Ccr6 pe 11a9

Cell surface labeling was performed using standard protocols. Intracellular labeling was performed using the True-Nuclear Transcription Factor Buffer Set according to the manufacturers’ instructions. The following anti-human mAbs were obtained from Biolegend: CD4-APC (OKT4), CD8α-PE (TuGh4), CD161-Alexa Fluor 647 (HP-3G10), CD69-PE (FN50), CD3-PE/Cy7, Brilliant Violet-711, or Alexa-700 (UCHT1), CD137-biotin (n4b4-1), CXCR3-Brilliant violet 421 (G025H7), CD83-biotin (HB15e) and TRAV1-2- PE (10C3). CD86-FITC (2331), CCR4-PECy7 (1G1) and CCR6-PE (11A9) mAbs were from BD Pharmingen. All these mAbs were used at 5 µg/ml. Biotinylated mAbs were revealed with streptavidin-PE, -Alexa Fluor 488, or -Brilliant violet 421 (2 µg/ml, Biolegend). The MR1-specific mAb clone 26.5 (mouse IgG2a) was provided by Ted Hansen, Marina Cella and Marco Colonna, Washington University School of Medicine, St. Louis (MO) (Lepore et al., 2014 (link)). Unlabeled MR1-specific mAbs were revealed with goat anti-mouse IgG2a-PE (2 µg/ml, Southern Biotech). Samples were acquired on LSR Fortessa flow cytometer (Becton Dickinson). Cell sorting experiments were performed using an Influx instrument (Becton Dickinson). Dead cells and doublets were excluded on the basis of forward scatter area and width, side scatter, and DAPI staining. All data were analyzed using FlowJo software (TreeStar).

Immunophenotyping of lymphocytes was performed by multicolor flow cytometry. Heparinized whole blood samples were immediately stained for 15 minutes with the following antibodies: CD3-APC/Cy7 (UCHT, BioLegend; San Diego, CA, USA), CD4-HorizonV500 (RPA-T4, BD Biosciences; San Jose, CA, USA), CD45RA-BV421 (HI-100, BioLegend), CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), CXCR3-APC (1C6, BD Biosciences), CCR6-PE (11A9, BD Biosciences), CCR7-PE/Cy7 (G043H7, BioLegend), PD-1-FITC (EH12.2H7, BioLegend), CD19-BV421 (HIB19, BioLegend), CD20-APC (2H7, BioLegend), CD27-APC/Cy7 (O323, BioLegend), and CD38-FITC (HIT2, BioLegend). Circulating Tfh cells were defined as CD3⁺CD4⁺CD45RA^-CXCR5⁺ cells, and Tfh1, Tfh2, or Tfh17 cell subsets as CXCR3⁺CCR6^- cells, CXCR3^-CCR6^- cells, or CXCR3^-CCR6⁺ cells among Tfh cells, respectively [19 (link), 20 (link)]. Activated Tfh cells were defined as the CCR7^lowPD-1^high cells among Tfh cells [26 (link)]. CD19⁺CD20^-CD27⁺CD38⁺ cells were defined as plasmablasts. Red blood cells were lysed with FACS Lysing Solution (BD Biosciences). All samples were analyzed with a FACS Aria III (BD Biosciences), and data were analyzed with FlowJo v.7.6.4 Software (Tree Star, Stanford University, CA, USA). Proportions of lymphocyte subsets were determined by the combination of surface marker staining, with exclusion of doublets by forward and side scatter.

Flow cytometry analysis was performed to identify PBMC subpopulations and surface expression of CCR6. One million cells from each of the 40 subjects were incubated with human Fc Receptor binding inhibitor from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min. Cell were further stained for 30 min at room temperature with the following anti-human antibodies: CD8a PerCP (HIT8a), CD14 Pacific Blue (HCD14) and CD19 APC (HIB19) from BioLegend (San Diego, CA, USA), CD4 FITC (RPA-T4) from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) and CCR6 PE (11A9) or isotype control IgG1κ PE (MOPC-21) and CD16 APC-H7 (3G8) from BD Bioscience (Franklin Lakes, NJ, USA). After incubation with fix/lysis buffer from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at room temperature, cells were washed twice in FACS buffer (PBS with 0.1% BSA) and resuspended in PBS. Compensation controls were prepared using Anti-mouse Igκ/Negative Control Compensation Particle Set from BD Bioscience (Franklin Lakes, NJ, USA) according to the manufacturer’s recommendations. Flow cytometry was performed on a BD FACS Canto II flow cytometer with FACS Diva software from BD Bioscience (Franklin Lakes, NJ, USA) (10,000 events per sample) and samples were analysed using FlowJo v10 software from Flow Jo, LLC (Ashland, OR, USA).