T Regulatory Cells + Foxp3 + CD25 +CD4 Determinant Controlling the ...

The Journal of Immunology

Selective Availability of IL-2 Is a Major Determinant Controlling the Production of CD4ⴙCD25ⴙFoxp3ⴙ T Regulatory Cells1 Aixin Yu and Thomas R. Malek2 The development and maintenance of T regulatory (Treg) cells critically depend on IL-2. This requirement for IL-2 might be due to specificity associated with IL-2R signal transduction or because IL-2 was uniquely present in the niche in which Treg cells reside. To address this issue, we examined the capacity of IL-7R-dependent signaling to support Treg cell production and prevent autoimmunity in IL-2R␤ⴚ/ⴚ mice. Expression of transgenic wild-type IL-7R or a chimeric receptor that consisted of the extracytoplasmic domain of the IL-7R ␣-chain and the cytoplasmic domain of IL-2R ␤-chain in IL-2R␤ⴚ/ⴚ mice did not prevent autoimmunity. Importantly, expression of a chimeric receptor that consisted of the extracytoplasmic domain of the IL-2R ␤-chain and the cytoplasmic domain of IL-7R ␣-chain in IL-2R␤ⴚ/ⴚ mice led to Treg cells production in the thymus and periphery and prevented autoimmunity. Signaling through the IL-2R or chimeric IL-2R␤/IL-7R␣ in vivo or the culture of thymocytes from IL-2R␤ⴚ/ⴚ mice with IL-7 led to up-regulation of Foxp3 and CD25 on Treg cells. These findings indicate that IL-7R signal transduction is competent to promote Treg cell production, but this signaling requires triggering through IL-2 by binding to the extracytoplasmic portion of the IL-2R via this chimeric receptor. Thus, a major factor controlling the nonredundant activity of the IL-2R is selective compartmentalization of IL-2-producing cells with Treg cells in vivo. The Journal of Immunology, 2006, 177: 5115–5121.

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xtensive redundancy in biological function and signal transduction has been noted for cytokines, i.e., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which use receptors that share the common ␥-chain (␥c)3 as one of their receptor subunits (1–5). Nevertheless, knockout mouse models have revealed very specific functions for each one of these cytokines in the development and regulation of cells within the immune system. IL-2 controls production of CD4⫹CD25⫹ T regulatory (Treg) cells; IL-4 promotes the development of TH2 effector cells; IL-7 is essential for T and B cell development; IL-9 is an important mast cell growth factor; IL-15 is necessary for NK and NKT cell development and T memory cells homeostasis; and IL-21 in conjunction with IL-4 is essential for IgG production. In face of the extensive redundancy by ␥c-dependent cytokines, there are three obvious factors that might contribute to the nonredundant activity of individual ␥c-dependent cytokine, i.e., selective availability of the cytokine, restricted expression of the cytokine receptor, and distinctive cytokine receptor signal transduction. However, it is not known the extent that any one of these factors might dominant to result in the specificity for particular cytokines for key processes in the immune system as revealed by the knockout mouse models.

Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL 33101 Received for publication April 17, 2006. Accepted for publication August 2, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grants RO1-CA45957 and RO1-AI055815 from the National Institutes of Health. 2 Address correspondence and reprint requests to Dr. Thomas R. Malek, Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, PO Box 016960, Miami, FL 33101. E-mail address: [email protected]

Abbreviations used in this paper: ␥c, common ␥-chain; DN, double negative; DP, double positive; Treg; T regulatory; MFI, mean fluorescence intensity.

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Copyright © 2006 by The American Association of Immunologists, Inc.

With respect to the IL-2R, expression of a functional high affinity IL-2R consisting of IL-2R␣ (CD25), IL-2R␤ (CD122), and ␥c (CD132) is readily detected on recently activated effector T cells and on CD4⫹CD25⫹ Treg cells (2). However, these cell populations also express receptors for other ␥c-dependent cytokines (6 – 8). For T cell immunity, effective immune responses do not require IL-2 in vivo, indicating that there is sufficient redundancy to overcome a lack of IL-2 (9 –15). Other ␥c-dependent cytokines might substitute for IL-2 as activated T cells readily respond to most ␥c-dependent cytokines. However, IL-2 remains the dominate cytokine for Treg cells (3). This nonredundant activity of IL-2 for Treg cells might be due to selective signal transduction by the IL-2R or to selective availability of IL-2. In the current study we directly assessed these possibilities by evaluating whether chimeric IL-2R and IL-7R prevented autoimmunity when expressed as transgenes in IL-2R␤-deficient mice. We show that IL-7R signaling is capable of supporting Treg cell production, but this only occurs when this signaling is induced through a chimeric receptor that depends upon IL-2 for its triggering. Thus, a major factor controlling the nonredundant activity of the IL-2R is compartmentalization of the IL-2-producing cells with Treg cell in vivo.

