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. 2016 Nov;17(11):1322-1333.
doi: 10.1038/ni.3540. Epub 2016 Sep 5.

An Essential Role for the IL-2 Receptor in T reg Cell Function

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Free PMC article

An Essential Role for the IL-2 Receptor in T reg Cell Function

Takatoshi Chinen et al. Nat Immunol. .
Free PMC article

Abstract

Regulatory T cells (Treg cells), which have abundant expression of the interleukin 2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. This feature indicates a key role for a simple network based on the consumption of IL-2 by Treg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the Treg cell lineage-specification factor Foxp3, which has confounded experimental efforts to understand the role of IL-2R expression and signaling in the suppressor function of Treg cells. Using genetic gain- and loss-of-function approaches, we found that capture of IL-2 was dispensable for the control of CD4+ T cells but was important for limiting the activation of CD8+ T cells, and that IL-2R-dependent activation of the transcription factor STAT5 had an essential role in the suppressor function of Treg cells separable from signaling via the T cell antigen receptor.

Figures

Figure 1
Figure 1
IL-2Rβ is indispensable for Treg cell function. (a) Histopathology of indicated organs of Foxp3CreIl2rbfl/wt and Foxp3CreIl2rbfl/fl mice. Scale bar, 100 μm. (b) Lymph node (LN) cellularity of indicated mice. (c) Cytokine production by splenic CD4+Foxp3 cells stimulated for 5 hr with anti-CD3/CD28. (d) IL-2R subunit expression by CD4+Foxp3+ cells from Foxp3CreIl2rbfl/wt (blue) and Foxp3CreIl2rbfl/fl (red) mice. (e) Intracellular phospho-STAT5 levels in Treg cells from the indicated mice unstimulated (US) or in vitro stimulated with rmIL-2 (1,000 U/ml) for 20 min. (f) The frequencies of Treg cells among LN CD3+CD4+ cells (left) and Foxp3 expression levels (MFI: mean fluorescence intensity) in the CD3+CD4+ Foxp3+ cells (right). (g-j) The analysis of healthy heterozygous female Foxp3Cre/wt mice. (g) YFP (Cre) expression and intracellular Foxp3 staining identify Treg cells with or without YFP-Cre expression. Gates shown are for CD3+CD4+ cells. (h) The frequencies of Foxp3+ cells among CD3+CD4+ cells (upper panel) and of Cre expressing cells among Foxp3+ cells (lower panel) in the indicated organs of Foxp3Cre/wtIl2rbfl/wt (black) and Foxp3Cre/wtIl2rbfl/fl (red) mice. (i) Foxp3 expression levels (MFI) in YFPFoxp3+ (upper panel) and YFP+Foxp3+ (lower panel) cells. (j) The expression of indicated markers in YFP+Foxp3+ cells. Cells were analyzed by flow cytometry (bj). 3–5 wk-old sex and age matched mice were analyzed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant (two-tailed unpaired Student's t test). Data are from one experiment representative of three independent experiments with similar results with three or more mice per group in each (b, c, f, h, i, j; each dot represents a single mouse; mean ± s.e.m.) or representative data of more than five (a) or ten (d, e, g) mice per group analyzed are shown.
Figure 2
Figure 2
Restoration of the suppressor activity of IL-2R-deficient Treg cells in the presence of an active form of STAT5. (a) The frequencies of Foxp3+ cells among CD3+CD4+ cells and CD122 and CD25 expression by CD3+CD4+Foxp3+ cells. (b) Intracellular phospho-STAT5 levels in LN Treg cells from the indicated mice unstimulated (US) or in vitro stimulated with rmIL-2 (1,000 U/ml) for 20 min. (c) In vitro IL-2 capture assay. Treg and non-Treg cells from the indicated mice were sorted and cultured for 2 hr with recombinant human IL-2 (hIL-2). The amount of residual hIL-2 in the media after 2 hr was measured using flow cytometry-based bead array analysis and shown as percent value. (d) The cell numbers of indicated CD4+ and CD8+ cell subset (both CD3+Foxp3) in the LNs of indicated mice (2 wk old). (e) The frequencies of naive (CD62LhiCD44lo) T cells among CD3+CD4+Foxp3 and CD3+CD8+Foxp3 cells (upper two panels) and the numbers of CD44hi activated CD3+CD4+Foxp3 and CD3+CD8+Foxp3 cells (lower two panels) in the LNs of indicated mice treated with IL-2 neutralizing antibodies or control IgG for 2 wks starting from day 5–7 after birth. (f) Analysis of the ability of IL-2R-sufficient and -deficient Treg cells to suppress the expansion of naive and activated/memory CD4+ and CD8+ T cells. CD4+Foxp3CD62LhiCD44lo (CD4+ naive), CD8+Foxp3CD62LhiCD44lo (CD8+ naive), and CD8+Foxp3CD62LhiCD44hi (CD8+ memory) T cells were sorted from Foxp3Cre mice and adoptively transferred (1 × 106 cells each) into T cell-deficient (Tcrb−/−Tcrd−/−) mice together with Treg cells (2 × 105 cells) sorted from the indicated