αcd49d

Whole blood was incubated with 1 μg/mL αCD28, 1 μg/mL αCD49d (BD Biosciences) and stimulated with 20 μg/ml PPD (SSI, Denmark), 5 μg/ml staphylococcal enterotoxin B (Sigma Aldrich), or no antigen (unstimulated). Samples were incubated at 37°C in 5% CO₂ for 6 h, 3 μg/ml Brefeldin-A (Sigma Aldrich) was added, and samples were incubated for another 6 h in a 37°C water bath. Samples were then treated with 2 mM ethylenediaminetetraacetic acid (Gibco), and red blood cells were lysed using FACS Lysing solution (BD Biosciences) and samples were frozen in PBS with 10% DMSO for batched ICS staining. Frozen samples were thawed, permeabilised and incubated with antibodies against CD3 (AF700), IFN-γ (PE-Cy7 (eBioscience); CD4 (APC), CD14 (Pacific blue), TNF-α (PerCP-Cy5.5), and IL-17 (AF488) (BioLegend); CD8 (APC-H7) (Becton Dickinson) and IL-2 (PE) (Beckman Coulter). Samples were acquired on an LSR II flow cytometer (BD Biosciences) and responses analyzed using FlowJo version 8.8.7 (Tree Star Inc., Ashland, USA). Cytokines were measured as the frequency of singlet CD14– CD3+ T cells, CD4+ T cells, and CD8+ T cells. Data are presented as percentages of cytokine-positive cells minus responses in unstimulated cells; the gating strategy is shown in Supplementary Figure 1.

PBMC were plated at 1 × 10⁶ live cells/well into 96‐well round bottom plates and stimulated with a final concentration of 50 μg/mL soluble antigens derived from heat inactivated (HIA) BP (K96243 and clinical isolates 199a and 207a) in the presence of co‐stimulatory molecules αCD28 and αCD49d (1 mg/mL, BD Biosciences, Franklin Lakes, NJ, USA) at a final concentration of 1 μg/mL each. R10 was used as negative control and 5 μg/mL staphylococcal enterotoxin B (Sigma) as positive control. In some experiments, PBMCs were treated with 0.3 μg/mL cyclosporine A (CsA, LKT Labs, St. Paul, MN, USA) in addition to antigenic stimuli and co‐stimulants. In all cases, PBMCs were incubated for 6 h at 37⁰C, 5% CO2, 95% humidity. Brefeldin A (Biolegend, San Diego, CA, USA) was added at a final dilution of 1:1000, 2 h after the addition of stimulants and PBMCs were incubated for further 4 h prior to flow cytometry staining as described below.