The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bayer, A. L.
Right arrow Articles by Malek, T. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bayer, A. L.
Right arrow Articles by Malek, T. R.
The Journal of Immunology, 2007, 178: 4062-4071.
Copyright © 2007 by The American Association of Immunologists, Inc.

Function of the IL-2R for Thymic and Peripheral CD4+CD25+ Foxp3+ T Regulatory Cells1

Allison L. Bayer, Aixin Yu and Thomas R. Malek2

Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL 33136


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-2 contributes to the production, function, and homeostasis of CD4+CD25+ Treg cells. However, it remains uncertain whether IL-2 is essential for the development of Treg cells in the thymus, their homeostasis in the periphery, or both. The present study was undertaken to investigate the contribution of IL-2 during thymic Treg cell development and its maintenance in peripheral immune tissue. Relying on genetic mouse models where IL-2R signaling was either completely blocked or selectively inhibited in peripheral CD4+CD25+ Treg cells, we show that the IL-2/IL-2R interaction is active in the thymus at the earliest stage of the development of Treg cells to promote their expansion and to up-regulate Foxp3 and CD25 to normal levels. Furthermore, CD4+CD25+Foxp3+ Treg cells with impaired IL-2-induced signaling persist in the periphery and control autoimmunity without constant thymic output. These peripheral Treg cells with poor responsiveness to IL-2 exhibited slower growth and extended survival in vivo, somewhat lower suppressive activity, and poor IL-2-dependent survival in vitro. Mixed thymic and bone marrow chimeric mice showed that wild-type-derived Treg cells were substantially more effective in populating peripheral immune tissue than Treg cells with impaired IL-2 signaling. Collectively, these data support the notion that normally IL-2 is a dominant mechanism controlling the number of thymic and peripheral Treg cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Normally, CD4+CD25+ T regulatory (Treg)3 cells function to suppress autoreactive T cells that escape thymic negative selection (1, 2, 3, 4). These cells also suppress antitumor immunity, regulate immune responses to certain infectious diseases, and can be induced to inhibit transplant rejection reactions. Treg cells are characterized by the selective expression of the transcription factor Forkhead box P3 transcription factor (Foxp3) (5, 6, 7) and bear the high affinity IL-2R comprised of CD25 (IL-2R{alpha}), CD122 (IL-2Rbeta), and CD132 ({gamma}c) (8, 9, 10). The IL-2/IL-2R interaction has been ascribed to be an important step in the production and maintenance of Treg cells. Mice deficient in IL-2, IL-2R{alpha}, or IL-2Rbeta exhibit a rapid, lethal autoimmunity remarkably similar to that of mice lacking functional Foxp3, and all of these animals contain severely reduced numbers of CD4+CD25+ Treg cells (5, 6, 7, 11, 12). Correcting IL-2 production or responsiveness in the context of IL-2/IL-2R deficiency restored Treg cell production and normalized most of the abnormalities associated with this autoimmune syndrome (11, 13, 14, 15).

A critical issue raised from these studies is whether IL-2 functions as an essential cytokine for the development of Treg cells in the thymus, their homeostasis in the periphery, or both. Primarily, two observations support a role for IL-2 during thymic development. First, thymic-specific expression of IL-2Rbeta in IL-2Rbeta–/– mice (referred to as Tg–/– in this report) increased the number of thymic Treg cells, leading to a normal compartment of peripheral Treg cells that prevented lethal autoimmunity (11). Second, an anti-IL-2 blockade decreased the production of Treg cells within the thymus (16).

