Anti ifn γ pe or per cp 5.5 xmg1 2

Mononuclear cells were isolated from recipient spleen or liver as previously described and stained for surface markers and intracellular cytokines using standard flow cytometric protocols [10 (link),11 (link)]. Stained cells were analyzed using FACSDiva software, LSR II (BD Biosciences, San Jose, CA), and FlowJo (Tree Star, Ashland, OR).The following Abs were used for cell-surface staining: anti-CD4–V450, -APC, and -PEcy7 (BD Biosciences), anti-CD8-PEcy5, -APCcy7 and -AF700 (BD Biosciences,); anti–CD45.1-FITC, - and -APC (BD Biosciences). Intracellular staining was carried out using anti-IFN-γ-PE or Per-cp 5.5 (XMG1.2; BD Biosciences), anti–IL-4–PE (11B11; BD Pharmingen), anti–IL-5–PE (TRFK5; BD Pharmingen), anti-Foxp3–PE (FJK-16s; eBioscience).

Recipient splenocytes and thymocytes were isolated and stained for surface receptors and intracellular cytokines using standard flow cytometric protocols as previously described [22 (link), 24 (link), 26 (link)]. The following Abs were used for cell-surface staining: anti-CD4–FITC, or–V450, anti-CD8α–FITC, or–allophycocyanin-cy7, anti-B220–v450 (RA3-6B2), anti-CD80–FITC, anti- CD86–FITC, anti-CD40 APC, Biotin-anti-CD29 (β1 integrin), anti-CD229.1–Biotin or PE, anti- CD5.1-FITC, purchased from BD Biosciences. Intracellular staining was carried out using anti–IFN-γ–PE or Per-cp 5.5 (XMG1.2; BD Biosciences), anti–IL-17–allophycocyanin (17B7; eBioscience), anti–IL-4–PE (11B11; BD Pharmingen), anti–IL-5–PE (TRFK5; BD Pharmingen), anti-TNFα–PE, or PE-Cy7 (MP6-XT22; BD Pharmingen), anti-Foxp3–PE (FJK-16s; eBioscience), and the appropriate isotype controls. Cell isolates were analyzed using Diva software, LSR II (BD Biosciences,San Jose, CA), FACS Verse (BD Biosciences, San Jose, CA), and FlowJo (TreeStar, Ashland, OR).