Rainbow calibration particles

Cryopreserved PBMC were thawed, washed with phosphate buffered saline (PBS) and stained with anti-CD3 and anti-CD56 fluorochrome-labeled antibodies for detection of NK cells by fluorescence activated cell sorting (FACS). In addition, cells were stained with different combinations of the following fluorochrome-labeled antibodies: anti-CD27, anti-CD57, anti-CD62L, anti-CD69, anti-CD127, anti-NKG2A, anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp46, anti-CD107a and anti-INF-γ (Table 2). Isotype controls were used to set up gates and to determine positive and negative populations, respectively (S1 Fig). Dead cells were excluded by use of Zombie Aqua Fixable Viability Kit (Biolegend, Fell, Germany). After incubation of the cells with 1 to 10 μl of antibody solution (depending on antibody) and washing with PBS, samples were analyzed on a FACSCanto II flow cytometer using CellQuest Pro (BD Biosciences) and FlowJo 7.5 software packages (TreeStar Inc., Ashland, OR, USA). To ensure consistent cytometer settings Rainbow Calibration Particles (Biolegend, Fell, Germany) were used. Fig 1 depicts the complete gating strategy for identification of NK cells as well as CD56^dim and CD56^bright subsets (Fig 1).

The expression of KIR receptors (KIR2DL1, 2DS1, 2DL2/S2, 2DL3 and 3DL1) on CD3+CD8+ T cells and CD3–CD56+ NK cells in peripheral blood was evaluated as a percentage of positive cells using LSR-II and DIVA Software (BD, San Diego, CA, USA), as previously described [18 (link),20 (link)]. LSR-II photomultiplier (PMT) voltages were adjusted daily using rainbow calibration particles (BioLegend, San Diego, CA, USA). Fluorescence compensation was finely adjusted using negative events for each fluorochrome as a reference. The staining protocol consisted of a 11-color/12-monoclonal antibody (mAb) panel: CD3 AmCyam (clone SK7, BD), CD4 PE-CF594 (RPA-T4, BD), CD8 APCCy7 (SK1, BD), CD16 PacBlue (3G8, BD), CD56 BV711 (NCAM16.2, BD), CD158a,h PECy7 (EB6, BD, recognizes both KIR2DL1 and 2DS1), CD158b1/b2,j PE-Cy5 (GL183, Beckman-Coulter, Brea, CA USA, recognizes KIR2DL2, 2DL3 and 2DS2), CD158a FITC (143211, R&D Systems, MN, USA; KIR2DL1), CD158b2 APC (180701, R&D Systems, KIR2DL3), CD158e APC (DX9, R&D Systems, KIR3DL1), CD226 PE (11A8, BioLegend), and NKG2A biotin (REA110, Miltenyi Biotech, BergischGladbach, Germany).
The gating strategy used to identify total lymphocytes, CD3+, CD4+, and CD8+ T lymphocytes, as well as CD16^−/+CD56⁺⁺ (CD56^bright) and CD16⁺CD56⁺ (CD56^dim) NK cells is shown in Figure S2.

Apparent dissociation constants (K_D) were measured by calibrated flow cytometry on a Guava easyCyte 6HT instrument (Merck Millipore, Burlington, MA, USA) with 3.0 to 3.4 μm Rainbow Calibration particles (BioLegend, San Diego, CA, USA) as calibration control [26 (link)]. After normalization, data points were fitted to a one-site specific binding model. Expression and purification of molecules is outlined in supplementary methods.