Other studies clearly indicate that IL-2 functions to control the number of peripheral Treg cells. In this regard, anti-IL-2 was especially effective in blocking Treg cell expansion within neonatal lymph nodes (LN) (16) and caused autoimmune gastritis in BALB/c mice and a rapid onset of autoimmunity in NOD mice that was accompanied by decreased numbers of Treg cells (17). Furthermore, the adoptive transfer of CD4+CD25+ Treg cells into IL-2Rbeta–/– mice led to an engraftment of donor Treg cells in peripheral immune tissue that fully prevented lethal autoimmunity, and this depended upon donor cells expressing a functional high affinity IL-2R and the host producing IL-2 (11). In many other studies, the conclusion that IL-2 is essential for peripheral Treg cells relied on the behavior of Treg cells in mixed bone marrow or T cell chimeric mice using donor cells from IL-2-, IL-2R{alpha}-, or IL-2Rbeta-deficient mice (13, 14, 15). For some experiments, however, it was not possible to discern whether the reconstitution of Treg cells was due to their production from precursor cells or their expansion from a small pool of preexisting mature Treg cells. In other studies, even though mature Treg cells were transferred, it was not apparent whether IL-2 simply increased their number or also enhanced their function. Nevertheless, all of these data together with the observation that the short-term treatment of adult mice with anti-IL-2 decreased Treg cells in peripheral immune tissue (18) support the idea that IL-2 is an important cytokine for the initial production and subsequent maintenance of Treg cells in the periphery.

Two recent studies have shown that there are polyclonal or TCR-transgenic CD4+ T cells that express Foxp3 in IL-2- or IL-2R{alpha}-deficient mice and that such cells were more numerous in the thymus than in peripheral immune tissues (19, 20). These data support the notion that commitment to Foxp3+ Treg cell lineage is IL-2 independent and have led to the conclusion that the main nonredundant role of IL-2 lies in peripheral Treg cell expansion and survival rather than in thymic Treg cell production. Nevertheless, Foxp3+ thymocytes may still depend upon IL-2 because the polyclonal thymic IL-2–/– Foxp3+ T cells were at a 2-fold reduced number and expressed a lower level of Foxp3 and CD25 when compared with wild-type (WT) littermate control mice (20). These data, the effectiveness of thymic-driven IL-2Rbeta to support Treg cell production and prevent autoimmunity in IL-2Rbeta–/– mice, and the presence of a substantial population Treg cells in the periphery of Tg–/– mice raise unresolved questions concerning IL-2 in Treg cell production. With these issues in mind, the present study was undertaken to investigate the contribution of IL-2 during thymic Treg cell development and maintenance in peripheral immune tissue. Our data indicate that the IL-2/IL-2R interaction is active and essential in the thymus to promote Treg cells expansion and regulate the expression of Foxp3 and CD25. Furthermore, although Treg cells are maintained in the periphery with greatly impaired IL-2R signaling, IL-2 remains the dominant mechanism controlling the number of peripheral Treg cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 and Thy1.1 mice were obtained from The Jackson Laboratory. Congenic B6.SJL-Ptprca Pepcb (CD45.1+) C57BL/6 mice were bred and maintained in the animal facility at the University of Miami (Miami, FL). IL-2Rbeta–/– mice, backcrossed for at least 12 generations to C57BL/6 mice, have been previously described (21). These mice were maintained by using breeding pairs that were homozygous IL-2Rbeta–/– mice rendered autoimmune free by neonatal adoptive transfer of purified CD4+ or CD4+CD25+ Treg cells (11). The thymic targeted transgenic wild-type (WT) IL-2Rbeta expressed in IL-2Rbeta–/– mice on the C57BL/6 genetic background (designated Tg–/– in this study) has been previously described (22).

Cell purification

CD4+CD25+ Treg cells were purified from the spleen first by the depletion of CD8+ T cells and B cells followed by the positive selection of CD25+ cells as previously described (11). Single positive (SP) CD4 thymocytes were prepared by first depleting CD8+ cells with negative selection using anti-CD8 MACS microbeads (Miltenyi Biotec) followed by FACS sorting of CD4+ and CD8– thymocytes using the BD Biosciences FACSAria sorter and Diva software. The purity of Treg cells and the SP thymocytes was >90 and >99%, respectively. SP thymocytes were labeled with CFSE as previously described (16). T cell-depleted bone marrow was prepared by cells from femurs and tibias, and the T cells were removed by incubation with the Thy-1.2 mAb and the Low-Tox-M rabbit complement (Accurate Chemical & Scientific) for 45 min at 37°C. Cells were then washed one time with Complete medium and twice with HBSS.

