Ifn γ apc b27

Intracellular cytokine staining was used to confirm epitopes identified by ELISpot. Thawed PBMCs were rested overnight and stimulated the next day for 6 h in culture medium (RPMI 1640, 10% FCS, 2 mM glutamine, 100 IU ml⁻¹ penicillin/streptomycin) using selected peptides (10 μg ml⁻¹) plus anti-CD28 and anti-CD49. After 2 h, Brefeldin A (1 μl/ml) was added and after 6 h cells were washed and stained with live/dead marker (Invitrogen, USA); IFN-γ APC (B27, #554702), TNF PE Cy7 (MAb11, #557647), interleukin-2 FITC (5344.111, #340448; all BD Biosciences); and relevant surface markers. Dilutions at which antibodies were used are shown in Supplementary Table 8. The frequencies of expression profiles for single, double and triple cytokine producers were calculated using the Boolean combination gating option in FlowJo software (FlowJo LLC, USA).

PBMCs (1–3 × 10⁶ cells) were stimulated with 2 μg ml⁻¹ of the cognate peptide pools for 6 h in RPMI containing 10% human serum in the presence of 5 μg ml⁻¹ of GolgiPlug (10 μg ml⁻¹, BD Biosciences). Negative controls received an equal concentration of DMSO. Cells were stained with the following surface marker-specific antibodies for 30 min at 4°C: APC Cy7 anti-CD3 (SP34.2; BD Biosciences), BV421 anti-CD4 (OKT4; BioLegend), and CD8-BV570 (clone RPA-T8; BioLegend). Following fixation and permeabilization, cells were stained with ECD anti-CD69 (clone TP1.55.3; Beckman Coulter), PE anti-IL-2 (MQ1-17H12; BD Biosciences), IFNγ-APC (B27; BD Biosciences), and FITC anti-TNFα (Mab11; BD Biosciences). The Aqua LIVE/DEAD kit (Invitrogen) was used to exclude dead cells. Samples were acquired on an LSRII flow cytometer and analyzed using FlowJo version 9.6.3 (Treestar, Inc.).