Figure 5: XLP1 NK cells have defects in NF-κB activation and IFN-γ expression in response to coactivation by NKG2D and 2B4.

(a) Total lysates of primary expanded NK cells from representative normal or XLP1 patient donors were immunoblotted for SAP and actin. (b,c) Primary rested NK cells after expansion from normal or XLP1 patient donors were mixed with 221 or K562 cells in the presence of fluorochrome-conjugated anti-CD107a mAb for degranulation assay. After incubation for 2 h, cells were stained with fluorochrome-conjugated mAb to CD56, and the level of CD56⁺CD107a⁺ NK cells was measured using flow cytometry. (b) Representative result is shown. (c) Percent increase of CD107a⁺ NK cells obtained from normal or XLP1 donors after stimulation with target cells relative to CD107a⁺ NK cells without target cells (ΔCD107a⁺ cells). Values represent the mean±s.e.m. (d) Primary rested NK cells after expansion from normal or XLP1 patient donors were stimulated with the indicated receptors for 5 min. Lysates were immunoblotted for the indicated phosphorylations of p65. The normalized intensities of the phosphorylated p65 relative to p65 are presented. Representative result (left) and statistical bar charts (right) are shown. Values represent mean±s.e.m. (e,f) Primary rested NK cells after expansion from normal or XLP1 patient donors were mixed with P815 target cells as indicated. After incubation for 6 h, cells were stained with fluorochrome-conjugated mAb to CD56 and analysed by flow cytometry after intracellular staining of IFN-γ. (e) Representative result is shown. (f) Percent increase of IFN-γ⁺ NK cells from individual normal or XLP1 patient donors after stimulation with the indicated receptors relative to IFN-γ⁺ NK cells without stimulation (ΔIFN-γ⁺ cells) is presented. Values represent mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (two-sided Student’s t-test). Statistical bar charts in d show pooled data from three different donors.