Materials and Methods Chimeric receptor constructs The IL-2R␤ cDNA in pSKII was digested with ClaI and NcoI to liberate the portion of the cDNA that encoded the leader peptide, extracytoplasmic and transmembrane domains, and the first six amino acids of the cytoplasmic tail. A PCR fragment corresponding to these regions of the IL-7R␣ cDNA was synthesized and cloned into this vector yielding 7␣/2␤ (see Fig. 2A) that was then subcloned into pENTR. The IL-2R␤ cDNA in pENTR was digested with NcoI and XhoI to release a DNA fragment encoding all but six amino acids of the cytoplasmic tail adjacent to the transmembrane region. This construct deletes all known species conserved tyrosine residues required for IL-2R signaling (16, 17), although one nonspecies conserved tyrosine is still present in the short segment remaining from the 0022-1767/06/$02.00

5116 IL-2R␤ cytoplasmic tail. A PCR fragment corresponding to the cytoplasmic tail of IL-7R␣ was cloned into this vector yielding 2␤/7␣. Both chimeric receptors were verified by DNA sequencing. The 7␣/2␤ and 2␤/7␣ cDNAs were then subcloned into a vector containing the CD2 promoter and enhancer (provided by P. Love, National Institutes of Health, Bethesda, MD) that was modified to contain the Gateway cassette to facilitate recombination based cloning using pENTR vectors containing the insert of interest (Invitrogen Life Technologies). The DNA fragment containing the CD2 mini gene and the 7␣/2␤ or 2␤/7␣ inserts were purified for production of transgenic mice.

Mice C57BL/6 IL-2R␤⫺/⫺, IL-7R␣⫺/⫺, and littermate control mice were maintained in our breeding colony. In some experiments C57BL/6 mice were used as wild-type (WT) controls and were obtained from The Jackson Laboratory. C57BL/6 transgenic WT 2␤ (2, 18) and WT 7␣ (19) mice on the IL-2R␤⫺/⫺ and IL-7R␣⫺/⫺ genetic backgrounds were previously described. The transgenic mice expressing the 7␣/2␤ and 2␤/7␣ chimeric receptor were prepared by injection of the purified DNAs into (B6 x SJL)F2 oocytes. The resulting founder mice were backcrossed to autoimmune-free C57BL/6 IL-2R␤⫺/⫺ (2) or IL-7R␣⫺/⫺ mice for three to five generations, as required.

Monoclonal Abs and FACS analysis Monoclonal Abs to mouse CD4, CD8, CD122, CD127, CD69, CD62L, and CD25 were obtained from BD Biosciences and were used for surface FACS staining. The mAb to Foxp3 (FJK16s) was obtained from eBioscience and was used in intracellular FACS analysis using a kit for this purpose according to the manufacturer’s instructions. FACS analysis was usually performed on a LSR I analyzer using CellQuest software (BD Biosciences). In some experiments when Foxp3 was enumerated, FACS analysis was performed on a LSR II analyzer using DIVA software (BD Biosciences). To enumerate Treg cells, from 100,000 –300,000 events were typically analyzed after gating on viable cells based on forward vs light scatter gating. To increase the fraction of Treg cells, sometimes CD8⫹ thymocytes were depleted before FACS staining and analysis by using anti-CD8 magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions.

