Article Figures & Data
Figures
- FIGURE 1.
IL-2Rβ and γc chains are required for the development of CD4+Foxp3+ cells. A, Shown are CD4+CD8−-gated thymocytes (THY), total splenocytes (SPL), or LN cells stained with Abs to CD4 and Foxp3. Numbers to the right of the outlined areas represent the percentage of cells in the designated gate. B, Shown are average total Foxp3+ cell numbers from C57BL/6 (▪) and IL-2−/− (□) mice. C, Thymii, spleens, and LN were harvested from 4-wk-old C57BL/6 or IL-2Rβ−/− mice and stained with Abs to CD4 and Foxp3. Flow cytometry plots shown depict CD4+CD8−-gated thymocytes, lymphocyte-gated splenocytes, and LN cells . Numbers indicate the percentage of cells in the gate. D, Total CD4+Foxp3+-gated cells from both C57BL/6 (▪) and IL-2Rβ−/− (□) mice. E, Thymii and spleens were harvested from 6- to 8-wk-old IL-7Rα−/− and γc−/− mice and stained with Abs to CD4, CD25, and Foxp3. Numbers indicate the percentage of CD4+CD8− thymocytes or total splenocytes inside the depicted gate. F, Total numbers of CD4+Foxp3+ cells from IL-7Rα−/− (▪) and γc−/− (□). Error bars, SEM. Data in each case are representative of three or more independent experiments using between two and five mice per group. Mice used were between 4 and 5 wk of age, except as otherwise noted. The p values for all figures were determined by a two-tailed Student’s t test.
- FIGURE 2.
IL-2 and IL-15 act as redundant factors to promote the development of Foxp3+ cells. A, Shown are CD4+CD8−-gated thymocytes (THY) or total splenocytes (SPL) stained with Abs to CD4 and Foxp3. Numbers to the right of the outlined areas represent the percentage of cells in the designated gate. B, Shown are average total CD4+Foxp3+ cell numbers from C57BL/6 (▪), IL-15−/− (□), and IL-2−/− × IL-15−/− (▩) mice. Error bars, SEM. Mice used in these experiments were between 4 and 5 wk of age. Data are representative of nine WT C57BL/6 and IL-15−/− mice and four IL-2−/− × IL-15−/− mice.
- FIGURE 3.
Foxp3 expression rescues Treg development in IL-2Rβ−/− mice. A, Spleens were harvested from 5- to 8-wk-old LMC, IL-2Rβ−/−, foxp3tg, and IL-2Rβ−/− × foxp3tg mice and stained with Abs to CD4 and Foxp3. B, CD4+-gated T cells were stained with Abs to CD69 and CD62L. C, Shown are spleens from 5-wk-old LMC, foxp3tg, IL-2Rβ−/−, and IL-2Rβ−/− × foxp3tg mice. Data are representative of five mice. D, One hundred thousand CD4+CD25− responder T cells from C57BL/6 mice were mixed with equal numbers of allogeneic bm12 splenocytes to induce proliferation. FACS-sorted CD4+ cells from foxp3 transgenic mice (□), IL-2Rβ−/− × foxp3tg mice (▦), or CD4+CD25+ T cells from C57BL/6 mice (▪) were cultured at the indicated Treg to responder cell ratio. Data are presented as mean [3H]thymidine incorporation of quadruplicate cultures and error bars represent SEM. Data presented are representative of three independent experiments.
- FIGURE 4.
STAT5 signaling is necessary for the development of Tregs. A and B, Left panels, show thymocytes (A) or splenocytes (B) from LMC (CD4-Cre− × STAT5a/bFL/+ or STAT5a/bFL/FL) and CD4-Cre+ × STAT5a/bFL/FL mice stained with Abs to CD4 and CD8. Center and middle panels, CD4-gated thymocytes (A) or splenocytes (B) stained with Abs to CD25 or Foxp3. C, Shown are total numbers of CD4+Foxp3+ T cells from either LMC (□) or CD4-Cre × STAT5a/bFL/FL mice. D, Shown are histograms of CD4-gated T cells from LMC or CD4-Cre × STAT5a/bFL/FL mice stained with Abs to CD62L or CD69. E and F, Thymocytes (E) or splenocytes (F) were stimulated with 1000 U/ml IL-2 and 30 ng/ml IL-7 for 30 min and then stained with Abs to CD4, CD8, and CD25. Cells were then fixed, permeabilized, and stained for phopsho-STAT5. Shown is a representative example of three or more experiments.
- FIGURE 5.
STAT5b activation restores Treg development in the thymus of IL-2Rβ−/− mice. Shown are CD4+CD8−-gated thymocytes (A) or splenocytes (B) stained with Abs to CD4, CD25, and Foxp3 from WT controls; STAT5b-CA transgenic mice; IL-2Rβ−/− mice; and STAT5b-CA × IL-2Rβ−/− mice. C, Shown are total numbers of CD4+Foxp3+ T cells in WT controls, STAT5b-CA transgenic mice, IL-2Rβ−/− mice, and STAT5b-CA × IL-2Rβ−/− mice. Error bars, SEM. Data are representative of three separate experiments.
- FIGURE 6.
STAT5 signaling is sufficient to mediate IL-2Rβ-dependent Treg development. A, Schematic representation of IL-2Rβ constructs used for retroviral reconstitution experiments. B, Spleens and thymii from recipient mice were harvested at 9 wk postreconstitution and stained with Abs to IL-2Rβ (open histograms). Shaded histograms represent Ab staining on IL-2Rβ-deficient mice. C and D, Foxp3 and CD62L levels of expression were analyzed on CD4+-gated cells. Numbers above the outlined areas represent the percentage of cells in the designated gate. Data are representative of three independent recipient mice per group.
- FIGURE 7.
STAT5 binds to the foxp3 promoter in Tregs. A, Schematic representation of the foxp3 gene. Arrows, Locations of PCR primers used for quantification of ChIP assays. Asterisks, The conserved STAT5 binding sites shown in B and analyzed by ChIP assays in C. B, Alignment of selected regions of foxp3 gene among various species. C, Real-time PCR quantification of genomic DNA obtained from CD4+CD25+ T cells immunoprecipitated with either anti-STAT5 (▪) or anti-MEK-1. Left panel, The amount of PCR product normalized to input control of the foxp3 promoter region containing the STAT5 binding sites noted in A. Middle panel, The amount of PCR product normalized to input control of the foxp3 intron 1 region containing the STAT5 binding sites noted in A. Right panel, the amount of PCR product normalized to input control of the foxp3 intron 11 region noted in A. Input samples amplified DNA from all regions tested. Error bars, SEM. Data are representative of two independent experiments. D, Comparison of STAT5 binding to the foxp3 promoter sites in CD4+CD25+ vs CD4+CD25− T cells. Error bars, SEM. Data are representative of two independent experiments.
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