Figure 1.
Preferential phosphorylation of STAT-5 in foxp3+ cells in adult C57BL/6 mice.
(A) The gating strategy is indicated by the arrows. Left panel: total thymocytes were stained with CD4 and CD8 antibodies. The indicated gate defines CD4+CD8− cells. Middle panel: CD25 and foxp3 expression in CD4+CD8− cells. Right panel: pSTAT-5 expression in CD25+foxp3+CD4+CD8− cells. (B–C) Frequencies of pSTAT-5+ cells within thymic CD4+CD8− cells of the indicated CD25/foxp3 phenotype in the thymus (B) and in the mesenteric lymph nodes (C) in 6- to 13- week-old mice. Each dot represents a single mouse. Results are compiled from 4 to 11 independent experiments. The p values are indicated in each panel (***p<0.001; *p<0.05; NS not significant).
Figure 2.
Kinetics of pSTAT-5 expression during Treg cell differentiation in the neonatal thymus of C57BL/6 mice.
(A–B) Frequencies (A) and absolute numbers (B) of CD4+CD8− cells of the indicated phenotype defined by CD25 and foxp3 expression in the thymus of C57BL/6 neonates at the indicated days after birth. For sake of clarity, analysis of CD25−foxp3− cells which encompass >90% of all CD4+ T cells, is not included in the figure (C–D) Frequencies (C) and absolute numbers (D) of pSTAT-5+ cells within CD4+CD8− thymic subsets defined by CD25 and foxp3 expression in C57BL/6 neonates at the indicated days after birth. The p values are indicated in each panel (***p<0.001; **p<0.01; *p<0.05; NS not significant). Results shown are the compilation of 2 independent experiments with 6 to 8 mice analyzed per day. For sake of clarity, only the p values comparing CD25+foxp3− and CD25+foxp3+ cells are shown.
Figure 3.
Preferential STAT-5 phosphorylation in foxp3+ thymocytes in vitro correlates with CD122 expression.
(A) Frequency of pSTAT-5+ cells in CD25+foxp3+CD4+CD8− cells of the thymus and of the mesenteric lymph node (mLN) fixed with formaldehyde immediately after sacrifice (0 min.) or after 60 minute incubation on ice (60 min.). (B) Frequency of pSTAT-5+ cells in thymocytes of the indicated CD25/foxp3 phenotype after in vitro incubation with the indicated cytokines for 60 min. (Med; medium only). Results shown are compiled from 2 to 8 independent experiments. (C) Representative profile of CD122 expression in the indicated thymic subsets defined by CD25 and foxp3 expression. Note the higher expression of CD122 in foxp3+ cells (D) Median fluorescence intensity of CD122 expression in thymic subsets defined by CD25 and foxp3 expression. Each dot represents a single mouse. Results shown are compiled from 2 independent experiments. The p values are indicated in the figure (***p<0.001; **p<0.01).
Figure 4.
Enhanced selection of Mls-1a–specific T cells into the regulatory lineage in the thymus of C57BL/6×DBA/2 F1 neonates.
(A) Representative flow cytometry analysis of CD25 and foxp3 expression in BV6+ or BV6− cells among CD4+CD8− thymocytes of a F1 mouse at day 3 after birth. (B–E) Frequencies and absolute numbers of CD25+foxp3+ (B–C) or CD25+foxp3− cells (D–E) were determined in CD4+CD8− cells within the indicated TCR-expressing cells (BV6, BV11, BV8.1/8.2) from 3 to 21 mice at the indicated age after birth in B6D2 mice. Results shown are compiled from 17 independent experiments.
Table 1.
Self-specific nTreg are not enriched in pSTAT-5+ cellsa).