Experimental animals

Thymectomies were performed on anesthetized adult mice by opening the chest and removing the thymus with suction. At sacrifice, the thorax was examined and partially thymectomized mice were excluded from analysis. BrdU incorporation in vivo was accomplished by giving mice BrdU in their drinking water (0.8 mg/ml) for up to 10 days. Adoptive transfer into 1–2 day old IL-2Rbeta–/– neonates was performed by i.v. injection with the indicated number of cells in 50 µl of HBSS. Bone marrow chimeras were generated by single 9.0-gray (Gy) or split-dose 11.0-Gy (5.5 Gy in the afternoon and 5.5 Gy the following morning) total body irradiation of Thy1.1 recipients and 24 h later infusing i.v. a 1:1 ratio (5.0 x 106 total cells) of T cell-depleted bone marrow from congenic CD45.1+ C57BL/6 and Tg–/– mice. Chimeras were maintained on acidified/antibiotic water (pH 2.2; 100 mg/L neomycin sulfate and 10 mg/L polymyxin B sulfate).

Abs and FACS analysis

Purified anti-lymphotoxin-beta (LTbeta), 7-aminoactinomycin D (7-AAD), biotin-conjugated mAbs to CD69 and CD103, Cy-Chrome-conjugated mAb to CD4, PE-conjugated mAbs to CD45RB, CD25 (PC61), and Thy1.2, PE-streptavidin, PerCP-conjugated mAbs to CD4 and CD8, PerCP-streptavidin, allophycocyanin-conjugated mAb to CD4 and Thy1.2, PE-Cy7 conjugated mAb to CD25 (PC61), allophycocyanin-streptavidin, Alexa 647-phospho-STAT5a, and FITC-conjugated mAb to BrdU were purchased from BD Biosciences. Biotin-conjugated Ab to glucocorticoid-induced TNFR (GITR) was purchased from R&D Systems. FITC-anti-CD4 (clone GK1.5), FITC-anti-CD45.1, and biotin-conjugated mAbs to CD62L (clone Mel14) and CD25 (clone 7D4) were prepared in our laboratory. Foxp3 staining was performed according to manufacturer’s instructions (eBioscience). FACS analysis of LTbeta was revealed by three-step staining consisting of sequential incubations with anti-LTbeta, biotin-anti-hamster IgG, and Cy-Chrome-streptavidin. For phospho-STAT5 staining, spleen cells were cultured at 37°C in medium for 30 min, incubated with IL-2 (10 ng/ml) at 37°C for the indicated time, and fixed with paraformaldehyde (1.5% final concentration) at 37°C for 10 min. After centrifugation the cells were permeabilized in 100% methanol and maintained on ice for 30 min, after which they were washed twice in PBS containing 0.5% BSA and 0.02% NaN3 and stained for phospho-STAT5a and the appropriated surface markers. For an analysis of BrdU incorporation, cells were incubated with BD Cytofix/Cytoperm buffer (BD Biosciences) for 20 min at 4°C and washed with Dulbecco’s PBS (2.7 mM KCl, 0.370 mM KH2PO4, 1.4 M NaCl, and 0.800 mM Na2HPO4) containing 3% FBS, 0.1% saponin, and 0.09% NaN3 (wash buffer) followed by incubation for 10 min at 4°C with Cytoperm Plus buffer (BD Biosciences) with one wash. Cells were incubated again with Cytofix/Cytoperm buffer for 5 min at 4°C, washed once, and then incubated with FITC-conjugated anti-BrdU for 20 min at room temperature followed by one wash. FACS analysis was performed as previously described (16) using a BD Biosciences LSR1 cytometer and CellQuest software or a BD Biosciences LSRII cytometer and Diva software.

In vitro Treg cell culture

The suppressor activity of purified CD4+CD25+ Treg cells was previously described (11). Briefly, Treg cells (0.3–5 x 104) were cultured with CD4+CD25– T cells (5 x 104) and mitomycin-treated, T cell-depleted splenic cells (5 x 104) (23) in a 96-well round-bottom microtiter plate with anti-CD3 (0.1–0.25 µg/ml) for 72 h. [3H]Thymidine was added during the last 4–6 h of culture. For cell survival, purified CD4+CD25+ Treg cells were cultured (5 x 105/well) in 48-well microculture plates with or without IL-2 (10 ng/ml) for 96 h. Dead cells were enumerated by 7-AAD staining. LTbeta and CD25 expression was assessed at various times in parallel or separate experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-2/IL-2R interaction is active in the thymus

