IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development - PubMed (original) (raw)
IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development
Kieng B Vang et al. J Immunol. 2008.
Abstract
Common gamma chain (gammac)-receptor dependent cytokines are required for regulatory T cell (Treg) development as gammac(-/-) mice lack Tregs. However, it is unclear which gammac-dependent cytokines are involved in this process. Furthermore, thymic stromal lymphopoietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing CD4(+)Foxp3(+) Tregs in the thymus express the IL-2Rbeta, IL-4Ralpha, IL-7Ralpha, IL-15Ralpha, and IL-21Ralpha chains, but not the IL9Ralpha or TSLPRalpha chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in CD4(+)Foxp3(+) Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were capable of inducing the conversion of CD4(+)CD25(+)Foxp3(-) thymic Treg progenitors into CD4(+)Foxp3(+) mature Tregs in vitro. To examine this issue in more detail, we generated IL-2Rbeta(-/-) x IL-7Ralpha(-/-) and IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice. We found that IL-2Rbeta(-/-) x IL-7Ralpha(-/-) mice were devoid of Tregs thereby recapitulating the phenotype observed in gammac(-/-) mice; in contrast, the phenotype observed in IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice was comparable to that seen in IL-2Rbeta(-/-) mice. Finally, we observed that Tregs from both IL-2(-/-) and IL-2Rbeta(-/-) mice show elevated expression of IL-7Ralpha and IL-15Ralpha chains. Addition of IL-2 to Tregs from IL-2(-/-) mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the predominant role in Treg development, but that in its absence the IL-7Ralpha and IL-15Ralpha chains are up-regulated and allow for IL-7 and IL-15 to partially compensate for loss of IL-2.
Figures
Fig. 1. γ_c_-cytokine-family receptor expression on CD4+Foxp3+ SP thymocytes and CD4+Foxp3+ splenocytes
Thymus and spleen cells from 5–9 week old C57Bl/6 mice were harvested and stained with antibodies to CD4, CD8 and Foxp3, to identify distinct thymic and splenic T cell subsets, as well as with antibodies to IL2Rβ, IL4Rα, IL7Rα, IL9Rα, IL15Rα, IL21Rα and TSLPRα. Shown are flow cytometry histograms of CD4 single positive (SP) thymocytes (left column) and CD4+ splenocytes (right column). Gray filled in histograms represent staining of the corresponding CD4+Foxp3+ population with isotype control antibody. Solid lines and broken lines represent staining of CD4+Foxp3+ and CD4+Foxp3− cells, respectively. A representative example of 4 independent experiments is depicted (n=17).
Fig. 2. Cytokine stimulation and Phospho-STAT5 expression on CD4+Foxp3+ thymocytes and splenocytes
Single cell suspensions of thymocytes or splenocytes from Foxp3-GFP mice were serum starved for 30 minutes and then stimulated with IL2, IL4, IL7, IL9, IL15, IL21 or TSLP for 20 minutes. Cells were then stained with antibodies to CD4, CD8, CD25 and phospho-STAT5 as described in the methods section. Shown are histograms of phospho-STAT5 expression in CD4SP thymocytes (left column) and CD4+ splenocytes (right column). Gray filled in histograms represent staining of unstimulated Foxp3-GFP+ cells. Solid lines and broken lines represent staining of stimulated Foxp3-GFP+ and Foxp3-GFP− cells, respectively. A representative example of 2 independent experiments is depicted (n=3 mice). Similar results were obtained when using CD25 to identify Tregs in C57Bl/6 mice (n=13 mice, data not shown).
Fig. 3. IL2, IL7 and IL15, but not TSLP, induce the conversion of CD4+CD25+Foxp3− thymic Treg progenitors into CD4+Foxp3+ Tregs
Sorted CD4+CD25+ Foxp3− thymic Treg progenitors from Foxp3-GFP reporter mice were stimulated with 10 U/mL IL2, 5 ng/mL IL7, 100 ng/mL IL15 and 50 ng/mL TSLP in culture overnight. After 24 hours of stimulation, cells were stained with antibodies to CD4 and CD25. Shown are contour plots of Foxp3 versus CD25 for CD4-gated cells (95% of cells were CD4+) after stimulation with medium alone (top left), TSLP (top middle), IL2 (top right), IL7 (bottom left) or IL15 (bottom right). A representative example of 3 independent experiments is depicted.
Fig. 4. Treg development in _IL4R_α−/− and _IL2R_β−/− _x IL4R_α−/− mice
Thymus and spleen were harvested from 4–5 week old LMC, _IL4R_α−/−, _IL2R_β−/− and _IL2R_β−/− _x IL4R_α−/− mice. Cells were stained with antibodies to CD4, CD8 and Foxp3 to identify Tregs. Shown are flow cytometry plots of thymus (top panel) or spleen cells (bottom panel) gated on CD4+ T cells. A representative example of 6 independent experiments is depicted.
Fig. 5. Treg development in _IL2R_β−/− and _IL2R_β−/− _x IL7R_α−/− mice
A. Thymus and spleen were harvested from 4–5 week old LMC, _IL7R_α−/−, and _IL2R_β−/− _x IL7R_α−/− mice. Cells were stained with antibodies to CD4, CD8 and Foxp3 to identify Tregs. Shown are flow cytometry plots of thymus (top panel) or spleen cells (bottom panel) gated on CD4+ T cells. A representative example of 6 independent experiments is depicted. B. Shown are bar graphs representing total numbers of CD4+Foxp3+ Tregs in the thymus (left panel) and spleen (right panel). Error bars represent standard error of the mean; n= 6 _IL7R_α−/− and 6 _IL2R_β−/− _x IL7R_α−/− mice; p-values were calculated using two-tailed students t-test.
Fig. 6. Expression of IL2Rβ, IL7Rα and IL15Rα on CD4+Foxp3+ Tregs from _IL2_−/− and _IL2R_β−/− mice compared to wild type littermate controls
Splenocytes were isolated from 4–5 week old LMC, _IL2_−/− and _IL2R_β−/− mice and stained with antibodies for CD4, CD8, and Foxp3 to identify splenic Tregs. Shown are CD4+Foxp3+ gated cells stained for IL2Rβ (left panels), IL7Rα (middle panels) and IL15Rα (right panels). Gray histograms represent staining of CD4+Foxp3+ cells with isotype control antibody. Solid lines represent histograms of CD4+Foxp3+ T cells from _IL2_−/− (top panel) or _IL2R_β−/− (bottom panel) mice; broken lines represent staining of CD4+Foxp3+ Tregs from WT LMC mice. A representative example of 3 independent experiments is depicted (n= 6 _IL2_−/− and 3 _IL2R_β−/− mice).
Fig. 7. Expression of IL7Rα and IL15Rα on CD4+Foxp3+ Tregs in _IL2_−/− mice after ex vivo IL2 stimulation
CD4+ T cells were isolated as described in the methods section and cells were stimulated with 100 units/mL of IL2 for 8 or 24 hours. Cells were then stained for Foxp3, IL7Rα and IL15Rα and analyzed by flow cytometry. Dark unshaded histogram represents IL7Rα and IL15Rα expression after IL2 stimulation for the times indicated. Shaded histograms represent non-stimulated controls. Shown is a representative example of six independent experiments for IL7Rα expression and two independent experiments for IL15Rα expression.
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