Characterization of Th17 Cell Subsets

The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter), HIV-p24 PE (KC57) (Beckman Coulter), CD3 Pacific blue (UCHT1), CD4 PerCP/Cy5.5 (RPA-T4) (BioLegend), CD4 Alexa Fluor 700 (RPA-T4), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), RORC2 Alexa Fluor 647 (Q31-378), K_i-67 BUV395 (B56), IL-17A PE (eBio64DEC17), and IFN-γ Alexa Fluor 700 (B27). The Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies) was used to exclude dead cells. Intracellular staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences), and intranuclear staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were analyzed with the BD-LSRII cytometer, BD LSRFortessa and BD-Diva (BD Biosciences), and FlowJo version 10 (Tree Star, Inc.). The positivity gates were placed using fluorescence minus one strategy (8 (link), 20 (link)). For fluorescence-activated cell sorting (FACS), memory CD4 T cells from the PBMCs of ART+ PLWH were isolated by negative selection using magnetic beads. CCR6⁺RORC2⁺, CCR6⁺RORC2⁻, and CCR6⁻RORC⁻ T cells were sorted by FACS (BDAria II; BD Biosciences) using the Abs CD3 Pacific blue (UCHT1), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), and RORC2 Alexa Fluor 647 (Q31-378).