In vitro T cell culture The capacity of purified CD4⫹CD25⫹ splenic T cells to inhibit the proliferation by CD4⫹ T cells (5 ⫻ 104/well) to anti-CD3 (0.25 ␮g/ml) in the presence of mitomycin-treated T cell-depleted splenic accessory cells (5 ⫻ 104/well) was performed by culturing in 96-well round-bottom plates for 72 h culture as previously described (2). Cytokine-induced proliferation by anti-CD3 activated T cells has been previously described (18). [3H]Thymidine was added during the last 4 –5 h of culture. Thymocytes (1 ⫻ 106/well) from

FIGURE 1. Constitutive T lineage expression of the IL-7R does not prevent autoimmunity in IL-2R␤⫺/⫺ mice. A, FACS analysis of CD127 (IL-7R␣) expression (thick line histogram) of the indicated T cell subsets in nontransgenic WT or 7␣ transgenic IL-2R␤⫺/⫺ mice. Control staining (thin line histogram) is shown. B, Effect on autoimmunity as assessed by determining body weight, the occurrence of hemolytic anemic, and the presence of autoreactive T cells as indicated by CD4⫹ T cells with an activated CD69⫹ phenotype. The data represent the mean ⫾ SD. Values shown in graph (right) represent the number of mice per group evaluated at 8 wk of age. Not significant (NS) using t test in comparison to IL-2R␤⫺/⫺ mice.

IL-2 AND Treg CELL PRODUCTION IL-2R␤-deficient mice were cultured in medium alone, mouse IL-2 (10 ng/ml), or mouse IL-7 (10 ng/ml) for 24 h. The 7␣/2␤ chimeric receptor construct was subcloned into pCIneo expression vector (Promega) and used for stable transfection of the IL-2-dependent CTLL cell line. The transfected cells (5 ⫻ 103/ well) were cultured in 96-well flat-bottom plates with grades doses of mouse IL-2 or human IL-7 for 24 h. [3H]Thymidine was added during the last 4 –5 h of culture.

Results IL-7R does not substitute for IL-2R to prevent autoimmunity in IL-2R␤⫺/⫺ mice The IL-7R is selectively expressed on thymic subsets from WT mice such that it is readily detected on the most immature CD4⫺CD8⫺ double negative (DN) thymocytes, but is down-regulated such that it is undetectable on more mature DN and double positive (DP) thymocytes. During a later stage of development, the IL-7R is up-regulated on subsets of CD4⫹ and CD8⫹ single positive thymocytes (19) (Fig. 1A). Signal transduction by the IL-7R overlaps with the IL-2R in that both receptors activate the STAT5 and PI3K signaling pathways, but unique signaling has also been attributed to each receptor (20, 21). Because IL-7 is essential for thymic development, there must be IL-7-secreting cells within the thymus. Therefore, we tested whether IL-7R signaling might prevent autoimmunity in IL-2R␤⫺/⫺ mice, if there were not the normal constraints controlling IL-7R expression. We previously characterized WT IL-7R␣ transgenic mice that upon breeding to IL-7R␣-deficient mice reconstituted thymic T cell development (19). This IL-7R␣ (7␣) transgene was expressed in the thymus of IL-2R␤⫺/⫺ mice such that this expression in conjunction with the endogenous IL-7R gene led to IL-7R␣ (CD127) expression on all thymocytes and mature T cells, except some DN thymocytes (Fig. 1A). However, this wide expression of the IL-7R was unable to prevent autoimmunity as assessed by the inability of these mice to gain weight, the presence of hemolytic anemia, and peripheral lymphocyte with an activated phenotype (Fig. 1B). These findings raised the possibility that IL-7R signal transduction is unable to support Treg cell production in IL-2R␤⫺/⫺ mice.

The Journal of Immunology IL-2-dependent triggering of IL-7R signaling prevents autoimmunity in IL-2R␤-deficient mice An alternative explanation for the results described was that IL-7R signaling is potentially productive for Treg cell production but IL-7 was unavailable to trigger the transgenic IL-7R that was expressed on virtually all thymocytes and mature T cells. To directly address this possibility, chimeric cytokine receptors were constructed in which the extracellular domain of IL-2R␤ was linked to the cytoplasmic domain of IL-7R␣ (2␤/7␣), or reciprocally, the extracellular domain of IL-7R␣ was linked to the cytoplasmic domain of IL-2R␤ (7␣/2␤) (Fig. 2A). We reasoned that when these chimeric cytokine receptors were expressed as transgenes in T lineage cells in IL-2R␤⫺/⫺ mice, the 2␤/7␣ receptor would support Treg production, if the main problem was that IL-7 was unavailable to induce IL-7R signaling. Alternatively, the 7␣/2␤ receptor might selectively support Treg cell production, if IL-7 was available but there was a requirement for unique IL-2R signaling. To definitively assess expression the 7␣/2␤ and the 2␤/7␣ transgenic chimeric receptors, these transgenic mice were backcrossed onto the IL-7R␣⫺/⫺ and IL-2R␤⫺/⫺ genetic background, respectively. Using cells from nontransgenic cytokine receptor-deficient

