PMC

Foxp3 expression following allogeneic MLR. Cells were analyzed by flow cytometry after an 8-day MLR. BrdU incorporation was determined on gated CD4+CD25+ or CD8+CD25+ T cells (A; top panel). Gated CD4+ or CD8+ T cells were analyzed for the detection of CD25+Foxp3+ cells (A; bottom panel). Gated CD4+ T cells (B; top panel) or CD8+ T cells (B; bottom panel) were analyzed for the expression of CD44, CD62L, Foxp3. The CD44+ and CD62L+ T cells were determined by gating on CD4+Foxp3+ or CD8+Foxp3+ T cells. Representative data are shown from two donors in duplicate experiments.

T cell proliferation in the presence of inducible CD4+FoxP3+ T cells. To perform a co-culture suppression assay, responder T cells were labeled with CFSE and cultured in the absence or presence of different ratios of inducible FoxP3+ T cells (20:1 and 2:1) for 3 days in the presence of anti-CD3/CD28 Abs. Gated CD8+ T cells showed CFSE dilution (A, left panel). Responder CD4+ T cells that expressed FoxP3 due to a 3-day activation were also gated and analyzed for CFSE dilution (A, right panel). Cells obtained from a co-culture suppression assay (A, left panel) were also stained for Annexin V in order to determine apoptosis in responder CD8+ T cells (B, left panel) and the B/I-activated CD4+FoxP3+ T cells (B, right panel).

Foxp3 expression following T cell activation. T cells were isolated from healthy volunteers and split into two groups. Control group remained unactivated and cultured in the presence of IL-2 for 3 days (A; top panel) and another group was activated with B/I for 16 h and cultured in the presence of IL-2 for 3 days (A; middle panel) or 6 days (A; bottom panel). Absolute numbers of CD4+ and CD8+ T cells on pooled samples were determined on days 0, 3, and 6 post-culture by flow cytometry analysis (B). Expression of FoxP3 and CD25 were determined in freshly isolated CD4+ T cells (day 0) and after a 3-day stimulation with anti-CD3/CD28 Abs (C). Freshly isolated and B/I-activated T cells were subjected to flow cytometry to determine T cell phenotypes (D; top panel); Foxp3+ effector and memory T cells were determined in gated CD4+Foxp3+ cells or gated CD8+Foxp3+ cells (D; bottom panel). Representative data are shown from two donors in duplicate experiments.