Expanded Helios+ thymocyte populations in apoptosis-deficient mice. (A) CD24/CCR7 phenotype of non-Tg DP and CD4SP thymocytes showing the gates used to define the CD4SP subsets SP1 (top left), SP2 (top right), and SP3 (bottom right), which were examined for Qa-2 expression (middle); summary (right) shows the percentage of Qa-2+ cells within each CD4SP subset compiled from two experiments. Error bars show SD. (B) Phenotypic progression within the CD4SP stage. Non-Tg B6 mice were either left uninjected or injected i.p. with 1 mg BrdU either 1, 2, 3, 4, 5, 6, or 7 d before flow cytometry on thymocytes. Plots show labeling for TCRβ versus BrdU on all thymocytes (top row). BrdU+ thymocytes were analyzed for expression of CD4 versus CD8 (middle row), and BrdU+ CD4SP cells were analyzed for CD24 versus CCR7 expression (bottom row). Data are representative of three experiments. (C) Helios+ thymocyte populations expand in apoptosis-deficient mice with natural TCR repertoires. Representative plots show phenotype of Foxp3− thymocyte subsets in wild-type, Bim−/−, and Vav-BCL2 Tg mice (denoted far left). The first column shows quadrant gates defining four populations on the basis of CD4 and CD8; in the third column, the CD4SP population is further divided into the subsets SP1–SP4. The fourth through eighth columns display the Helios/Bim phenotype of populations denoted above the plots. Graphs (bottom) show number of Helios+ (left) or Helios− (right) Foxp3− thymocytes within each subpopulation as defined using gates represented in the plots above, compiled from eight separate experiments that used a total of 13 Bim−/− and 8 B6.Vav-BCL2 Tg mice. Numbers in plots indicate percentage of cells in gates shown, and columns in graphs show mean and SD. The wild-type group comprises pooled data from 16 B10.BR mice and 10 B6 mice. No statistically significant differences existed between wild-type B10.BR and B6 mice. Paired (in A) or unpaired (in C) Student’s t test p-values: ***, P < 0.001; **, P = 0.001–0.01; *, P = 0.01–0.05.
