Murine Cytomegalovirus Is Regulated by a Discrete Subset of Natural Killer Cells Reactive with Monoclonal Antibody to Ly49h

Daniels, Keith A.; Devora, Gene; Lai, Wayne C.; O'Donnell, Carey L.; Bennett, Michael; Welsh, Raymond M.

Immunofluorescence Staining and Intracellular IFN-␥ Assay. Leukocytes were isolated and stained for the intracellular accumulation of IFN-␥ using reagents and the protocol of BD PharMingen. One million cells were cultured in Falcon 96-well U-bottomed plates (Becton Dickinson) with RPMI media (Life Technologies) containing GolgiPlug (BD PharMingen) at 0.2 l per sample in the presence or absence of 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 h at 37 Њ C. Unless otherwise stated, data are shown for samples incubated in the absence of PMA and ionomycin. Cells were washed using FACS ® buffer (PBS with 2% FCS and 0.02% sodium azide) and incubated for 15 min at 4 Њ C in 100 l of FACS ® buffer plus 9 l normal mouse serum and 1 l of anti-Fc ␥ II receptor Ab to block nonspecific staining. Cells were washed and incubated at 4 Њ C for 30 min with combinations of anti-NK1.1-PE (BD PharMingen), anti-CD3-PerCP (BD PharMingen), and various anti-Ly49 mAb or isotype controls. These were isotype control mouse (m) IgG2a-FITC (BD PharMingen), isotype control rat (r) IgG2a-FITC (BD PharMingen), anti-Ly49D-FITC (a gift from Dr. John Ortaldo, National Cancer Institute, Frederick, MD), anti-Ly49G2-FITC (BD PharMingen), anti-Ly49C-biotin (BD PharMingen), and anti-Ly49H (1F8)-FITC produced by us. Samples were washed twice and, if necessary, were incubated with SA-PerCP (BD PharMingen) for 30 min at 4 Њ C. Otherwise they were fixed and permeabilized using 100 l of cytofix/cytoperm solution for 20 min at 4 Њ C. After fixation the samples were washed twice using perm/wash solution (200 l per sample) and stained with rat IgG1-APC mAb to mouse IFN-␥ (BD PharMingen) or with a control rat IgG1-APC (BD PharMingen) for 30 min at 4 Њ C. Samples were then washed two times using perm/ wash and once using FACS ® buffer before being transferred to tubes (model 2008; Falcon) for analysis on either a FACS ® 440 (Becton Dickinson) or a FACStar™ Plus (Becton Dickinson). For these analyses, 60-80,000 events were calculated, ensuring a sizable NK cell population for a valid analysis of NK subsets. All experiments used relevant control mAbs with isotypes equivalent to the anti-Ly49 and anti-IFN-␥ mAb, but in some figures the negative isotype control plots are not shown to maintain clarity and simplicity of the figure.