Anti cd49d and anti cd154 bv421 abs

The capture of activated CD4⁺ T cells was performed as previously reported^{19 (link)}. Briefly, PBMCs were thawed in complete RPMI 1640 medium at 2 × 10⁶ cells/ml and recovered 12 hours before stimulation. PBMCs were stimulated with an Mtb lysate (10 μg/ml) for 12 hours in the presence of 1 μg/ml purified anti-CD49d antibody and anti-CD154-BV421. After stimulation, cells were harvested and stained with antibodies to various cell surface markers, including anti-CD3-Alexa700, anti-TCR α/β-PE/Cy7, anti-CD4-PerCP/Cy5.5, anti-CD8-BV605, anti-CD69-APC/Cy7 abs from BioLegend; purified anti-CD49d and anti-CD154-BV421 abs from BD Biosciences. Dead cells were stained using LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit from Thermo Fisher scientific. Activated CD4⁺ T cells were either single-cell sorted into 96-well plate for single cell TCRα/β sequencing or bulk sorted into an individual tube for TCRβ library preparation.