(A, B) Purified peripheral blood neutrophils were stimulated with PBS (control) or IL-21 (100 ng/ml) for 4 hr, and IL21R mRNA was measured by real-time PCR and normalized to RPL7 expression (A), and IL-21R protein levels were measured by flow cytometry (B); left panel shows isotype control shaded and anti-IL21R black line, with a summary in the right panel). (C) RNA-Seq was performed on neutrophils after 4 or 24 hr incubation with PBS or IL-21 in the absence or presence of heat-killed S. aureus (106/ml). We used HKSA rather than live bacteria in order to allow analysis at 24 hr, as live bacteria would have overgrown the system by then. Genes differentially expressed (fold-change >2.0) are shown. Shown is a representative RNA-Seq analysis. (D – G) Human peripheral blood neutrophils were stimulated for 4 hr in vitro in the presence or absence of IL-21 and GZMA (D), GZMB (E), GNLY (F), and IFNG (G) mRNA levels were quantitated by RT-PCR and normalized to RPL7 expression. (H) Purified human neutrophils were stimulated with IL-21 for 4 hr, fixed, permealized, and stained for intracellular granzyme B protein (gated on CD66b+ cells); MFIs are summarized in right panel. (I) Granzyme B protein was measured by ELISA in supernatants from human peripheral blood neutrophils cultured for 24 hr in either the absence or presence of IL-21 (100 ng/ml). (J, K) Human neutrophils were incubated in vitro with either MRSA (J) or S. pyogenes (K) for 3 hr with PBS or IL-21 (100 ng/ml). MRSA and S. pyogenes CFU were quantitated by plating serial dilutions on blood agar plates. Results shown are representative of 3 independent experiments, except panel C shows one of two similar independent RNA-Seq experiments, each from a different donor.