Figure 2.
Two subsets of Foxp3+ TR defined by ICOS expression in periphery. a,c, Double staining of Foxp3 (red) and ICOS (blue) in the human tonsil (a) and lymph node (c) showing two Foxp3+ T cell populations: Foxp3+ICOS+ T cells and Foxp3+ICOS− T cells at a low magnification (100×). All scale bars, 50 μm. b,d, Immunofluorescence staining of Foxp3 (red) and ICOS (green) performed on the same section of tonsil (b) and lymph node (d) showing two Foxp3+ T-cell populations: nuclear Foxp3+ cytoplasmic ICOS+ TR and nuclear Foxp3+ cytoplasmic ICOS− TR. e, Human peripheral blood CD25high CD4+ TR were separated into ICOS+ and ICOS− subpopulations. Foxp3 expression in each subset was determined by flow cytometry. f, h, Phenotypical analysis of two subsets of TR in adult blood (f) and cord blood (h). Intranuclear Foxp3 and intracytoplasmic CTLA4 and the cell surface markers on sorted blood CD25high ICOS+ CD4+ TR and CD25high ICOS− CD4+ TR were determined by flow cytometry. g, Percentages of two subsets of TR in 6 thymus and 18 adult blood from healthy donors. Percentages of CD25high ICOS+ CD4+ TR and CD25high ICOS− CD4+ TR among CD4+ T cells were determined by flow cytometry. Bars represent the group means. Statistical significance was determined using paired Student’s t-test. Similar results were observed in three (a–d) and five (e) experiments, and at least three experiments for each marker (f, h), and the results of a representative experiment are shown.