An essential role for the IL-2 receptor in Treg cell function

Abstract

Regulatory T cells (T_reg 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 T_reg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the T_reg 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 T_reg 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 T_reg cells separable from signaling via the T cell antigen receptor.

Figure 1

IL-2Rβ is indispensable for T_reg cell function. (a) Histopathology of indicated organs of Foxp3^CreIl2rb^fl/wt and Foxp3^CreIl2rb^fl/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 Foxp3^CreIl2rb^fl/wt (blue) and Foxp3^CreIl2rb^fl/fl (red) mice. (e) Intracellular phospho-STAT5 levels in T_reg 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 T_reg 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 Foxp3^Cre/wt mice. (g) YFP (Cre) expression and intracellular Foxp3 staining identify T_reg 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 Foxp3^Cre/wtIl2rb^fl/wt (black) and Foxp3^Cre/wtIl2rb^fl/fl (red) mice. (i) Foxp3 expression levels (MFI) in YFP⁻Foxp3⁺ (upper panel) and YFP⁺Foxp3⁺ (lower panel) cells. (j) The expression of indicated markers in YFP⁺Foxp3⁺ cells. Cells were analyzed by flow cytometry (b–j). 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

Restoration of the suppressor activity of IL-2R-deficient T_reg 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 T_reg 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. T_reg and non-T_reg 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 (CD62L^hiCD44^lo) T cells among CD3⁺CD4⁺Foxp3⁻ and CD3⁺CD8⁺Foxp3⁻ cells (upper two panels) and the numbers of CD44^hi 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 T_reg cells to suppress the expansion of naive and activated/memory CD4⁺ and CD8⁺ T cells. CD4⁺Foxp3⁻CD62L^hiCD44^lo (CD4⁺ naive), CD8⁺Foxp3⁻CD62L^hiCD44^lo (CD8⁺ naive), and CD8⁺Foxp3⁻CD62L^hiCD44^hi (CD8⁺ memory) T cells were sorted from Foxp3^Cre mice and adoptively transferred (1 × 10⁶ cells each) into T cell-deficient (Tcrb^−/−Tcrd^−/−) mice together with T_reg 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 T_reg mediated suppression. (g) The frequencies of STAT5b-CA-expressing CD4⁺ and CD8⁺ T_eff cells within total CD4⁺ and CD8⁺ T_eff cells 3 wks after a transfer of in vitro TAT-Cre recombinase treated CD4⁺Foxp3⁻ and CD8⁺Foxp3⁻ T cells sorted from Foxp3^CreRosa26^Stat5bCA 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 T_reg cells (red bars) or with 2 × 10⁵ control (black bars) or STAT5b-CA-expressing T_reg cells (blue bars) sorted from Foxp3^Cre or Foxp3^CreRosa26^Stat5bCA mice, respectively. As a control, CD4⁺Foxp3⁻ and CD8⁺Foxp3⁻ T cells sorted from Foxp3^CreRosa26^wt mice were similarly treated with TAT-Cre and transferred to assess the susceptibility of STAT5b-CA⁻ T_eff cells to T_reg 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

Increased proliferative and suppressor activity of T_reg cells expressing an active form of STAT5. (a–g) Analysis of Foxp3^Cre-ERT2 (black dots) and Foxp3^Cre-ERT2Rosa26^Stat5bCA (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⁺ T_reg cells. (d) The expression of indicated markers in splenic CD4⁺Foxp3⁺ T_reg 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 II^hi) 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 (a–f). *, 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

Potent suppressor function of T_reg cells expressing an active form of STAT5. (a–c) 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⁺ T_reg 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-2K^b-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 (b–e). Sex and age matched Foxp3^Cre-ERT2 (black) and Foxp3^Cre-ERT2Rosa26^Stat5bCA (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 (a–c) 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, Rosa26^Stat5bCA) mice per group are shown (d). Each dot represents a single mouse (b–e). mean ± s.e.m. (a–e).

Figure 5

RNA-seq analysis of T_reg 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 Foxp3^Cre-ERT2Rosa26^Stat5bCA mice and do not express STAT5b-CA. Only “Stat5bCA T_reg” 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 T_reg 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 T_reg vs. STAT5b-CA expressing T_reg cells. (d) The empirical cumulative distribution function (ECDF) for the log₂ fold change of all expressed genes in STAT5b-CA vs. control T_reg cells, is plotted along with ECDFs for the subsets of genes up- or down-regulated by inflammatory activation in T_reg cells (upper graph), or the subsets of genes up- or down-regulated in a TCR-dependent manner in CD44^hi T_reg 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 T_reg 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 T_reg 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

Augmented STAT5 signaling in T_reg cells increases the conjugate formation between T_reg 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 T_reg and non-T_reg 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 Tcra^fl/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 T_reg 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 (TCRβ^loCD3^lo) or TCR-sufficient T_reg cells (3 × 10⁵ cells) sorted from the indicated mice that had been treated with tamoxifen for 2 wks. The frequencies of T_reg cells and Foxp3 expression levels in T_reg cells (upper two panels), the expression of the indicated molecules in T_reg 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 (a–d). *, 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.).