Anti cd56 apc

TILs were collected and stained with the following antibodies: anti-CD3 (Pcy5), anti-CD4 (PE), anti-CD8 (ECD), and anti-CD56 (APC) (Biolegend, CA).CD27 and CD28 surface expression was further tested after incubating TIL in complete medium without rhIL-2 for 48 hours. Isotype-matching antibodies were used as controls. After incubation at 4°C for 30 min with the above coupled antibodies and being washed with PBS, TILs were fixed with 4% paraformaldehyde and analyzed using CytomicsFC500 Flow Cytometry (Beckman Coulter Inc., Fullerton, CA). Data analysis was performed using the CXP Analysis software (Beckman Coulter). For cytokine production assays, TILs were stimulated with paraformaldehyde (PMA) (Sigma, 50 ng/ml) and ionomycin (Sigma, 500 ng/ml) for 4 h in the presence of Brefeldin A (Biolegend; 10 mg/ml). Cells were then collected, washed and fixed with 4% PMA for 5 min at room temperature (RT) and permeabilized with permeabilization buffer (eBioscience, San Diego, CA) for 10 min at RT. Cells were then labeled with anti-IFN-γ (APC), anti-TNF-α (FITC), and anti-IL-4 (APC) and analyzed by flow cytometer.

Fresh whole-blood leukocytes were analyzed by flow cytometry to identify and quantify the major leukocyte populations. Briefly, 200 μl EDTA-stabilized venous blood was stained with a panel of fluorochrome-conjugated antibodies (anti-CD45-PerCP [Clone: HI30; BioLegend], anti-CD3-APC/Cy7 [Clone: OKT3; BioLegend], anti-CD4-BUV395 [Clone: SK3; BD Biosciences], anti-CD8-BUV737 [Clone: SK1; BD Biosciences], anti-CD19-PE [Clone: HIB19; BioLegend], anti-CD14-Alexa Fluor 488 [Clone: HCD14; BioLegend], anti-CD16-PE/Cy7 [Clone: 3G8; BioLegend], anti-CD15-BV605 [Clone: W6D3; BioLegend], and anti-CD56-APC [Clone: HCD56; BioLegend]) for 15 min at room temperature in BD Truecount tubes. Red blood cells were lysed (BD FACS Lysing solution), and leukocytes were analyzed immediately on a BD Fortessa flow cytometer with BD FACSDiva software. Absolute cell numbers were calculated according to the manufacturer’s instructions (Truecount Tubes, BD Biosciences).
For the characterization of both rare and abundant leukocyte populations in the blood, cryopreserved PBMCs (4–8 × 10⁵ cells per individual) from the PD-L1-deficient siblings, their heterozygous mother, a healthy adult, and age-matched controls, and the previously described PD-1-deficient child (Ogishi et al., 2021 (link)) were analyzed by spectral flow cytometry as previously described (Ogishi et al., 2023 (link)).

NK cell degranulation assays were performed as elsewhere described (37 (link), 38 (link)). Briefly, PBMCs (1x10⁶/ml) were incubated with P815 cells (2x10⁶/ml) in a total volume of 200 μl in a 96 well plate. P815 cells were supplemented with 5mg/ml of anti-CD16 mAb (Clone 3G8, eBiosciences). After 3 hours of incubation at 37°C, cells were recovered and stained using following antibodies: anti-CD3 FITC, anti-CD56 APC, and anti-CD107 PE (Biolegend, clone H4A3). GolgiStop was not included in these assays. Cells were acquired on FACSCanto II (BD bioscience) and analyses with the use of FlowJo 7.6.5 software (Tree Star, Ashland, OR). Gates were set to exclude CD3+ lymphocytes. Thereafter, the percentage of cells positive for CD107a was obtained after gating in CD3-CD56+ lymphocytes. The basal percentages for CD107a were obtained from PBMCs incubated with P815 cells with no agonist mAb. Degranulation was represented as the fold increase for CD107a, which is the difference between the percentage of NK cells expressing CD107a at surface after stimulation with P815 cells supplemented with agonist mAb, and the percentage of NK cells expressing CD107a at NK cell surface after incubation with P815 cells with no agonist mAb. NK cell degranulation assays were performed in 32 acute leukemia patients and 10 age-matched healthy controls.

Peripheral blood mononuclear cells (PBMCs) from 47 healthy controls and 151 MS patients were isolated from blood samples collected in EDTA tubes using Ficoll–Hypaque density gradient centrifugation and cryopreserved in fetal calf serum with 10% dimethyl sulfoxide until analysis. Sample staining for flow cytometry analysis was performed using anti-NKG2A (clone Z199) indirectly labeled with a secondary goat anti-mouse PE-Cy7 (Biolegend) and the following fluorochrome-conjugated antibodies: anti-CD3-PerCP (BD Pharmigen), anti-CD56-APC (Biolegend), antiCD16-eFluor450 (eBioscience), and NKG2C-PE (R&D Systems). For the analysis of FcRγ (MS, n = 139; controls n = 47) and PLZF expression (MS, n = 86; controls n = 26), cells were treated with a fixation/permeabilization kit (BD Biosciences) followed by incubation with anti-FcRγ-FITC (Millipore) and anti-PLZF-PE CF594 (BD Biosciences). Samples were acquired in LSRFortessa (BD Biosciences) and data were analyzed using FlowJo software (Tree Star, Oregon, USA), using the gating strategy shown in Figure 1.

Fresh CSF cells and PBMCs were isolated and immunophenotyped within 6 hours of sample collection. Briefly, CSF cells were isolated by centrifugation at room temperature at 500g for 10 minutes from whole CSF and washed with PBS. PBMCs were isolated by layering whole blood on Ficoll-Paque PLUS (Cytiva), followed by centrifugation at room temperature at 1,000g for 25 minutes with minimal acceleration and deceleration, harvesting, and washing. CSF cells and PBMCs were then incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain (L34975, Invitrogen), anti–CD3-V500 (561416, BD Biosciences), anti–CD14-V500 (561391, BD Biosciences), anti–CD19-BV510 (562947, BD Biosciences), and anti–CD56-APC (362503, BioLegend) for 30 minutes on ice. After washing, they were analyzed on a BD LSR Fortessa flow cytometer. NK cell frequency was calculated as CD56⁺CD3^–CD19^–CD14^– out of total live lymphocyte singlets.