FIGURE 2. Production and characterization of chimeric IL-2R␤ and IL7R␣. A, Schematic of the chimeric receptors. B–D, Transgenic chimeric receptor expression. FACS analysis was performed using mAbs to the extracellular portion of the chimeric 7␣/2␤ and 2␤/7␣ receptors from the indicated CD4 and CD8 subsets within the thymus (B) or spleen and lymph nodes (LN) (C) from IL-7R␣⫺/⫺ or IL-2R␤⫺/⫺ mice, respectively, or after gating on CD4⫹ Foxp3⫹ T cells in the thymus and lymph nodes (D).

5117 littermate mice as the negative control, FACS analysis revealed that virtually all thymocytes (Fig. 2B), with the exception of stage 1 pro-T cells (data not shown), and the large majority of peripheral CD4⫹ and CD8⫹ T cells (Fig. 2C) expressed the 7␣/2␤ chimeric receptor. The 2␤/7␣ receptor was expressed at very low levels on DN and DP thymocytes, but its expression increased to readily detectable levels on virtually all CD4 and CD8 single positive thymocytes (Fig. 2B) and peripheral T cells (Fig. 2C). Both transgenic receptors were not detected on CD4⫺CD8⫺ spleen cells that include both B cells and macrophages (Fig. 2C), demonstrating that expression of the chimeric receptors was limited to T lineage lymphoid cells. Both transgenic chimeric receptors were detected on CD4⫹Foxp3⫹ thymocytes and peripheral T cells (Fig. 2D). This analysis also indicated that CD4⫹Foxp3⫹ WT cells expressed detectable levels of endogenous IL-7R␣ and IL-2R␤. IL-2R␤⫺/⫺ mice on the C57BL/6 genetic background exhibit rapid lethal autoimmunity such that these mice typically die between 8 and 12 wk of age (20, 21). Past work demonstrated that thymic-specific transgenic expression of WT IL-2R␤ in IL-2R␤deficient mice prevented this autoimmunity such that these transgenic mice live an outwardly normal disease-free life span (18). When the 7␣/2␤ transgenic chimeric receptors were expressed in IL-2R␤-deficient mice, all animals exhibited severe lethal autoimmunity. When such mice were examined early in life, these mice exhibited symptoms of this autoimmunity that were comparable to that observed in nontransgenic IL-2R␤⫺/⫺ mice, including hemolytic anemia, increased lymph nodes cellularity, and peripheral CD4 T cells with an activated phenotype as judged by expression of CD69 and a high portion of cells that were CD62Llow (Fig. 3A). In marked contrast, most 2␤/7␣ transgenic IL-2R␤⫺/⫺ mice did not exhibit obvious symptoms of autoimmunity and readily thrived. When analyzed between 6 and 39 wk of age, 10 of 14 2␤/7␣ transgenic IL-2R␤⫺/⫺ mice were disease-free, while four mice exhibited symptoms of autoimmunity. Although these latter four animals had peripheral T cells with a somewhat activated phenotype and lower hematocrit, these values were typically more pronounced and observed at an earlier age in nontransgenic IL2R␤⫺/⫺ or 7␣/2␤ transgenic IL-2R␤⫺/⫺ mice. Therefore, it is likely that IL-2R␤ signaling optimally reconstituted functional Treg cells in IL-2R␤⫺/⫺ mice. However, the 2␤/7␣ receptor was remarkably efficacious when compared with the 7␣ and the 7␣/2␤ transgenes, either of which did not affect the course of this autoimmune syndrome. Thus, there is sufficient redundancy between IL-2R and IL-7R signaling to support Treg production, without ruling out a potential role for specific IL-2R signals for optimal production. Because the decrease in Treg cells in IL-2R␤⫺/⫺ mice is most striking in peripheral lymphoid tissue (2), lymph nodes of control and transgenic IL-2R␤⫺/⫺ mice were examined for CD4⫹CD25⫹ T cells by FACS analysis. The 7␣/2␤ transgenic IL-2R␤⫺/⫺ mice resembled IL-2R␤-deficient mice in that their lymph nodes contained a low percentage of CD4⫹ T cells that expressed CD25 and these cells expressed CD25 at a relatively low level as measured by the mean fluorescence intensity (MFI) (Fig. 3B). The lymph nodes of 2␤/7␣ transgenic IL-2R␤⫺/⫺ mice, however, contained a higher proportion of CD4⫹ T cells that expressed CD25 and these cells expressed CD25 at a relatively high level, when compared with cells from IL-2R␤⫺/⫺ mice. These values, moreover, approached that observed when examining CD4⫹CD25⫹ T cells from the lymph nodes of WT mice. Purified CD4⫹CD25⫹ T cells from 2␤/7␣ transgenic IL-2R␤⫺/⫺ mice suppressed anti-CD3-induced T cell proliferation to a level that was almost comparable to that mediated by WT CD4⫹CD25⫹ Treg cells (Fig. 3C). Collectively, these data indicate that signaling through the cytoplasmic tail of the IL-7R ␣-chain is competent to prevent autoimmunity and produce CD4⫹CD25high