In mice Foxp3 is the most specific known marker of Treg cells (5, 6, 7). Because our previous characterization of the activity of thymic IL-2Rbeta on Treg cell production relied upon the analysis of CD4 cells that coexpressed CD25 (11), the thymus and periphery of C57BL/6 (WT) and Tg–/– (4- to 16-wk-old) and 1- to 3-wk-old IL-2Rbeta-deficient mice were re-evaluated for the presence of Foxp3+ T cells, as these latter animals do not yet exhibit severe symptoms of autoimmunity. As previously reported for IL-2- and IL-2R{alpha}-deficient mice (19, 20), Foxp3-expressing CD4+ T cells were detected in the thymus (Fig. 1, A and C) and LN of IL-2Rbeta–/– mice (Fig. 1, B and D). However, based on their relative abundance WT and Tg–/– mice contained ~2- to 3-fold more CD4+ Foxp3+ cells in the thymus and 4- to 7-fold more Foxp3+ cells in the LN than IL-2Rbeta–/– mice, and the level of Foxp3 was ~2-fold higher for the cells from WT and Tg–/– mice (Fig. 1, C and D). This greater proportion of Treg cells was not related to differences in the ages of the mice analyzed, as the proportion of SP CD4+Foxp3+ thymocytes and LN T cells was similar in 2- to 3-wk-old and adult WT and Tg–/– mice (data not shown). This lower proportion of Treg cells in IL-2Rbeta–/– mice reflected a lower total number of Treg cells for the thymus, as thymic cell recoveries were similar for all mice after 2 wk of age (mean ± SE: 114.1 ± 9.6 x 106, 119.9 ± 10.5, and 119.0 ± 14.5 for WT, Tg–/–, and IL-2Rbeta–/– respectively). However, even though the proportion of Treg cells in the IL-2Rbeta–/– LN was very low, its cellularity varied widely between individual IL-2Rbeta–/– mice, as hyperproliferation represents an early symptom of autoimmunity such that ~50% of IL-2Rbeta–/– mice contained LN with a total number of Foxp3+ cells that was comparable to that detected in LN from WT mice. This has been noted previously for IL-2- and IL-2R{alpha}-deficient mice (20).


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 1. IL-2/IL-2R interaction is active in the thymus. A and B, Thymocytes (A) or LN cells (B) from individual C57BL/6 (WT), Tg–/–, or IL-2Rbeta–/– mice were subjected to FACS analysis for the indicated markers after gating on CD4+Foxp3+ T cells. Shown are representative FACS histograms for the indicated surface marker in the thymus and LN. C and D, Number of Foxp3+ cells (left panels) and MFI values (right panels) in the thymus (C) and LN (D). Data are means ± SEM for 6–10 mice per group.

 
The phenotype of the CD4+ Foxp3+ cells in the thymus (Fig. 1A) and LN (Fig. 1C) was largely similar with respect to CD69, CD62L, and CD24 (heat-stable Ag (HSA)), although there appeared to be a slight increase in the fraction of CD69+ Treg cells from IL-2Rbeta-deficient mice. Notably, most CD4+ Foxp3+ cells from the thymus and periphery of WT and Tg–/– mice expressed relatively high levels of CD25 (IL-2R{alpha}), whereas those cells were largely CD25neg in IL-2Rbeta–/– mice. Even though Treg cell number and expression of CD25 and Foxp3 were very similar in WT and Tg–/– mice, one important difference was that essentially all Foxp3+ Treg cells in WT mice expressed IL-2Rbeta (CD122), whereas CD122 was readily detected on only a small subset of thymic Tg–/– Foxp3+ cells.

