Foxp3 236a e7

PBMCs were thawed and cultured in R10. Counts and viability were acquired using the TC-20 Automated Cell Counter (Bio-Rad Laboratories, Inc.). T-cells and Tregs were stained with Live/Dead Fixable Aqua Dead Cell Stain Kit from Molecular Probes (OR, USA), followed by cell surface staining with the appropriate cell panel. Surface markers examined for phenotype staining protocol include: CD197 (150503), CD39 (TU66), from BD Biosciences (CA, USA); CD3 (OKT3 and HIT3a), CD4 (OKT4), HLA-DR (L243), CD38 (HIT2), CD25 (BC96), CD127 (A019D5), ICOS (C398.4A), CD14 (HCD14), from Biolegend, Inc. (CA, USA); CD8 (SK1) from eBioscience, Inc. (CA, USA); CD45RA (2H4), HLA-DR (Immu-357), CD3 (UCHT1), and CD14 (RM052) from Beckman Coulter (Marseille, France). Intracellular markers examined include: CTLA-4 (BNI3) from BD Biosciences (CA, USA), and FoxP3 (236 A/E7) from ebioscience, Inc. (CA, USA). Immediately following staining, samples were analyzed using a LSRII flow cytometer (BC Biosciences, CA, USA) with FlowJo software (Treestar, Inc, OR).

Antibodies used for flow cytometry are listed in Supplementary Table S1. IHC staining on patient tissue was performed using FOXP3 236A/E7 from eBioscience (#14–4777), CD8 Ab-1 (#MS-457-R7) from Thermo Fisher Scientific, and CD4 4B12 (#PA0427) from Leica Biosystems.

We stained human PBMCs with monoclonal antibodies to CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), CD25 (M-A251; BD Biosciences), CD45RA (ALB11; Beckman Coulter), CCR7 (MAB197; R&D Systems), Helios (22F6; BioLegend), Foxp3 (236A/E7; eBioscience), IL4 (MP4-25D2; BD Biosciences), IL-17 (eBio64DEC17; eBioscience), IFN-γ (B27; BD Biosciences) and a Fixable Viability Dye eFluor780 (eBioscience). The cytokine-producing capacity of T cell subsets was assessed after PBMCs were stimulated (at a density of 5 × 10⁵ cells per 100 μL) for 5 h with 50 ng/ml PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich) and 2 mg/ml of ionomycin (Sigma-Aldrich) in the presence of 10 mg/ml of brefeldin A (Sigma-Aldrich). Cells were fixed and made permeable with the Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer's instructions (eBioscience). For flow cytometric analysis of human intestine, IEL and LPL were stained with monoclonal antibodies to CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), IL-7Rα (HIL-7R-M21; BD Pharmingen), and c-Kit (104D2; BD Biosciences).

Peripheral blood mononuclear cells (PBMCs) were labeled with a combination of the following mAbs: CD3 (SP34–2), CD4 (L200), CD8 (SK1), CD21 (B-ly4), CD27 (M-T271), CD28 (CD28.2), CD95 (DX2), and IgM (G20–127) (BD Pharmingen, San Jose, CA), CD20 (2H7) (Biolegend, Inc., San Diego, CA) and FOXP3 (236A/E7) (eBioscience, Inc., San Diego, CA). Based on previous reports, which suggest that long-term allograft survival is associated with a predominance of immature B lymphocyte reconstitution,^{12 (link),13 (link)} CD3^-CD20⁺CD21⁺IgM⁺ or CD3^-CD20⁺CD21^-CD27⁺ B cells representing immature or mature B cell phenotypes, respectively, were monitored before and after conditioning. The fluorescence of the stained samples was analyzed using FACSverse (BD Biosciences, San Jose, CA) and Accuri flow cytometers (BD PharMingen), and FlowJo software (Tree Star, Inc., Ashland, OR).