Flojo v 10

Peripheral blood was collected in EDTA- or heparin-containing vacutainers. Red blood cells were lysed from 200 µl of EDTA-blood using RBC lysis buffer (eBioscience). Cells were washed with FACS buffer (PBS containing 2% FBS), then equally distributed in five tubes and incubated for 1 h at 4°C with primary antibodies (Ab), as described below. After washing with FACS buffer, cells were incubated for 30 min at 4°C with secondary antibodies, washed again with FACS buffer, and incubated for 30 min at 4°C with conjugated antibodies. After washing, cells were re-suspended in FACS buffer and immediately analyzed on LSR Fortessa Flow Cytometer (BD) using FACS Diva v.6.1.3 software (BD). NK cells were defined as CD56⁺ CD3⁻ CD20⁻ CD14⁻ cells. Final analysis on the expression of NK cell markers was performed using FloJo v.10.2 (TreeStar). The antibodies used to define NK cell phenotype were either commercially available or were generated in the laboratories of Alessandro Moretta and Silvia Parolini.
To analyze the expression of NK cell surface markers, five different tubes were prepared (Table 2), each of which contained FITC-conjugated antibodies directed against CD3, CD20, and CD14, to gate out T cells, B cells, and monocytes. Furthermore, tube #1 contained isotype controls, whereas tubes #2–5 contained antibodies to NK cell markers.

Natural killer cell degranulation activity was tested against the K562 erythroleukemia human cell line. In particular, PBMCs derived from patients and from healthy donors were obtained upon Ficoll separation of heparinized blood samples, and incubated with or without 100 U/mL recombinant human IL-2 (NIH) at 37°C overnight. Cells were then incubated with target cells at an effector:target ratio of 1:3 in a final volume of 200 µl in round-bottomed 96-well plates at 37°C and 5% CO2 for 4 h in culture medium supplemented with anti-CD107a-PE (BD Biosciences Pharmingen, San Diego, CA, USA) monoclonal antibody. Cells were then surface-stained with FITC anti-CD3, PC5 anti-CD56, FITC anti-CD14 (Beckman Coulter), FITC anti-CD20, and APC/Cy7 anti-CD16 (BD Biosciences Pharmingen, San Diego, CA, USA) Ab for 30 min at 4°C. The cells were washed, and the proportion of CD3⁻ CD14⁻ CD20⁻ CD56⁺ cells expressing CD107a was analyzed immediately on LSR Fortessa Flow Cytometer (BD) using FACSDiva v6.1.3 software (BD Biosciences, Mountain View, CA, USA). Final analysis was performed using FloJo v.10.2 (TreeStar). The threshold to define CD107a expression in cells co-cultured with K562 target cells (in the presence or absence of IL-2) was set up on cells cultured with IL-2 alone, without K562 cells.

To detect intracellular production of IFN-γ, PBMCs from patients and healthy donors were incubated overnight at 37°C with IL-12 (0.5 ng/ml), or IL-12 (0.5 ng/ml) and IL-18 (0.1 ng/ml) combined. Surface staining was done by incubating the cells with FITC anti-CD3, PC5 anti-CD56, FITC anti-CD14 (Beckman Coulter), FITC anti-CD20, and APC/Cy7 anti-CD16 (BD) mAbs for 30 min at 4°C. Cells were then washed, fixed, and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences Pharmingen). IFN-γ production was detected by subsequent intracellular staining with PE-conjugated anti-IFN-γ (BD Biosciences Pharmingen). After washing, the proportion of CD3⁻ CD14⁻ CD20⁻ CD56⁺ cells expressing IFN-γ was immediately analyzed on LSR Fortessa Flow Cytometer (BD) using FACSDiva software (BD). Final analysis was done using FloJo v.10.2 (TreeStar).

Intracellular content of perforin was analyzed in a limited number of patients with SCID/OS/AS due to RAG/NHEJ defects and in healthy infants. Briefly, PBMCs were first stained with a mixture of FITC-conjugated mAbs directed against CD3, CD20, and CD14, as well as with PC5-labeled anti-CD56 mAb, and incubated for 30 min at 4°C. After treatment with 200 µl of Cytofix/Cytoperm (BD-Bioscience, Pharmigen CA, USA) for 20 min at 4°C, cells were washed with 1 ml of saponin (0.1% solution in PBS), and then stained with 5 µl of purified R-PE-labeled anti-perforin mAb (Ancell). After washing, the proportion of CD3⁻ CD14⁻ CD20⁻ CD56⁺ cells expressing perforin and the mean fluorescent intensity (MFI) of perforin were immediately analyzed on LSR Fortessa Flow Cytometer (BD) using FACSDiva software (BD). Final analysis was done using FloJo v.10.2 (TreeStar).