Thymic CD4+ Foxp3+ cells can be divided into two subsets based on CD4 and CD8 expression (24). We found for all three mouse strains that ~25% of these Foxp3+ cells were CD4+CD8+ double positive (DP) thymocytes that expressed CD24 while the remaining cells were CD4+CD8– SP thymocytes (Fig. 2, A and B) that largely lacked CD24. These data are consistent with the notion that the DP Foxp3+ thymocytes are precursors to the CD4 SP cells, because CD24 (HSA) is readily detected on immature developing thymocytes but not on mature SP cells. For the Tg–/– Foxp3+ thymocytes a normal level of CD122 was largely contained to the most immature DP CD24+ cells. In fact, almost all of the mature Tg–/– CD4 SP Foxp3+ thymocytes expressed an essentially undetectable level of CD122, indicating that it is highly unlikely that these Treg cells express a functional IL-2R as they exit the thymus to seed peripheral immune tissue. By comparison with thymocytes from IL-2Rbeta-deficient mice, transgenic expression of CD122 in only the most immature Foxp3+ thymocytes of IL-2Rbeta–/– mice resulted in increasing the number of Foxp3+ cells (Fig. 2B) as well as the up-regulation of Foxp3 (Fig. 2C) and CD25 (Fig. 2D) in both the DP and SP Treg subsets to levels equivalent to that found in WT mice. Although we noted some day-to-day variation in the mean fluorescence intensity (MFI) for Foxp3 (Fig. 2C), when we considered the increase in Foxp3 levels for SP CD4+ Foxp3+ cells in samples compared on the same day with those of Foxp3+ cells from the IL-2Rbeta–/– thymus, there was a significant (p < 0.05) 1.89 ± 0.19 and 1.86 ± 0.22-fold higher level of Foxp3, respectively, for cells from the WT thymus and the Tg–/– thymus using a repeated measures one-way ANOVA followed by Tukey’s multiple comparison test. Thus, the expression of a functional high-affinity IL-2R at a very early stage of Treg cell development by itself appears to be sufficient for the normal production of Treg cells.


Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 2. Thymocytes from individual C57BL/6 (WT), Tg–/–, or IL-2Rbeta–/– adult mice were subjected to FACS analysis after gating on CD4+CD8+ or CD4+CD8– Foxp3+ cells. Shown are representative FACS histograms for the indicated surface marker (A), the number of Foxp3+ cells per one million thymocytes (B), MFI values for Foxp3 expression (C), and the percentage of Foxp3+ thymocytes for CD25 expression (D). Data in B and D are means ± SEM for 6–10 mice per group and the data in C are for three mice per group.

 
Constant thymic output does not account for IL-2R-independent peripheral Treg cell homeostasis

Peripheral Foxp3+ Treg cells in adult Tg–/– mice lack readily detectable expression of CD122 (Fig. 1C) and do not undergo IL-2-dependent expansion upon adoptive transfer to IL-2Rbeta–/– mice (11, 23). Nevertheless, adult Tg–/– mice contain a normal number of peripheral Treg cells that effectively control autoimmunity. Thus, once there is IL-2-dependent thymic Treg cell production in Tg–/– mice, it appears that IL-2 is not required for the peripheral homeostasis of Treg cells. Alternatively, constant thymic output might compensate for an apparent lack of IL-2 signaling by peripheral Tg–/– Treg cells. To assess this possibility, we enumerated the persistence of CD4+CD25high T cells, of which the majority are Foxp3+ (Fig. 3A), in the LN of normal and Tg–/– mice following adult thymectomy. From 2- to 8 wk post-thymectomy no obvious reduction was noted in the fraction of CD4+CD25high T cells from both groups of mice (Fig. 3B). Furthermore, the thymectomized Tg–/– mice lacked the early symptoms of autoimmune disease associated with IL-2 or IL-2R-deficiency such as lymphoadenopathy and an activated phenotype (Fig. 3, C and D). This finding indicates that the persistence of Treg cells in the periphery of Tg–/– mice is not due to constant seeding by recent thymic emigrants. The level of BrdU incorporation over 4 days by peripheral WT Treg cells was similar for sham-treated and thymectomized mice (not shown). This finding suggests that Treg cells did not undergo markedly increased proliferation to account for their persistence after adult thymectomy and that the BrdU labeling largely reflected a proliferation of Treg cells that was not substantially affected by recent thymic emigrants. Thus, Treg cells can survive long term in the periphery of adult mice without normal expression of a functional IL-2R and without a need for replacement by newly produced cells.


Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 3. Persistence of Treg cells in the periphery of adult mice is not due constant thymic output. A, Representative FACS dot plot for CD4 and CD25 expression with a corresponding histogram for Foxp3 expression from gated CD4+CD25high population in LN cells from C57BL/6 (WT) and Tg–/– mice. B–D, WT or Tg–/– mice (8–10 wk of age) were subject to adult thymectomy. At the indicated time post-thymectomy the LN were analyzed for the presence of CD4+CD25high T cells (B), their overall cellularity (C), and the percentage of activated CD69+ CD4+ T cells (D). Unmanipulated WT and Tg–/– adult mice served as controls. Data are means ± SEM for 2–4 animals per group per day.

