(A) Freshly isolated thymocytes were analyzed by 4-color flow cytometry for expression of CD4, CD8, CD25 and TCR. The histograms depict TCR and CD25 levels of cells electronically gated on CD4 and CD8 as indicated. (B) The percentage of CD25⁺ among CD4⁺CD8⁻TCR^hi thymocytes in several inbred strains of mice (n≥10 per strain) was calculated using gates indicated in A. Mean values (± SD) are shown. Statistical significance between B6 and other strains is indicated (***p<0.001, NS, not significant, Student’s t-test). (C) CD25⁺TCR^hiCD4⁺CD8⁻ (“Treg”)/CD4⁺CD8⁺ (“DP”)(upper panel) and CD25⁻TCR^hiCD4⁺CD8⁻ (“Teff”)/CD4⁺CD8⁺ (“DP”) ratios (lower panel) were determined for B6, SJL, and DBA/2 mice. Depicted are mean values ± SD (n≥4)(**p<0.01, ***p<0.001, Student’s t-test). (D) Thymocytes from DBA/2, SJL and B6 mice were labeled with Abs specific for CD4, CD8, CD25 and either TCR, HSA, CD69, GITR, or Foxp3. CD4⁺CD8⁻ cells were electronically gated and analyzed for expression of indicated surface markers. (E) Percentage of FoxP3 expressing thymocytes among CD4SP thymocytes in the indicated mouse-strains. Mean values (± SD) are shown (n=4). Statistical significance is indicated (***p<0.001, *p<0.05, Student’s t-test). (F) PBMC and thymocytes from different inbred strains were stained with anti-TCR, anti-CD4, anti-CD8 and anti-CD25 antibodies and analyzed by flow cytometry. The percentage of CD25⁺ cells among CD4⁺CD8⁻TCR^hi cells was calculated. Mean values (± SD) are shown.

BrdU was continuously administrated to mice in their drinking water. At indicated timepoints, thymocytes were analyzed by 4-color flow cytometry using anti-CD4, anti-CD8, anti- CD25 and anti-BrdU Abs. The percentage of BrdU⁺CD25⁺ among CD4⁺CD8⁻ (“CD4SP”) cells was calculated. Each point represents one mouse. Statistical significance of the difference between the two mouse strains was calculated for each day using Student’s t-test (**p<0.01, ***p<0.001).

Thymocytes from indicated mouse strains were stained with anti-CD4, anti-CD8, anti-CD25 and anti-TCR Abs and analyzed as described in the legend to . Mean values (± SD) are shown (n≥5). Statistical significance was calculated using Student’s t-test (**p<0.01, ***p<0.001).

(A) Lethally irradiated B6D2F1 hosts were reconstituted with bone marrow cells from B6 (CD5.2) and DBA/2 (CD5.1) at a 1:1 ratio. Six weeks later, thymocytes were analyzed by 4- color flow cytometry for expression of CD4, CD8, CD25 and CD5.1, using indicated electronic gates. (B) Similar experiments were performed using B6SJLF1 recipients injected with B6 (CD45.2) and SJL (CD45.1) bone marrow cells. (C) Quantitative analysis of CD25⁺ thymocyte development in mixed chimeras. Bar graphs depict mean values (± SD), n≥5. Statistical significance was calculated using Student’s t-test (**p<0.01, ***p<0.001).