AICL upregulation and concomitant NKp80 downregulation by PMA-treated CD3⁻CD56⁺ PBMC. Freshly isolated human PBMC were stimulated with PMA and subsequently, at the indicated times, probed for expression of AICL or NKp80. (A-B) Kinetics of cell surface expression of AICL and NKp80, respectively, by NK cells among PMA-treated PBMC as determined by flow cytometry. PBMC were cultured with PMA for up to 5 days and stained for AICL (mAb 7F12), for NKp80 (mAb 5D12), or with isotype controls at various time points. One representative experiment out of 4 is shown. (A) Histograms show CD3⁻CD56⁺ cells stained with mAb 7F12 (upper) or mAb 5D12 (lower) (open histograms), and isotype controls (gray) at various time points. (B) Kinetics of AICL (●) and NKp80 (▪) surface expression on CD3⁻CD56⁺ cells among PMA-stimulated PBMC. MFI of AICL or NKp80 stainings was set in relation to those of isotype controls. (C) AICL glycoproteins in lysates of unstimulated PBMC (ø) or PBMC stimulated with PMA for various times were detected by immunoblotting with mAb 7G4. Actin detection served as loading control. Experiments with PBMC of 3 different donors gave comparable results. (D) Lysates of PBMC stimulated for 2 days with PMA were either left untreated (ø) or treated with endoglycosidase H or PNGase F, as indicated, before immunoblotting with mAb 7G4.