A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells

Abstract

Interleukin-2 receptor (IL-2R) signaling is essential for T regulatory (Treg) cell development and homeostasis. Here, we show that expression of IL-2Rbeta chains that lack tyrosine residues important for the association of the adaptor Shc and the transcription factor STAT5 in IL-2Rbeta-deficient mice resulted in production of a normal proportion of natural Treg cells that suppressed severe autoimmunity related with deficiency in IL-2 or IL-2R. These mutant IL-2Rbeta chains supported suboptimal and transient STAT5 activation that upregulate the transcription factor Foxp3 to normal amounts in natural, but not induced, Treg cells. Nevertheless, gene expression profiling revealed many targets in peripheral natural Treg cells that were IL-2 dependent and a substantial overlap between the Treg cell IL-2-dependent gene program and the Treg cell transcriptional signature. Collectively, these findings demonstrate that a critical, and perhaps minor, subset of IL-2-dependent targets is indexed to a low IL-2R signaling threshold and that a substantial proportion of the Treg cell gene program is regulated by IL-2.

Figure 1. Characterization of lymphocyte cell populations within individual transgenic lines

(A) Scheme represents mutant IL-2Rβ transgenes. Domains of IL-2Rβ are listed above the model, where TM refers to transmembrane. (B) IL-2Rβ expression by representative transgenic lines. Thymus and LNs were obtained from the indicated transgenic IL-2Rβ^−/− mice and were assessed for CD122 (IL-2Rβ) expression after gating on the indicated populations of CD4⁺ and CD8⁺ cells. For reference, CD122 expression by normal WT C57BL/6 (B6) cells was included. DN and DP are CD4^neg CD8^neg or CD4⁺ CD8⁺ thymocytes, respectively. Data are representative of two founders/transgenic line. The numbers to the right of the dot plots represent the % of cells within the designated gated regions. (C) Thymic and LN cellularity and (D) splenic NK cells for the indicated transgenic lines. Data are mean ± SEM of ≥6 mice/group <16 wks of age.

Figure 2. Autoimmune status of transgenic IL-2Rβ^−/− mice expressing mutant IL-2Rβ

The indicated IL-2Rβ transgenic mice were assessed for peripheral T cells with an activated (A) CD44^high, (B) CD69⁺, or (C) CD62L^low (C) phenotype or for (D) hemolytic anemia. Controls were IL-2Rβ^+/− or IL-2Rβ^−/− littermate mice. Data are mean ± SEM of ≥7 mice/group <16 wks of age, except for 2Rβ^WT (n=3) (A-C) and representative of at least 5 mice/group in (D). Autoimmune status was also assessed as a function of age for the indicated transgenic mice by enumerating (E) the % of CD4⁺ T cells that were CD69⁺ or CD62L^low. The line in (E) represents the value for two standard deviations above of the mean for littermate control mice. (F) Hematoxylin-eosin fixed sections of the indictaed tissues were examined for inflammatory infiltrates.

Figure 3. Natural and iTreg cells from mice expressing mutant IL-2Rβ

(A) CD4⁺ Foxp3⁺ T cells within the thymus and LNs of the indicated mice were examined for CD25 (IL-2Rα) and CD122 (IL-2Rβ) expression. (B) % (n ≥5 mice/group) and levels of Foxp3 (n≥3 mice/group) and (C) the level of CD25 (≥3 mice/group) for Foxp3⁺ T cells in the thymus and LNs. Levels are expressed as mean fluorescent intensity (MFI). The MFI of staining by control IL-2Rβ^+/− was normalized to a value of 1 and used to compare staining by T cells bearing mutant IL-2Rβ. (D) Production of Foxp3⁺ T cells after culture of spleen cells from the indicated mice (n≥2) with TGFβ and IL-2. Data are mean ± SEM for mice <16 wks of age.

Figure 4. Signaling by CD4⁺Foxp3⁺ T cells bearing mutant IL-2Rβ

Thymocytes and spleen cells from the indicated mice were cultured in medium for 30 min and then stimulated with an excess of IL-2 (10 ng/ml) for the indicated time. (A) Representative pSTAT5 and pS6 staining by normal B6 CD4⁺Foxp3⁺ T cells. (B) Summary of all experiments for pSTAT5 activation by WT Treg cells where the MFI of pSTAT5 staining at 15 min by thymic Treg cells was normalize to a value of 1. (C) Representative comparison of IL-2-dependent pSTAT5 by Treg cells from B6 and mutant IL-2Rβ transgenic mice. (D) Summary of all comparisons where the MFI of pSTAT5 staining at 15 min by control IL-2Rβ^{+/+ or +/−} was normalize to a value of 1 and used to compare staining by Treg cells bearing mutant IL-2Rβ. Data are the mean ± SEM of 2–5 mice/group <16 wks of age.

Figure 5. Proliferative and functional responses by T blasts bearing mutant IL-2Rβ

Spleen cells from the indicated mice were culture with anti-CD3 for 48 hr. (A) The activated T cells were washed and re-cultured in the indicated level of IL-2 for 24 hr. ³H-thymidine was added during the last 4 hr of culture. Each data point represents the mean of at least 3 separate experiments, where the highest response was normalized to 100%. The activated CD4⁺ and CD8⁺ spleen cells were stained for (B) CD25, (C) granzyme B (Gzmb), or (D) CD69. The MFI of staining by control IL-2Rβ^{+/+ or +/−} was normalized to a value of 1 and used to compare staining by T cells bearing mutant IL-2Rβ. Data are the mean ± SEM of ≥3 mice/group <16 wks of age.

Figure 6. IL-2-dependent signaling by T blasts bearing mutant IL-2Rβ

(A) Spleen cells from the indicated mice were cultured with anti-CD3 for 48 hr. The T cells were isolated by magnetic bead sorting using anti-Thy-1.2, cultured in IL-4 for 24 hr, and then washed and “rested” in medium for 4 hr. These T cells were cultured with IL-2 for the indicated time and cytoplasmic extracts were prepared. Western blots were probed with mAbs to the indicated phospho-tyrosine proteins. mAb to unmodified STAT5 served as a loading control. Data are representative of 2 experiments. (B) The indicated anti-CD3 activated spleen cells were cultured in medium for 4 hr and then stimulated with IL-2 for the indicated time followed by staining for pSTAT5 and pS6. (C) Summary of all experiments where the MFI of pSTAT5 staining at 15 min by control IL-2Rβ^{+/+ or +/−} was normalized to a value of 1 and used to compare staining by T cells bearing mutant IL-2Rβ. Data are the mean ± SEM of ≥3 mice/group <16 wks of age.

Figure 7. IL-2-dependent mRNA in Treg and T effector cells

(A) IL-2-dependent genes (>2-fold) from Treg and CD4⁺ T effector cells that overlap with each other and the Treg cell gene signature. (B) IL-2 dependent targets (dark curves) that overlap with the Treg cell transcriptional signature (light shaded curves). The overlapping Treg (upper graph) and T effector (lower graph) targets vs. all targets of the Treg transcriptional signature plotted as a function of their correlation to Foxp3 expression as reported by (Hill et al., 2007). (C) Functional groups of IL-2-dependent (>2-fold) differentially expressed genes by Treg and CD4⁺ T effector cells. Shaded genes represent those transcripts that overlap between IL-2-dependent Treg and T effector cells. Underlined genes were identified in the Treg cell transcriptional signature.