 
IL-2R signaling by peripheral Tg–/– Treg cells

Careful analysis of FACS histograms for IL-2Rbeta expression by LN Foxp3+ T cells from Tg–/– mice suggests a possible very low expression on peripheral Treg cells, as the staining of IL-2Rbeta was slightly higher than that found for Foxp3+ T cells from IL-2Rbeta-deficient mice (Fig. 1B). To further explore the possible significance of this expression, IL-2-induced STAT5 activation was tested for WT and Tg–/– CD4+CD25+ T cells by FACS analysis using a mAb specific for phosphotyrosine-STAT5. We first established that this method was suitable for this analysis because, when conventional activated T cells were stimulated with IL-2, staining by this Ab or Western blot analysis each yielded similar results for the induction of phosphotyrosine-STAT5 (data not shown). Rapid and sustained IL-2-dependent induction of phosphotyrosine-STAT5 was observed for WT CD4+CD25high cells, whereas low and transient phosphotyrosine-STAT5 induction was seen for CD4+CD25high cells from Tg–/– mice (Fig. 4A). The IL-2 dependency of this induction was shown by the lack of phosphotyrosine-STAT5 staining for cells cultured in the absence of IL-2 (Fig. 4A) and by the capacity of anti-IL-2 to inhibit this induction (data not shown). Thus, the very low expression of IL-2Rbeta on Tg–/– Treg cells induced low but detectable IL-2-dependent signaling when compared with that of WT Treg cells.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 4. IL-2R signaling by Tg–/– peripheral Treg cells. Spleen cells (A) or purified CD4+CD25+ T cells (B–D) from WT and Tg–/– mice were cultured in medium or IL-2 (10 ng/ml) for the indicated time. A, IL-2 induction of phosphotyrosine STAT5 (pSTAT5) after gating on CD4+CD25high T cells where the numbers in the histograms represent the percentages of positive cells and are representative of four mice. B, Viability of CD4+CD25+Foxp3+ cells by staining with 7-AAD after culture for 96 h. C and D, Expression of CD25 at 96 h (C) and LTbeta at 24 h (D) by viable 7-AAD– cells. Data in B and C are means ± SEM of 3–4 mice per group and data in D are representative of three mice/group.

 
Past studies have demonstrated that Tg–/– Treg cells do not undergo IL-2-dependent expansion in vivo upon adoptive transfer to IL-2-sufficient neonatal mice (11). Thus, the weak IL-2R signal in Tg–/– Treg cells is not sufficient to support IL-2-driven Treg cell growth in vivo. Furthermore, IL-2 promoted the survival of WT but not Tg–/– Treg cells (Fig. 4B) when analyzed in vitro according to previous approach used by Bensinger et al. (25). When the viable cells were examined, IL-2- and STAT5-dependent up-regulation of CD25 only occurred for the WT cells (Fig. 4C). Identical results for the up-regulation CD25 were noted in other experiments when CD25 was examined 24 and 48 after culture initiation (data not shown). Because IL-2 has been shown to induce the expression of LTbeta on T cells, which also depends upon the activation of STAT5 (26, 27), we compared the ability of IL-2 to induce LTbeta on purified WT and Tg–/– Treg cells. Cell surface LTbeta was induced on WT but not Tg–/– Treg cells by IL-2 over a range of IL-2 concentrations (Fig. 4D). Although the functional significance of the induction of LTbeta on WT Treg cells by IL-2 remains to be elucidated, these data and past work demonstrate that Tg–/– Treg cells are unable to mediate important down-stream responses dependent upon IL-2 signaling.

