6G and, with a lower affinity, Ly6C (Fleming et al., 1993). As RB6-8C5 recognizes a different epitope on Ly6C than the monoclonal anti-Ly6C (AL-21) antibody (Ribechini et al., 2009), we used CD11b and Ly6C to monitor the percentage of both neutrophils (CD11b^high Ly6C^inter, red circle) and monocytes (CD11b^inter Ly6C^high, black circle). (B) 6 d after treatment, splenocytes from rat IgG2b- and RB6-8C5–treated (top) or, Rat IgG2a- or 1A8-treated mice (bottom), were stimulated by incubation for 4 h on anti-NK1.1, anti-NKp46, or isotype control mAb-coated plates or with a mixture of PMA and ionomycin. NK cell intracellular IFN-γ production was measured by FACS. Experiments were repeated 6–20 times. n = 3–6 mice per group. Statistical significance was determined in a Mann-Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Percentages of neutrophils (red circles) and monocytes (black circles) in the blood and the BM of rat IgG2a- or 1A8-treated mice. Data are representative of 6–10 independent experiments. n = 3–4. (D) 6 d after anti-CD4 antibody (GK1.5) or isotype control antibody (rat IgG2b) injection, the presence of CD4⁺ cells among CD3⁺ T cells was monitored in the spleen of rat IgG2b- or GK1.5-treated mice. (E) 6 d after depletion, splenic NK cells were stimulated for 4 h by incubation in plates coated with NK1.1, NKp46, or isotype control mAb, or with a mixture of PMA and ionomycin. The percentages of NK cells expressing intracellular IFN-γ are shown for control isotype-treated mice (shaded bars) and CD4-depleted mice (open bars). Experiments were performed twice. n = 2–3.