mice. CD4+Foxp3 and CD8+Foxp3 T cell numbers in the LNs of recipients 3 wks after transfer are shown. (g, h) Analysis of susceptibility of CD4+ and CD8+ T cells expressing an active form of STAT5 to Treg mediated suppression. (g) The frequencies of STAT5b-CA-expressing CD4+ and CD8+ Teff cells within total CD4+ and CD8+ Teff cells 3 wks after a transfer of in vitro TAT-Cre recombinase treated CD4+Foxp3 and CD8+Foxp3 T cells sorted from Foxp3CreRosa26Stat5bCA mice and transferred (1 × 106 cells each) into Tcrb−/−Tcrd−/− recipients. (h) The numbers and proportion (%) of IFN-γ-producing CD4+ and CD8+ T cells in the recipients without a co-transfer of Treg cells (red bars) or with 2 × 105 control (black bars) or STAT5b-CA-expressing Treg cells (blue bars) sorted from Foxp3Cre or Foxp3CreRosa26Stat5bCA mice, respectively. As a control, CD4+Foxp3 and CD8+Foxp3 T cells sorted from Foxp3CreRosa26wt mice were similarly treated with TAT-Cre and transferred to assess the susceptibility of STAT5b-CA Teff cells to Treg mediated suppression (open bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant (two-tailed unpaired Student's t test). Data are from one experiment representative of two (b, c, e, f) or three (a, d, g, h) independent experiments with similar results with two or more (a, b) or three or more (c, d, e, f, g, h) mice per group in each experiment (each dot represents a single mouse; mean ± s.e.m.).
Figure 3
Figure 3
Increased proliferative and suppressor activity of Treg cells expressing an active form of STAT5. (ag) Analysis of Foxp3Cre-ERT2 (black dots) and Foxp3Cre-ERT2Rosa26Stat5bCA (blue dots) mice three months after a single tamoxifen treatment. (a) The frequencies of Foxp3+ cells among CD3+CD4+ cells (upper graph) and the expression levels of Foxp3 in CD3+CD4+Foxp3+ cells (lower graph) in the indicated organs. Sp: spleen, SILPL: small intestine lamina propria lymphocytes. (b) Analysis of splenic CD4+ T cells for the expression of CD25 and Foxp3. (c) Analysis of splenic CD4+Foxp3+ Treg cells. (d) The expression of indicated markers in splenic CD4+Foxp3+ Treg cells. (e) Analysis of splenic CD3+CD4+Foxp3 (upper panels) and CD3+CD8+Foxp3 (lower panels) cells. (f) The expression of CD80 and CD86 on DCs (CD11c+MHC class IIhi) and B cells (B220+CD11c) in the LNs. (g) Serum and fecal IgA levels as determined by ELISA. Sex and age matched mice were analyzed. Cells were analyzed by flow cytometry (af). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed unpaired Student's t test). Data are from one experiment representative of two (a, d, f, g) independent experiments with similar results with five or more mice per group in each experiment (a, d, f, g; each dot represents a single mouse; mean ± s.e.m.) or representative data of more than ten mice per group analyzed are shown (b, c, e).
Figure 4
Figure 4
Potent suppressor function of Treg cells expressing an active form of STAT5. (ac) Analysis of EAE induced upon immunization with MOG peptide in CFA. (a) Average disease scores (n=10 each). (b) The frequencies Foxp3+ cells among brain-infiltrating CD3+CD4+ (left) and CD3+CD8+ (right) cells in mice shown in (a). (c) The numbers of the indicated brain-infiltrating cells in mice shown in (a). (d) T cell responses against Listeria monocytogenes in uninfected (UI) or infected (Inf; day 8 after infection) mice. The frequencies of Foxp3+ Treg cells among CD3+CD4+ cells (left). The frequencies of IFN-γ (middle) and TNFα (right) producing CD4+TCRβ+Foxp3 cells after 5 hr in vitro re-stimulation with heat-killed Listeria in the presence of DCs. (e) Anti-viral T cell responses in mice on day 8 after infection with non-replicating vaccinia virus. The frequencies of vaccinia B8R peptide-specific CD8+ T cells detected using H-2Kb-B8R tetramer (left), IFN-γ production by CD8+Foxp3 (middle) and CD4+Foxp3 (right) cells after a 5 hr in vitro stimulation with B8R peptide or a mixture of three vaccinia virus-specific peptides (ISK, A33R, and B5R). Cells were analyzed by flow cytometry (be). Sex and age matched Foxp3Cre-ERT2 (black) and Foxp3Cre-ERT2Rosa26Stat5bCA (blue) mice were challenged or infected as indicated 2-3 months after a single tamoxifen treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed unpaired Student's t test). Data are from one experiment representative of two independent experiments with similar results with ten (ac) or four (e) mice per group in each experiment, or pooled from four independent experiments with n=11 (UI), n=15 (Inf, control), or n=20 (Inf, Rosa26Stat5bCA) mice per group are shown (d). Each dot represents a single mouse (be). mean ± s.e.m. (ae).
Figure 5
Figure 5