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IL-2 AND Treg CELL PRODUCTION

FIGURE 3. Activity of chimeric IL-2R␤ and IL-7R␣ in IL-2R␤⫺/⫺ mice. A, Effect on autoimmunity as assessed by determining the occurrence of hemolytic anemic, lymphoproliferation as indicated by increased lymph node (LN) cellularity, and the presence of autoreactive T cells as indicated by CD4⫹ T cells with an activated CD69⫹ or CD62Llow phenotype. Data represent the mean ⫾ SD of 4 –14 mice per group. B, Capacity to reconstitute Treg cells as assessed by the presence of CD4⫹CD25high T cells as determined by the percentage (%) of CD4 cells that expressed CD25 and by the MFI of CD25 on CD4⫹CD25⫹ T cells. Data represent the mean ⫾ SD of 4 –14 mice per group. ⴱ, p ⱕ 0.004, with t test in comparison to IL-2R␤⫺/⫺ mice. Not significant (NS) with t test in comparison to IL-2R␤⫺/⫺ mice. C, Evaluation of the suppressive activity in vitro of purified CD4⫹CD25⫹ T cells from transgenic 2␤/7␣ IL2R␤⫺/⫺ mice. Data are derived from five independent experiments. The IL-2R␤⫺/⫺ and the 7␣/2␤ IL-2R␤⫺/⫺ mice were analyzed at 4 –7 wk of age, whereas WT and 2␤/7␣ IL-2R␤⫺/⫺ mice were analyzed between 6 and 39 wk of age. There were no age-related differences within these two groups of mice.

Treg cells in IL-2R␤-deficient mice provided that it was triggered by IL-2 in the context of the 2␤/7␣ receptor. Functional activity of the chimeric 7␣/2␤ An important consideration when evaluating the inability of the 7␣/2␤ transgenic receptor to affect autoimmunity in IL-2R␤⫺/⫺ mice was to verify the biological activity of this receptor. To that end, we first examined the activity of the 7␣/2␤ transgenic receptor when expressed in IL-7R␣-deficient mice. This chimeric receptor enhanced thymic development (Fig. 4A) leading to an increased number of CD4⫹ and CD8⫹ T cells in the periphery (Fig. 4B). This level of T cell reconstitution was similar to that seen when the 7␣/2␤ chimeric receptor was expressed in IL-7R␣⫹/⫺ mice (Fig. 4, A and B). The activity of the 7␣/2␤ chimeric receptor was lower than we have previously observed when the WT 7␣ transgene was

