Anti ifn γ 4s b3

PBMCs (1 ×10⁵) were incubated for 18–24 hrs with plate-bound ICAM-1-Fc (coated at 5 μg/ml) or with an isotype-matched negative control mAb (5 μg/ml MOPC-21, Sigma Aldrich). PBMCs were resuspended and Fc receptors were blocked by incubation in a solution of PBS containing 25% human AB serum (Atlanta Biologicals) for 20 minutes at 4°C, then cell-surface staining was performed for 30 minutes at 4 °C using the following human-specific antibodies or tetramers: anti-CD3 (OKT3, Biolegend), anti-CD4 (OKT4, Biolegend), anti-CD8α (HIT8a, Biolegend), anti-CD8β (SIDI8BEE, eBioscience), anti-CD11a (HI111, Biolegend), anti-CD56 (5.1H11, Biolegend), CD1d tetramer (PBS-57 loaded, NIH Tetramer Core Facility), MR1 tetramer (5-OP-RU loaded, NIH Tetramer Core Facility), anti-Vδ2 TCR (123R3, Miltenyi Biotec). Cells were fixed and permeabilized according to the manufacturer’s instructions using the BD Cytofix/Cytoperm kit (BD Biosciences), then stained with anti-IFN-γ (4S.B3, Biolegend), anti-PLZF (Mags.21F7, eBioscience), or the respective negative control mAbs suggested by the vendor. Cells were then washed, resuspended in PBS and analysed on an LSR II flow cytometer (BD Biosciences). Staining data were analyzed using FlowJo analysis software (Tree Star Inc).

For intracellular cytokine staining, T cells were activated for 3h with PMA and ionomycin in the presence of GolgiStop (BD Bioscience), followed by anti-IFNγ (4S.B3; Biolegend) or anti-IL-17 (eBio64Dec17; ebiosciences). Levels of IFNγ and IL-17 secreted by T cells stimulated for 4 days under Th1-, Th2- or Th17-polarizing conditions were measured using a Cytometric Bead Array (CBA) Flex Set system (BD Bioscience) and analyzed with FCAP Array Software (BD Bioscience). Cytokine levels in serum were determined by the University of Pittsburgh Cancer Institute Luminex Core Laboratory using MILLIPLEX NHP kits from Millipore (Billerica, MA), according to the manufacturer’s instructions.

Surface stain monoclonal antibodies (mAbs) include: anti-CD4 [L200], anti-CD49d α4 integrin [9F10] and anti-CD95 [DX2] (BD Biosciences, San Jose, CA); anti-CD14 [TUK4], anti-CD20 [HI47] and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Grand Island, NY); anti-CD28 [CD28.2] (Beckman Coulter, Pasadena, CA); anti-CD8 [2ST8.5H7] (Custom, mAb from Serotec, conjugation kit from Invitrogen).Intracellular stain mAbs include: anti-CD3 [SP34-2] and anti-TNF-α [MAb11] (BD Biosciences); anti-IFN-γ [4S.B3] and anti-IL-2 [MQ1-17H12] (Biolegend, San Diego CA).