Foxp3 alexa fluor 647

Frequencies of activation (HLA-DR, CD38) and maturation (CD27, CD45RO) markers on CD4 and CD8 T cells were determined as previously described [21 (link)] in fresh, anti-coagulated whole blood. CD4^pos T lymphocytes were classified as “naïve” (CD27^posCD45RO^neg), ‘‘central memory-like" (CD27^posCD45RO^pos) and effector memory (CD27^negCD45RO^pos) CD4 T cells. Gating strategies are given in the supplement. Blood samples were incubated for 10 minutes with CCR5 PECy7 (natutec, Frankfurt, Germany) followed by 30 minutes incubation using the following fluorochrome labeled monoclonal antibodies for cell surface staining (mABs): CD3 Pac Blue (Becton Dickinson; New Jersey, USA), CD4 Per-CP Cy5.5 (eBioscience, San Diego USA), CD8 V500 or CD8 Amcyan, CD25 phycoerythrin (PE)-Cy7 (eBioscience), CD27 APC-H7, CD45RO APC, HLA-DR FITC and CD38 PE (all from BD). Intracellular forkhead-box-protein P3 (FoxP3) was detected using FoxP3-Alexa Fluor 647 (ebioscience). Stained cells were then finally fixed with 2% paraformaldehyde prior to acquisition. Acquisition was performed on a FACS CANTO II (BD). Compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Flow cytometry data was analyzed using FlowJo (version 9.5.3; Tree Star Inc.).