CD25+CD4+ from B6 mice or CD25–CD4+ cells from B6.SJL mice (Ly5.1+), isolated by magnetic bead sorting, were stimulated with 100 ng/ml anti-CD3 and 100 ng/ml anti-CD28 in goat-anti-hamster IgG (Jackson ImmunoResearch) coated 12-well plates. IL-2 (100 U/ml) was added to CD25+CD4+ cells on day 0 and to CD25–CD4+ cells on day 1 of culture. On days 1 and 2 following stimulation, cells were “spinfected” (90 min, 2500 rpm, 37°C) with retroviral supernatants, supplemented with 5 ug/ml polybrene, 10 mM HEPES and IL-2, from transfected phoenix-E cells transfected with MigR1, MigR1-PDE3B or MigR1-PDE3B-H801A. On day 5 of culture, aliquots of cells were stained for Foxp3. Cultures of CD25+CD4+ cells routinely contained >70% Foxp3+ cells, >55% of which were transduced (GFP+), while cultures of CD25–CD4+ cells contained >35% transduced cells. CD25–CD4+ cells were spinfected only on day 1 because retroviral transduction of these cells was more efficient than for TR. Cells transduced with the same vectors were mixed and transferred into TCRalpha-deficient recipients. Three weeks after transfer cells were analyzed by flow cytometry and isolated by FACS sorting for gene expression analysis.
TelTest Stat-60 RNA extraction.
RNA was converted to biotinylated cRNA (2 rounds of amplification) using commercially available reagent kits (MessageAmp II, Ambion, Austin, TX) and were hybridized to Affymetrix mouse 430 2.0 microarrays.
Standard hybridization to Affymetrix 430_2.0 arrays.
Standard scanning of Affymetrix 430_2.0 arrays.
GITR expression and Foxp3 expression correlated well in these experiments. Transduced cells were dectected by the MigR1 marker gene, GFP. GFP+ GITR+ CD4+ Ly5.1- cells were sorted from recipient mice for RNA.