αifn γ pecy7

Directly conjugated monoclonal antibodies were obtained as follows: (i) αMIP‐1β‐phycoerythrin (PE), αCD107a‐PE and αCD69‐PE (BD Biosciences, Oxford, UK); (ii) αTCR Vβ 13.1‐fluorescein isothiocyanate (FITC) and αTCR Vβ 17‐FITC (Beckman Coulter, High Wycombe, UK); (iii) live/dead fixable Aqua dead stain (Molecular Probes, Invitrogen, UK); (iv) αCD3‐Pacific blue, αCD3‐PEcyanin 7 (Cy7), αCD4‐allophycocyanin (APC) H7, αCD8‐APC H7, αCD19‐Brilliant Violet 521, αIFN‐γ‐PECy7, αTNF‐α‐PerCPCy5.5, αIL2‐APC, αperforin‐Brilliant Violet 421 (clone D48) and αgranzyme B‐AlexFlour 647 (Biolegend, London, UK); and (v) αCD3‐PCP (Miltenyi Biotech). Soluble peptide‐MHCI (pMHCI) monomers were generated and biotinylated as described previously 26, 35 and tetramers for SLLMWITQC/HLA A2 and YLEPGPVTV/HLA A2 were produced by adding streptavidin conjugated with APC (Molecular Probes, Invitrogen) 35. The HLA type of lymphocytes isolated from blood bags was determined using directly conjugated αHLA A2‐FITC (Serotec, Oxford, UK).

Flow cytometry staining was performed with panels of antibodies specific for activated/memory T cells (αCD3-Biotin, αCD4-FITC, αCD8-APCeF780, αCD62L-PeCy7, αCD44-APC; all from BioLegend) in PBS with 0.5% FCS 2 mM EDTA for 15 min. For intracellular staining cells will be stimulated with a mix containing 10 µg/ml Brefeldin A (Sigma), 50 ng/ml PDBu (Sigma) and 500 ng/ml ionomycin (Sigma) in RPMI-1640 (PAN Biotech) plus 10% FCS (Biochrom) plus 1% penicillin/streptomycin (Lonza) plus 2 mM L-glutamine (Lonza) for 4 h. The cells were then formalin-fixed, permeabilized (0.05% Triton X-100 in PBS) and stained for cytokines (αIL-2-PE, αIFNγ-PeCy7), and transcription factors (αFOXP3-FITC) or perforin (αPerforin-APC) for 1 h. All antibodies were from Biolegend. Cells were analyzed with Gallios and Cytoflex S flow cytometers (Beckman Coulter) and FlowJo Software (Beckton Dickinson).