Anti cd107a apc

A total of 100,000 SNAP-CAR Jurkat or primary human T cell effector cells were co-incubated with 200,000 of the indicated target cells and antibody concentrations for 24 h and assayed by flow cytometry for T cell marker gene expression. For primary cell assays, cells were stained with anti-CD69-PE (BD Biosciences) 1:100 or anti-CD69-BV711(BD Biosciences) 1:100, anti-CD62L-FITC (BD Biosciences) 1:100, and anti-CD107a-APC (BD Biosciences) 1:100 antibodies and for Jurkat effector assays, cells were stained with anti-CD62L-FITC (BD Biosciences) 1:100 and anti-CD25-APC (BD Biosciences) 1:100 antibodies. For flow cytometry CAR + cells were analyzed by gating for the TagBFP+ population. Supernatants from primary cell assays were also collected and analyzed for IFNɣ by ELISA (BioLegend). Absorbances were read using the SpectraMax i3 plate reader running the SoftMax Pro 7 software. All assays were performed in triplicate and average IFNɣ production was plotted with standard deviation.

CD3⁺ cells were depleted from the freshly thawed PBMCs using EasySep Positive Selection Kits II (Stemcell). Flowthroughs consisting of CD3⁻ cells were collected. The cells (5 × 10⁵) were then pulsed with 5 μg/ml of peptides or DMSO for 1 h in 37°C. After incubation, pulsed cells were washed twice before addition of autologous nasal cells together with anti-CD40L-PE (RRID: AB_314828; BioLegend) and anti-CD107a-APC (RRID: AB_1727417; BD). After 3 h of incubation at 37°C, the cells were washed in PBS stained with Zombie NIR Fixable Viability Kit to exclude dead cells in subsequent analysis. Then, they were washed in FACS buffer and stained with surface markers anti-CD3-BV605 (RRID: AB_2561911; BioLegend), anti-CD4-BV650 (RRID: AB_2744425; BD), anti-CD8-PE-Cy7 (RRID: AB_396852; BD), anti-CD69-AF700 (RRID: AB_493775; BioLegend), and anti-CD103-FITC (RRID: AB_10597744; eBioscience) diluted in FACS buffer for 30 min on ice. After two more washes in FACS buffer, cells were resuspended in PBS before acquisition with a Beckman Coulter CytoFLEX S analyzer.

The following antibodies for flow cytometry were used: anti-CD107a-APC (H4A3), anti-CD3-AF700 (UCHT1), anti-CD3-PE-CF594 (UCHT1), anti-CD56-PE-Cy7 (B159), anti-CD56-PerCP Cy5.5 (B159), anti-CD57-PE (NK-1), anti-CD45-PE-Cy5 (HI30), anti-NKG2D-BV605 (1D11), anti-NKG2D-PE-CF594 (1D11), anti-CD16-BV510 (3G8), anti-DNAM-1-BV786 (DX11), anti-PD-1-BV421 (MIH4), anti-IFNγ-BV650 (4S.B3), anti-CD14-BV605 (M5E2), anti-CD19-BV650 (SJ25C1) purchased from BD Biosciences; anti-DNAM-1-APC (11A8), anti-NKp46-PE-Cy7 (9E2), anti-TNFα-AF700 (Mab11), anti-CD96-APC (NK92.39) purchased from Biolegend; anti-NKp30-PE (Z25), anti-KIR2DL1/2DS1-PE Cy5.5 (EB6B), anti-KIR2DL2/L3/S2-PE (GL-183) purchased from Beckman Coulter; anti-NKG2A-AF700 (131411), anti-KIR3DL1-APC (DX9) purchased from R&D Systems; anti-NKG2A-FITC (REA110), anti-NKG2C-PE (REA205) purchased from Miltenyi; anti-TIGIT-APC (MBSA43) purchased from eBioscience. All these antibodies were used according to the manufacturers’ protocol. Prior to surface staining, NK cells were pre-stained with Live/Dead™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Flow cytometry was performed by using FACSCantoTM II (BD Biosciences) or Cytoflex (Beckman Coulter) and analyzed by FlowJo Software.

