Cd69 pe fn50

Cells from days 4, 6, and 8 were transferred into a 96-well round-bottom plate and washed twice with FACS buffer [PBS + 0.02% sodium azide (Roth, Germany) + 2 mM EDTA (Sigma-Aldrich) + 2% h.i. FBS]. Extracellular staining was performed with CD4 APC-Cy7 (RPA-T4; BD Biosciences), CD8 BV605 (RPA-T8; BioLegend), the dead cell marker Zombie Aqua (BioLegend), CD25 PE-Cy7 (BC96; BioLegend), CD69 PE (FN50; BD Biosciences) and incubated for 20 min at 4°C. All antibodies were used at pretested optimal concentrations. Cells were washed twice with FACS buffer. Approximately 500,000 cells were acquired on the same day using an LSRFortessaTM SORP (BD Biosciences, USA) equipped with the DIVA Software (Version 6, BD Biosciences, USA). The percentage of proliferating CD4⁺ cells was determined by assessment of CFSE-negative cells and activation by the percentage of CD69⁺ or CD25⁺.

Harvested cells were first washed with PBS, then labeled with 1000 times diluted Zombie NIR™ dye (Biolegend, San Diego, CA, USA) at room temperature for 20 min and washed with 0.1%BSA/PBS. For surface staining, cells were incubated with antibody mix at 4 °C for 15–20 min, then washed and resuspended with 1%PFA/PBS. For intracellular cytokine stainings, after surface staining, CytofixCytoperm™ (BD) was used to permeabilize cell membranes. Staining results were acquired either on CyAn ADP cytometer (Dako Cytomation) or LSRFortessa (BD); analysis was done using FlowJo software and R.
The following antibodies were used in this study: CD3-PB (clone UCHT1, BD), CD3-BV510 (UCHT1, BD), TCR γδ-APC (11F2, Miltenyi Biotec, Bergisch Gladbach, Germany), TCR Vγ9-PC5 (IMMU 360, Beckman Coulter, Brea, CA, USA), TCR Vδ2-FITC (IMMU 389, Beckman Coulter), CD4-V500 (RPA-T4, BD), CD4-BV510 (RPA-T4, BD), CD8-PC7 (SFCI21Thy2D3, Beckman Coulter), CD56-PE-CF594 (NCAM16.2, BD), CD69-PE (FN50, BD), IFNγ-V450 (B27, BD), TNFα-FITC (MAb11, BD), IL-17a-PE (eBio64DEC17, eBioscience). Dead cells were excluded (negative for Zombie NIR) and gated on CD3+ lymphocytes (Supplementary Figure S1). Gating CD3+Vγ9+ lymphocytes identify the vast majority of Vγ9Vδ2 T cells in adult blood [21 (link)].

The following monoclonal antibodies were used for FACS: CCR7 APC-Cy7 (G043H7, Biolegend, 353211), CD69 PE (FN50, BD, 555531), CD3 APC R700 (UCHT1, BD, 565119), PD-1 BV421 (EH12.2H7, Biolegend, 329920), HLA-DR BV605 (G46-6, BD, 562845), CXCR5 BB515 (RF8B2, BD, 564624), CD14 PE-Cy5 (61D3, abcam, ab25395), CD16 PE-Cy5 (3G8, Biolegend, 3012010), CD19 PE-Cy5 (HIB19, Biolegend, 302210), CD45RA PE CF594 (HI100, BD, 562298), CD8 PE-Cy5.5 (RPA-T8, eBioscience, 35-0088-42), and CD4 PE Cy7 (RPA-T4, Biolegend, 300512).