expressed in IL-7R␣⫺/⫺ mice and the WT 7␣ transgene did not affect thymic development when expressed in IL-7R␣⫹/⫹ mice (19). The partial reconstitution of T cell development by the 7␣/2␤ chimeric receptor might reflect decreased availability of IL-7 for DN thymocytes, as others have shown that constitutive transgenic expression of the IL-7R ␣-chain by most thymocytes use much of the IL-7 required by pro-T cells or pre-T cells thereby reducing thymopoiesis (22) or that IL-7 induced an IL-2R signal. Nevertheless, these data show that the 7␣/2␤ transgenic chimeric receptor is active in vivo and demonstrate that there is not strict requirement for unique IL-7R signaling for T cell development. To further examine the quality of the signal induced by 7␣/2␤ receptor, we compared proliferative responses by splenic antiCD3-induced T cell blasts to IL-2 and IL-7 (Fig. 4C). WT T cell blasts readily proliferate when recultured with ␥c-dependent

FIGURE 4. Functional activity of the chimeric 7␣/2␤ receptor. Total thymic number (A) and splenic T cell cellularity (B) after expression of the 7␣/2␤ receptor in IL-7R␣⫺/⫺ mice. Data represent the mean ⫾ SD of six to nine mice per group. All mice were analyzed between 7 and 10 wk of age. ⴱ, p ⱕ 0.003 and ⴱⴱ, p ⱕ 0.0001, from t test in comparison to IL-7R␣⫺/⫺ mice. C, Proliferation by T cell blasts from the indicated mice previously activated with anti-CD3 to IL-2 (50 U/ml), IL-4 (10 ng/ml), or IL-7 (10 ng/ml). Data are the mean ⫾ SD of four mice per group, except data in nontransgenic IL-7R␣⫺/⫺ mice (n ⫽ 2) ⫾ range.

The Journal of Immunology

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cytokines such that the strongest responses were stimulated by IL-2, followed by IL-4 and then IL-7. The few T cells from IL7R␣⫺/⫺ mice yielded T cell blasts that proliferated in an analogous, but reduced fashion, to IL-2 and IL-4, but not IL-7. Importantly, T cell blasts from IL-7R⫹/⫹ or IL-7R⫺/⫺ that expressed the 7␣/2␤ receptor proliferated to the same extent to both IL-2 and IL-7 and these responses were comparable to those induced by IL-2 for WT T cells. Furthermore, dose-response curves to IL-2 and IL-7 for 7␣/2␤ receptor-transfected CTLL cells, which express endogenous IL-2R but not IL-7R, were identical (Fig. 5). These experiments indicate that the 7␣/2␤ chimeric receptor induces a potent IL-2R-like signal, and therefore has the potential to support Treg cell production upon triggering by IL-7. Because IL-2-driven proliferation requires cooperative signaling that depends on STAT5 and Shc activation of MAPK and PI3K (17, 23), these data provide additional support that the signaling through the chimeric IL-2R␤ cytoplasmic domain functions normally. Chimeric and endogenous IL-7R signaling regulates CD4⫹CD25⫹ Foxp3⫹ Treg cells IL-2R␤⫺/⫺ mice contain greatly reduced numbers of CD4⫹CD25⫹ Treg cells, and this defect is corrected upon expression of WT IL-2R␤ within the thymus of IL-2R␤⫺/⫺ mice (2). However, as Foxp3 is a more specific marker of Treg cells (24), we compared the activity of transgenic IL-2R␤ (2␤) and the chimeric 2␤/7␣ receptors for the production of thymic and peripheral Foxp3⫹ Treg cells by FACS analysis. As previously reported (25), IL-2R␤⫺/⫺ mice contained a reduced fraction of CD4⫹ Foxp3⫹ T cells in both the thymus and peripheral lymph nodes (Fig. 6A) that largely do not express CD25 (Fig. 6B). Expression of transgenic WT 2␤ or chimeric 2␤/7␣ in IL-2R␤⫺/⫺ mice increased the number of thymic and peripheral CD4⫹ Foxp3⫹ T cells (Fig. 6A) and resulted in the majority of the Foxp3⫹ cells to expressed CD25 (Fig. 6B). However, the overall regulation of CD25 was somewhat less efficient for IL-2R␤-deficient mice that expressed the chimeric 2␤/7␣ receptor. Importantly, the level of intracellular Foxp3 for Treg cells from 2␤ IL-2R␤⫺/⫺ mice was comparable to Treg cells from WT mice as judged by the MFI, and these levels were on average 1.6- and 1.8-fold higher (n ⫽ 8, p ⬍ 0.02, t test) than that for the Foxp3⫹ cells from the thymus or lymph nodes of IL2R␤⫺/⫺ mice, respectively (Fig. 6A). In three of four experiments, the Foxp3⫹ cells from or 2␤/7␣ mice also showed a similar trend toward higher (on average 1.8-fold) Foxp3 levels. Some thymic Treg cells from WT and IL-2R␤⫺/⫺ mice express the IL-7R (Fig. 2D and data not shown). Therefore, we cultured thymocytes from IL-2R␤⫺/⫺ mice to test whether the endogenous IL-7R was biologically active in Treg cells (Fig. 7). When CD4⫹ Foxp3⫹ thymocytes were examined 24 h later after culture with

