Anti foxp3 206d

As described previously (26 ), PBMC were stained at 4°C with the following fluorochrome-labeled anti-human Abs: anti-CD3 (clone SP34-2), anti-CD4 (OKT4), anti-CD8 (RPA-T8), anti-CD25 (BC96), anti-CD28 (CD28.2), anti-CD45RA (5H9), anti-CD62L (SK11) and anti-CD127 (HIL-7R-M21) from BD Pharminogen (Franklin Lakes, NJ). Anti-Foxp3 (206D) was obtained from BioLegend (San Diego, CA). Anti-IL-17 (eBio64Dec17) and anti-Ki67 (20Raj1) were obtained from eBioscience (San Diego, CA). Data were acquired on a LSR II or LSR Fortessa (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Absolute counts of Tmem and Treg were determined using CountBright^™ absolute counting beads (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol.
Following ATG-mediated lymphocyte depletion, whole blood samples were collected once a week to determine T cell subsets. At one month post-infusion, inguinal lymph nodes (LN) were collected and lymphocyte phenotypic analysis performed. Data were analyzed with FlowJo software (Tree Star) and graphed with GraphPad Prism (Graph Pad Software, San Diego, CA).

PBMC were stained with human anti-CD4 (RPA-T4, Biolegend), and anti-TLR1 (GD2.F4, Biolegend), anti-TLR5 (624915, R&D Systems) or isotype control. For a subset, this was followed by intracellular staining using anti-FoxP3 (206D, Biolegend) and a FOXP3 Fix/Perm buffer set (Biolegend).