Examination of CD69-negative SP thymocytes in WT and IL-15Rα KO mice.

<p>Thymocytes of WT and IL-15Rα KO mice (n = 6) were immunostained for CD4, CD8, TCR, and CD69. The percentages of CD69-negative (A) and CD69-positive (B) CD8SP TCR^hi (left panels) and CD4SP TCR^hi thymocytes (right panels) among total TCR^hi cells were compared. By calculation, the cell numbers of CD69⁻ CD8SP TCR^hi thymocytes in WT and IL-15Rα KO mice were about 1.6×10⁶±3.8×10⁵ and 9.0×10⁵±2.8×10⁵, respectively. The cell numbers of CD69⁻ CD4SP TCR^hi thymocytes in WT and IL-15Rα KO mice were about 3.6×10⁶±1.3×10⁵ and 3.2×10⁶±1.4×10⁵, respectively. Data are presented as means ± SD and were analyzed by single-classification ANOVA. *<i>p</i><0.05; n.s., not significant.</p

<i>In vivo</i> proliferation of CD8SP TCR^hi and CD4SP TCR^hi thymocytes analyzed by BrdU incorporation.

<p>WT and IL-15Rα KO mice (n = 7) were injected i.p. with BrdU. Total thymocytes were isolated 1 hr later, and the degrees of BrdU incorporation by CD4SP TCR^hi and CD8SP TCR^hi cells was determined. (A) Upper panel; The BrdU incorporation of IL-15Rα KO CD8SP TCR^hi cells (3.2×10⁴±1.1×10³ cells) was compared with that of WT CD8SP TCR^hi cells (9.4×10⁴±3.1×10³ cells). Lower panel; The percentages of WT (1.6×10⁵±1.0×10⁵ cells) and IL-15Rα KO CD4SP TCR^hi (1.5×10⁵±0.7×10⁵ cells) thymocytes that incorporate BrdU. (B) Values of each experimental group are averaged and the means are indicated by horizontal lines. Data are presented as means ± SD. **<i>p</i><0.005; n.s., not significant. Single-classification ANOVA.</p

Comparison of large SP TCR^hi thymocytes between WT and IL-15Rα KO mice.

<p>Total thymocytes were isolated from WT and IL-15Rα KO mice (n = 4) and stained for CD4, CD8, and TCR. (A) Cells were gated into 10 sub-regions (R1 to R10) on the basis of FSC. (B) The frequency of CD8SP TCR^hi thymocytes in each specified regions is displayed as percentages of total thymocytes (upper panels) or percentages of total SP TCR^hi thymocytes (lower panels). Data are means ± SD values. **<i>p</i><0.005 *<i>p</i><0.05 Single-classification ANOVA.</p

<i>In vitro</i> stimulation of CD8SP thymocyte proliferation with plate-bound IL-15-IL-15Rα-Fc proteins.

<p>Total thymocytes were labeled with CFSE and then cultured in medium supplemented with IL-15, IL-21, both IL-15 and IL-21, or both IL-15 and IL-21 with pre-coated rmIL-15Rα-Fc fusion protein. The cultures were supplemented with the same amounts of cytokine 3 days later. Cells were harvested 5 days after the initiation of the culture and stained for CD4 and CD8. The extent of cell proliferation in CD8SP thymocytes was analyzed by measuring CFSE dilution. The data are representative of three independent experiments.</p

Characterization of thymocyte subpopulations in WT and IL-15Rα KO mice.

<p>Total thymocytes were isolated from WT and IL-15Rα KO mice (n = 5 per group) and subjected to flow cytometric analysis of the expression of CD4, CD8, and TCRβ. (A) Thymic subsets of WT and IL-15Rα KO mice were shown for their expression of CD4 and CD8. (B) Percentages of DN, DP, CD4SP, and CD8SP thymocytes among total thymocytes of IL-15Rα KO (○) mice and wild-type littermates (•) were compared. The horizontal line represents the mean. (C) CD4SP and CD8SP thymocytes were further gated to compare their TCR expression. CD8SP thymocytes consisted of TCR^neg/lo and TCR^hi cells, whereas the majority of CD4SP cells expressed high levels of TCR. (D) The percentage of CD8SP TCR^hi cells (left panel) and CD8SP TCR^neg/lo cells (right panel) among total thymocytes was compared between WT and IL-15Rα KO mice. Data were analyzed by single-classification ANOVA. n.s., not significant.</p