FIGURE 5. Dose-response characteristics of the chimeric 7␣/2␤ receptor. CTLL cells, which express endogenous IL-2R but not IL-7R, were transfected to express the 7␣/2␤ chimeric receptor. These cells were cultured with increasing amounts of mouse IL-2 or human IL-7 for 24 h. [3H]Thymidine was added during the last 4 h of culture.

FIGURE 6. Comparison of transgenic WT 2␤ and chimeric 2␤/7␣ receptors to support Treg cell production in IL-2R␤⫺/⫺ mice. A, Enumeration of CD4⫹ Foxp3⫹ T cells in the thymus and lymph node (LN) from the indicated WT or IL-2R␤⫺/⫺ mice. Representative FACS dot plots are shown, which were used to determine the number and percentage of CD4⫹ Foxp3⫹ cells. ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⱕ 0.001, with t test in comparison to IL-2R␤⫺/⫺ mice. B, Representative histograms are shown for CD25 expression after gating on CD4⫹ Foxp3⫹ T cells, as shown in A, for the thymus and lymph nodes. The percentage of CD4⫹ T cells that are CD25⫹ are shown below. Data represent the mean ⫾ SD of 6 –11 mice per group. The IL-2R␤⫺/⫺ mice were analyzed from 1 to 3 wk of age, whereas all other mice were examined from 2 to 8 wk of age. There were no agerelated differences within each group of mice.

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FIGURE 7. Activity of the endogenous IL-7R on Treg cells. Thymocytes from IL-2R␤⫺/⫺ mice (⬍3-wk-old) were cultured with medium alone or IL-2 (control) or with IL-7 overnight, and the expression of Foxp3 and CD25 was determined for CD4⫹ thymocytes. As a reference, the expression of these markers is shown for the input cells. Representative FACS dot plots are shown, which were used to determine the MFI for Foxp3 and the percentage (%) of Foxp3⫹ cells that expressed CD25 (n ⫽ 3). ⴱ, p ⬍ 0.002, with t test in comparison to either the input or control cultured cells.

IL-7, CD25 expression was induced and Foxp3 levels increased ⬃1.5-fold, as judged by the MFI when compared with the input cells. These phenotypic changes mirrored the in vivo consequences of transgenic expression of IL-2R␤ or 2␤/7␣ expression within the thymus of IL-2R␤⫺/⫺ mice (Fig. 6, A and B). Thus, IL-2R␤⫺/⫺ CD4⫹ Foxp3⫹ thymocytes have the potential to respond to IL-7.

