Ccr6 pe cy7

Regulatory T cells (Treg) were defined as CD4⁺CD25⁺CD127^low/- cells. Other CD4⁺T subsets were identified by differential expression of CCR4, CXCR3, and CCR6 as previously reported (7 (link), 9 (link)): CXCR3⁺CCR4⁻CCR6⁻ (Th1), CXCR3⁻CCR4⁺CCR6⁻ (Th2), CXCR3⁻CCR4⁺CCR6⁺(Th17), and CXCR3⁺CCR4⁻CCR6⁺ (Th1Th17) (Figure 1). In addition, Th17 cells were defined as the summation of CXCR3⁻CCR4⁺CCR6⁺ (Th17) and CXCR3⁺CCR4⁻CCR6⁺(Th1Th17).
The fluorochrome-conjugated antibodies used for polychromatic flow cytometry analysis were CD3-BV510 (Biolegend, USA, catalog no. 317332), CD4-APC-Cy7 (Biolegend, USA, catalog no. 317418), CCR4-APC (Biolegend, USA, catalog no. 359404), CXCR3-PE (Biolegend, USA, catalog no. 353706), CCR6-PE-Cy7 (Biolegend, USA, catalog no. 353418), CD25-BV421 (Biolegend, USA, catalog no. 302630), CD127-FITC (Biolegend, USA, catalog no. 351312), PE-Cy7-CCR7 (Biolegend, USA, catalog no. 353226) and CD62L-FITC (Biolegend, USA, catalog no. 304804). A viability dye, 7-AAD (Becton Dickinson, USA, catalog no. 559925), was used to exclude dead cells. Isotype antibodies were also used as negative controls for every detection to set proper gating for the receptor expression. Cells were analyzed by fluorescence-activated cell sorting (FACS), using the BD LSRII cytometer and FlowJo software.

Intracellular pSTAT1 and total STAT1 kinetics were determined by FACS analysis. 50 µl whole blood was stimulated according to BD Phosflow™ T Cell Activation Kit Instruction Manual in polystyrene round-bottom tubes (Becton Dickinson Falcon). Blood was stimulated with Imukin^® 100ng/ml for 0–180min at 37°C. Erythrocytes were lysed and leucocytes fixed with 1 ml of pre-warmed 1x lyse/fix buffer. The cells were then permeabilized by adding 300 µL of cold perm buffer III on ice for 30 min, stained with 750 µl stain buffer and incubated for 1 h in the dark at 4°C with following antibodies: anti-human CD14-FITC (BD Biosciences 345784), isotype STAT normal mouse IgG2a Alexa Fluor^® 647 (Santa Cruz Biotechnology sc-24637) and Alexa Fluor^® 647 mouse Anti-Stat1 (pY701) (BD Biosciences 612597). For Th1 and Th17 percentages PBMCs were stained with CD3 – BV605 (Biolegend), CD4 - APC-eFluor780 (eBioscience), CD8 – BV650 (eBioscience), CXCR3 – FITC (BioLegend), CCR6 – PE-Cy7 (BioLegend), CD45RA – PerCP (eBioscience) GZMB - e450 (BioLegend), CFSE - Invitrogen. All data were collected with LSR II (Becton Dickinson) and analyzed with FlowJo software (Treestar, Ashland, OR, USA).