α cd25 pe

Transgenic C57BL/6.Foxp3^GFP and wild type C57BL/6 mice were purchased from The Jackson Laboratory. Human CD2 transgenic C57BL/6.Foxp3^hCD2 [16 (link)] and 4-1BB deficient C57BL/6.4-1BB^-/- mice [17 (link)] were bred and cared for in University of Louisville’s specific-pathogen-free vivarium in accordance with NIH guidelines. All animal procedures were approved by the University of Louisville’s IACUC. Antibodies used include anti-CD3 agonistic Ab (clone 145-2C11, BD Pharmingen, Cat# 553058), anti-TGF-β (1,2,3) neutralizing Ab (clone 1D11, homemade), anti-IFN-γ blocking Ab (clone XMG 1.2, homemade), and anti-IL-2 neutralizing Ab (clone S4B6, BD Pharmingen, Cat# 554375). Fluorescently-labeled Abs to various cell surface markers were obtained commercially: α-CD4-APC (BD Pharmingen, 553051); α-CD25-PE (BD Pharmingen, 553866); α-CD25-PE-Cy7 (eBioscience, 25-0251-82); α-hCD2-PE (BioLegend, 300208). Where indicated Tconvs or Tregs were stained with 2.5μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, C1157). CTLL-2 cell line was obtained from ATCC (TIB-214). Human IL-2, SA-4-1BBL, and SA proteins were produced in our lab according to standard protocols as previously reported [10 (link),11 (link)].

Anti-CD11b-PacBlu, αCD11b-FITC, αCD4-PE, αCD4-APC, αCD11c-APC, αCD11c-PE, αTCRvα2-PE, and αCD3ε(145-2C11) were purchased from BioLegend (San Diego, CA, USA). Anti-Ly6C-PerCP and αFoxP3-APC were products of eBioscience (San Jose, CA, USA). Anti-Ly6G-PE, αNK1.1-PE, αCD8α-PacBlu, αCD25-PE, and αCD103-Alexa Fluor 647 were products from BD Biosciences (San Jose, CA, USA). Flt3L-Fc fusion protein was purchased from BioXCell (West Lebanon, NH, USA). Anti-IL-2 was purchased from R&D Systems (Minneapolis, MN, USA). Fc-GITR-L fusion protein was produced as described previously (9 (link)).