Discussion Recent data demonstrate that a critical nonredundant role for IL-2 is in the production and maintenance of CD4⫹CD25⫹ Foxp3⫹ Treg cells (2, 3, 25–34). This study clarifies that one key factor underlying this nonredundant behavior of IL-2 is selective compartmentalization of IL-2 in vivo. Given the protective activity of the 2␤/7␣ transgene, the 7␣ and the 7␣/2␤ transgenes or the endogenous IL-7R present on CD4⫹ Foxp3⫹ cells should have also functioned to up-regulate CD25 and Foxp3 and prevent autoimmunity in IL-2R␤⫺/⫺ mice, if IL-7 was available to trigger these receptors. Because the 7␣/2␤ mice were developed before the availability of the Foxp3 mAb, our analysis of Treg cells was limited to CD4⫹CD25⫹ T cells. As IL-7 signaling was shown to substantially increase CD25 expression on IL-2R␤⫺/⫺ Foxp3⫹ T cells (Fig. 7), it is highly unlikely that we failed to detect Treg cells in 7␣/2␤ mice. Thus, it is the presence of IL-2 in the cellular niches in which the Treg cells reside rather than IL-2R signaling per se that is a critical determinant for the production of Treg cells. Signaling through the cytoplasmic tail of IL-2R␤ or the 2␤/7␣ receptor within the thymus functioned to increase Treg cell numbers and up-regulate CD25 and Foxp3. Although we have not established the nature of this signaling, one candidate is activation of STAT5 as this represents one consequence of both IL-2R and IL-7R signaling and this transcription factor is required for optimal production of Treg cells (35, 36). The activity of these transgenic receptors was also sufficient for CD4⫹CD25⫹ Foxp3⫹ Treg cells to expand and persist in the peripheral immune compartment.

IL-2 AND Treg CELL PRODUCTION Our study indicates that an important issue for understanding Treg cell immunobiology will be to define the in vivo context in which IL-2-producing cells reside in relationship to Treg cells. As IL-2 has a very short half-life in vivo, it is likely that both the IL-2-producing cells and Treg cells are in close proximity. Thus far, detection of IL-2-secreting cells in vivo has been problematic because IL-2 is transiently produced and exists primarily as a secreted product, so the relevant cells are difficult to identify. There is some evidence, however, that IL-2-producing cells are present at a low frequency in both the thymus and peripheral immune compartment (37, 38). Within the T cell compartment IL-2 mRNA was most readily detected on nonregulatory CD4⫹CD25low cells, which suggests that recently activated T cells may be the source of IL-2 for Treg cells (28). It is intriguing to speculate that autoreactive T cells might provide IL-2 for Treg cells, establishing a regulatory loop between these two cell populations. However, dendritic cells and NKT cells, along with Ag-activated T cells, have also been shown to produce IL-2 in vivo (28, 39 – 41), and remain candidates for IL-2-producing cells for Treg cells in vivo. IL-2 has been shown to be important during Treg cell development within the thymus and for Treg cell maintenance and homeostasis in the periphery. It is evident in previous work (25) and this study that the WT thymus contains ⬃2-fold greater number Foxp3⫹ T cells than found in the thymus of IL-2/IL-2R-deficient mice, suggesting a role for IL-2 in the thymus. The observation that transgenic expression of WT 2␤ or 2␤/7␣ receptors increased the number and up-regulated CD25 and Foxp3 on thymic Treg cells provides direct data that these changes depend upon IL-2induced signaling within the thymus. The detection of these cells cannot be attributed to peripheral Treg cells that simply traffic back and reside in the thymus of IL-2R␤⫺/⫺ mice because such a scenario was not observed when peripheral WT GFP⫹ Treg cells were adoptively transferred into IL-2R␤⫺/⫺ mice (33). However, the reconstitution of CD4⫹CD25⫹ Foxp3⫹ Treg cells was even greater in the lymph nodes of transgenic 2␤ or 2␤/7␣ IL-2R␤⫺/⫺ mice. Although on face value this result implies that both transgenic cytokine receptors are more active in the periphery, it is important to note that the 2␤/7␣ chimeric receptor, but not the 2␤ transgene (2), is readily detected on mature peripheral Treg cells. Importantly, previous work showed that peripheral Treg cells from adult autoimmune-free 2␤ transgenic IL-2R␤⫺/⫺ mice do not express a functional IL-2R because these cells do not undergo IL-2dependent expansion or prevent autoimmunity upon transfer into newborn IL-2R␤⫺/⫺ mice (2). This finding suggests that either there is an IL-2-independent pathway for peripheral Treg cell homeostasis or, upon exit from the thymus, 2␤ transgenic Treg cells remain competent to receive a critical IL-2 signal to promote their expansion, perhaps in a manner analogous to that reported for neonatal Treg cell (33), before down-regulation of IL-2R␤. Experiments are currently in progress to distinguish between these possibilities.

Acknowledgments We thank Danny Barzana, Ben Boyter, and Lin Zhu for technical assistance and Allison Bayer for critically reading this manuscript.

Disclosures The authors have no financial conflict of interest.

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