RNA-seq analysis of Treg cells expressing an active form of STAT5. (a) Principal component analysis of RNA-seq datasets, using the top 15% of genes with the highest variance. Each dot represents an RNA sample from a single mouse. Note that “Stat5bCA T naive” cells are T naive cells sorted from tamoxifen-treated Foxp3Cre-ERT2Rosa26Stat5bCA mice and do not express STAT5b-CA. Only “Stat5bCA Treg” cells express STAT5b-CA in the four groups of cells presented. (b) Plot of gene expression (as log2 normalized read count) in control vs. STAT5b-CA expressing Treg cells. The diagonal lines indicate fold change of at least 1.5× or 0.67× fold. Significantly up- and down-regulated genes (defined as genes with at least 1.5× or 0.67× fold change, adjusted P-value ≤ 0.05, and expression above a minimal threshold based on the distribution of all genes) are colored red or blue, respectively, and their numbers are shown. (c) Heat map of selected genes. Three replicates are shown in order. P-values are for control Treg vs. STAT5b-CA expressing Treg cells. (d) The empirical cumulative distribution function (ECDF) for the log2 fold change of all expressed genes in STAT5b-CA vs. control Treg cells, is plotted along with ECDFs for the subsets of genes up- or down-regulated by inflammatory activation in Treg cells (upper graph), or the subsets of genes up- or down-regulated in a TCR-dependent manner in CD44hi Treg cells (lower graph). (e) Signaling Pathway Impact Analysis (SPIA) of KEGG pathways. The 6 most statistically significant pathways that show enrichment among differentially expressed (DE) genes in STAT5b-CA vs. control Treg cells are shown. The net pathway perturbation indicates the status of the pathway (positive = activated; negative = inhibited) based on the activating or inhibitory relationships of DE genes within the pathway. The size of the red circle is proportional to the degree of enrichment, and the FDR-adjusted global P-value reflecting both enrichment and perturbation is shown. (f) Network analysis of GO term enrichment among significantly upregulated genes in STAT5b-CA vs. control Treg cells. Upregulated genes were analyzed for over-represented GO terms using BiNGO in Cytoscape, and the resulting network was calculated and visualized using EnrichmentMap. Groups of similar GO terms (Supplementary Table 1) were manually circled. Line thickness and color are proportional to the similarity coefficient between connected nodes. Node color is proportional to the FDR-adjusted P-value of the enrichment. Node size is proportional to gene set size.
Figure 6
Figure 6
Augmented STAT5 signaling in Treg cells increases the conjugate formation between Treg cells and DCs and potentiates suppressor function in a TCR independent manner. (a) Analysis of in vitro conjugate formation between T cells and DCs. CFSE-labeled Treg and non-Treg cells from the indicated mice were co-cultured with graded numbers of CellTrace Violet-labeled CD11c+ DCs from C57BL/6J mice for 720 min in the absence (left panel) or presence (right panel) of rmIL-2 (100 IU/ml). Each dot represents a flow cytometric analysis of conjugate formation in a single well. (b) Expression of Foxp3 and TCRβ by CD4+ cells in the LNs and spleen of Tcrafl/wt heterozygous mice treated with tamoxifen for 2 wks. The frequencies of TCR-sufficient and -deficient Foxp3+ cells among CD4+ cells are summarized in the right panel. (c) T cell activation and pro-inflammatory cytokine production in the LNs of indicated mice treated with tamoxifen for 2 wks. The lower five panels show the expression of the indicated molecules in LN Treg cells. (d) The analysis of T cells transferred into Tcrb−/−Tcrd−/− recipients. WT CD4+Foxp3 and CD8+Foxp3 T cells (5 × 105 cells each) were transferred together with TCR-ablated (TCRβloCD3lo) or TCR-sufficient Treg cells (3 × 105 cells) sorted from the indicated mice that had been treated with tamoxifen for 2 wks. The frequencies of Treg cells and Foxp3 expression levels in Treg cells (upper two panels), the expression of the indicated molecules in Treg cells (middle three), and the numbers of cytokine producing CD4+ and CD8+ T cells (lower three) in the LNs of recipient mice 3 wks after the transfer are shown. Sex and age matched mice were analyzed. Cells were analyzed by flow cytometry (ad). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (modified ANCOVA in Prism software (a) or two-tailed unpaired Student's t test (b, c, d)). Data are from one experiment representative of three (a) or two (b, d) or four (c) independent experiments with similar results with three or more (b, c) or five or more (d) mice per group in each experiment (b, c, d; each dot represents a single mouse; mean ± s.e.m.).

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