Anti cd49

PBMCs and TMCs were stained with fluorescent monoclonal antibodies against markers previously associated with T_FH cells Table S2. Briefly, cells were thawed and rested in R10 medium for 3 h at 37°C in 5% CO₂ and either directly stained with the phenotypic panel as described below or used for Intracellular Cytokine Staining (ICS) by stimulating with SEB at 1 μg/ml or with pools of overlapping 18-mer HIV peptides (Gag and Env at 2 μg/ml for each peptide) in the presence of anti-CD28 and anti-CD49 at 1 μg/ml (BD Biosciences). After 1 h of incubation at 37°C, Brefeldin A and Monensin (5 μg/ml; BD biosciences) were added and the cells were incubated overnight (14 h), washed and stained in the dark for 20 min with antibody cocktail and live dead stain (Fixable Blue, Thermo) and fixed. ICS was performed by standard methods (42 ), using fix/perm solution (BD), 20% Goat serum for Fc-receptor blockade and antibodies listed as above. Rainbow beads were run at every experiment to ensure interexperimental consistency. Flow cytometry acquisition was performed on a BD LSRFortessa within 5 h of staining and analyzed using FlowJo version 9.9.5.

The Monoclonal antibodies (mABs) used for phenotypic analysis included Fluorescein isothiocyanate (FITC)-, Phycoerythrin (PE)-, Phycoerythrin Cyanine 5.5 and allophycocyanin (APC)-labeled anti-CD45, anti-CD3, anti-CD56, NKp30, NKp44 (Beckman Coulter, Brea, CA, USA), anti-CD16, NKp46, NKG2A (R&D System), anti-CD94, anti-CD69, anti-CD18, anti-CD49, anti-CD62L, anti-CD38 and CXCR4 (BD Pharmingen, San Jose, CA, USA). Phenotype evaluation was performed by direct immunofluorescence according to previously reported methods [29 (link)]. NK cells recovered from immunodeficient NOD/SCID gamma (NSG) mice were stained with anti-mouse CD45-APC-Cy7, anti-human CD45-PE-Cy7, CD3-PerCP-Cy5.5, CD56-FITC (Biolegend, San Diego, CA, USA) and CD94-APC (BD).