Analysis of Foxp3(+)epigenetics(-) T cells, Foxp3(-)epigenetics(+) T cells, and Foxp3(+)epigenetics(+) T cells. Results indicate regulatory T cell (Treg) ontogenesis requires two independent processes, expression of the transcription factor Foxp3 and establishment of Treg epigenetic programs induced by T cell receptor (TCR) stimulation. Overall design: GFP+CD4+ and GFP-CD4+ splenocytes were sorted from DEREG and DEREG/Scurfy mice. These cells were activated with anti-CD3/CD28 antibodies, and then transduced with Foxp3-expressing retrovirus (pGCSamIN, NGFR marker). NGFR+ T cells sorted were subjected to microarray analysis (Affymetrix, mouse genome 430 2.0 array). To normalize the experimental conditions, Tregs (GFP+ T cells from DEREG) and Tconv (GFP- T cells from DEREG) were also activated and transduced with empty vector. Two replicates each.