Anti cd94

Cells were pre-incubated with 1 µg/ml of FcR blocker [Miltenyi-Biotech]. For intracellular cytokine staining, cells were first stimulated with PMA [0.1 µg/ml] and ionomycin [1 µg/ml] in the presence of Brefeldin A [3 µg/ml] for 4 h at 37°C before being surface-stained, fixed, and permeabilised [eBioscience kit]. The following antibodies were used for flow cytometry staining: intracellular staining panel including anti-CD45, anti-CD3, anti-CD8, anti-IL-17A, anti-IL-22, anti-TNF-α [all from Biolegend], and anti-IFN-γ [eBioscience] antibodies; CCR panel including anti-CXCR3 and anti-CCR4 [BD Biosciences], anti-CCR6 and anti-β7 [Biolegend], anti-CCR9 [eBioscience] and anti-CCR10 [R&D Systems] antibodies; ILC panel including anti-lineage [CD34, TCRαβ, TCRγδ, BDCA-2, FCεR1a, CD123, CD1a, CD11c, CD14, CD3, CD19, CD16], anti-CD45, anti-c-Kit, anti-CRTH2, anti-CD127 [all from Biolegend], anti-CD56 and anti-CD94 [BD Pharmingen]. All antibody cocktails contained a fluorescent dye [Invitrogen/Life technologies] to discriminate dead cells during analysis. Stained cells were acquired on an LSRII flow cytometer and analysis was performed using FlowJo software [Tree Star]. For critical discrimination, cell populations were gated against corresponding isotype or fluorescence minus one [FMO] control antibody panels.

Fluorescent monoclonal antibodies (mAbs) were purchased as follows: anti-CD8, anti-CD56, anti-CD94, anti-NKG2D, anti-CTLA-4 and anti-TNF from BD Bioscience (San Jose, CA); anti-CD127, anti-CD3, anti-CD28 and anti-Eomesodermin (Eomes) from eBioscience (San Diego, CA); anti-PD-1, anti-Tbet and anti-IFNγ from BioLegend (San Diego, CA); anti-NKG2A from R&D Systems (Minneapolis, MN); anti-Vδ1 TCR (clone REA173) from Miltenyi Biotec (San Diego, CA); anti-Vδ2 TCR (clone B6), anti-Vγ9 TCR (clone B3) and pan-γδ TCR (clone B1) from BioLegend (San Diego, CA). Dead cells were excluded using Aqua dead cell stain kit (Life Technologies). The phycoerythrin- or allophycocyanin-labeled CD1d tetramers were kindly provided by the NIH Tetramer Facility at Emory University (Atlanta, GA).