To measure NK cell capacity to degranulate, purified NK cells were cocultured at 1:1 ratio with target cells (C1R/C1R-MICA) at 37°C for 6 h, in the presence of anti-CD107a-APC (BD Biosciences). Brefeldin A (1 μg/ml; Sigma-Aldrich) and Monensin (1 μg/ml; Sigma-Aldrich) were added after 1 h. Where indicated, an anti-NKG2D blocking Ab or control Ab mAb (0.5 μg/ml; eBiosciences) was added at the onset of culture. NKG2D blockade did not affect NK cell viability.

The selective CXCR2 antagonist SB225002 and 7-AAD were purchased from Sigma-Aldrich. Monoclonal antibodies (mAbs) anti-CD138/FITC, anti-CD38/APC, anti-CD107a/APC, anti-CD3/FITC, anti-CD56/PE, anti-CD45/APCH7, anti-CD90/PeCy5, anti-CD105/APC, anti-CD146/BV395, anti-CD106/PE and anti-CD73/V450 were purchased from BD Biosciences. mAb anti-CXCR2/PE was purchased from Miltenyi Biotec. mAb anti-DNAM-1 (DX11) was purchased from Serotec-Biorad (Hercules, CA, USA). Conjugate anti-PVR/PeCy7 and anti-CXCR1 (MAB330-SP) were purchased from R&D Systems (Minneapolis, MN, Canada). Anti-PVR (SKII.4) was kindly provided by Prof. M. Colonna (Washington University, St Louis, MO, USA). Anti-MHC class I (W6/32) was purchased from ATCC. Allophycocyanin (APC)-conjugated with goat anti mouse antibody was purchased from Jackson Immuno-Research Laboratories (Cambridgeshire, United Kingdom).

In 12-well cell culture plates, PTA-6967 cells (1,000,000/well) were incubated for 6 hours at 37°C, 5% CO₂ in NK cell media containing 5 µL anti-CD107a-APC (BD #560664) as well as PMA (50 ng/mL) and ionomycin (1 µg/mL), or rFVIIIFc (250 nM). After one hour, monensin (0.67 µL/mL) and Brefeldin A (1 µL/mL) were added to block endocytic trafficking. Cell samples were harvested, washed and stained for viability (Fixable Viability Dye eFluor506; eBioscience #65-2860) and surface markers (CD3-FITC (BD #561806), CD16-PE-Cy5 (BD #555408), CD56-PE-Cy5 (BD #557747)). Samples were analyzed using a BD LSRII cytometer and FlowJo version 10 software.

The NAE inhibitor MLN4924/Pevonedistat was purchased from Selleckchem.Com (Houston, Texas, USA). The TGFβ type I receptor inhibitor SB431542 and Cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). The final concentration of DMSO in all experiments was <0.1%. Daratumumab and Elotuzumab mAbs were provided by the “Policlinico Umberto I” hematology center pharmacy.
The following monoclonal antibodies (mAbs) were used for immunostaining: anti-CD14-APC-H7 (MφP9), anti-CD19-APC-H7 (HIB19), anti-CD45-PE-Cy™7 (HI30), anti-CD3-BV510 (HIT3a and UCHT1), anti-CD56-BV421/PE (NCAM16.2), anti-CD16-PE-Cy™7 (3G8), anti-CD107a-APC (H4A3), anti-CD138-FITC/PE (MI15), anti-CD38-APC (HIT2), anti-IFN-γ-PE (4S.B3), anti-Perforin-Alexa Fluor® 488 (δG9), anti-Granzyme B-PE (GB11), anti-NKG2D-APC (1D11), anti-DNAM1-FITC (DX11), anti-NKp30-Alexa Fluor® 647/BB700 (p30-15), anti-TIM-3-BB515 (7D3), anti-PD-1-BV421 (EH12.1) were purchased from BD Biosciences (Franklin Lakes, New Jersey, U.S.A.). Anti-NKp46-PE (9E2) and anti-SLAMF7-APC (162.1) were purchased from BioLegend (San Diego, California, U.S.A.) . Anti-TNFα-FITC (cA2), REA Control Antibody-APC (REA293), anti-TGFβ RII-APC (REA903) were purchased from Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany). Anti-TGFβ_1/2/3 (1D11) monoclonal antibody was used as blocking antibody [R&D System (Minneapolis, MN, USA)].