Recombinant cd70 fc

T_reg progenitors were isolated as previously described^{9 (link)}. Briefly, 6-8 week old Foxp3-GFP thymii were dissected, dissociated, and pooled (up to 8 mice per experiment). CD4SP cells were enriched by magnetically depleting with biotinylated anti-CD8, CD11B, CD11C, CD19, CD45R/B220, MHC class II, NK1.1, and Ter119 antibodies (eBioscience) followed by secondary labeling with streptavidin-conjugated microbeads (Miltenyi Biotec). Enriched CD4SP cells were stained with fluorchrome-conjugated anti-CD4, CD25, and streptavidin prior to sorting CD4⁺CD25⁺GFP⁻ cells using a BD Facs Aria sorter (BD Biosciences). Purified T_reg progenitors were incubated in complete RPMI and supplemented with human IL-2 (1 Unit/mL, from the NIH repository at Frederick National Laboratory) with or without 100 nM recombinant GITR-L–Fc, OX40-L–Fc (both from Imgenex, San Diego, CA), recombinant murine TNF (Peprotech, Rocky Hill, NJ), recombinant CD70-Fc (Sino Biologicals, Beijing, China), soluble CD70 (Enzo Life Sciences, Farmingdale, NY), agonist CD27 mAb (RM27-3E5)^{47 (link)}, or recombinant mouse CD30-L and 4-1BBL (the latter two of which were crosslinked using 5 ug/ml anti-poly His antibody, both from R&D Systems, Minneapolis, MN). After 72 hours, cells were harvested, stained with anti-CD4 and CD25 antibodies and analyzed by flow cytometry for the percentage of cells that express GFP after incubation.