The relative effectiveness of peripheral Tg–/– Treg cell homeostasis

The long-term maintenance of peripheral CD4+CD25+Foxp3+ Treg cells in Tg–/– mice raised the possibility that minimal transient IL-2R signaling and/or an IL-2-independent pathway is the dominant mechanism for the homeostasis of peripheral Treg cells in normal mice. To assess this possibility, two distinct experimental approaches were used that compared the persistence of mixtures of WT and Tg–/– Treg cells in immune tissues. In one line of investigation, a 1:1 mixture of CFSE-labeled CD4 SP thymocytes from congenic CD45.1+ WT and CD45.2 Tg–/– mice was adoptively transferred into 2-day-old syngeneic IL-2Rbeta–/– mice. The population of Foxp3+ cells in the donor cells was determined before transfer (Fig. 5A) and was shown to contain almost 2-fold more Tg–/– Foxp3-expressing CD4 thymocytes (CD45.1–) than WT control thymocytes (CD45.1+). This skewing was anticipated, because Tg–/– mice have a higher proportion of thymic Treg cells when compared with WT mice (Fig. 2B). When recipient LN were examined 4 days after adoptive transfer, the donor Treg cells were detected based on gating on Foxp3+ cells and then by enumerating CFSE and CD25 so as not to include recipient Foxp3+ IL-2Rbeta-deficient cells, which mostly do not express CD25 (see Fig. 1B). In this short time after transfer, CD45.1 WT Treg cells now dominated such that after correcting for the input ratio, WT Treg cells outnumbered Tg–/– cells by 5.3 ± 0.8:1 (n = 3) (Fig. 5B). Based on the CFSE staining, the Tg–/– Treg cells proliferated less and expressed a lower level of Foxp3 and CD25. Similar analysis of an uninjected IL-2Rbeta neonatal mouse confirmed that there were essentially no recipient-derived CD45.2+ Foxp3+ T cells within the gate used to identify the donor cells (Fig. 5C). Thus, in this competitive setting WT Foxp3+ thymocytes dominated the engraftment of neonatal LN.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 5. Tg–/– Foxp3+ thymocytes fail to compete with WT Foxp3 thymocytes in the engraftment of neonatal LN. IL-2Rbeta–/– neonates (1- or 2-day old) were adoptively transferred with a 1:1 ratio of CFSE-labeled SP CD4 thymocytes (0.8 x 106 cells) from congenic CD45.1+ C57BL/6 and Tg–/– mice (CD45.1–) and sacrificed 4 days later for FACS analysis. A, FACS dot plot of CD4 and Foxp3 expression with a corresponding dot plot indicating input WT (CD45.1+) and Tg–/– (CD45.1–) of gated CD4+Foxp3+ thymocytes. B and C, FACS analysis of LN cells from adoptively transferred (B) and untreated (C) IL-2Rbeta–/– mice. Shown are FACS dot plot for CD45.1 and Foxp3 expression with a corresponding dot plot for CD25 and CFSE from gated CD45.1+Foxp3+ and CD45.1–Foxp3+ LN cells. Corresponding histograms indicate CFSE staining from gated CD25+CFSE+ of the CD45.1+Foxp3+or CD45.1–Foxp3+ cells. Data are representative of three IL-2Rbeta-deficient mice that received mixed thymocyte donor cells.

 
One drawback with the preceding experiment is that it is difficult to specifically follow Tg–/– cells in neonatal IL-2Rbeta–/– mice. Therefore, a second approach that we used was to generate mixed bone marrow chimeras using a 1:1 mixture of T cell-depleted bone marrow from congenic CD45.1+ WT and Tg–/– mice as the donor cells. By using lethally irradiated Thy-1.1 recipients, donor cells (Thy-1.2+) were readily distinguished from the host T cells, whereas WT- and Tg–/–-derived donor cells were distinguished from each other by use of the congenic marker CD45.1. When examined 8–9 wk after bone marrow transplantation, the thymus and LN of the recipient mice were fully reconstituted based on cellularity (data not shown) and normal percentages of double negative, DP, and SP CD4, SP CD8, and CD4 and CD8, respectively (Fig. 6A). Furthermore, the CD4 T cell compartment of spleen and LN contained a normal percentage of CD25+ or Foxp3+ cells (Fig. 6B). When the donor origin of the cells within the thymus was determined, WT (CD45.1+Thy1.2+) thymocytes slightly outnumbered Tg–/– cells (CD45.1–Thy1.2+) for all thymic subsets, including CD4+CD25+Foxp3+ cells (Fig. 6, C and D). This modest difference likely reflects the composition of the precursor pool within the donor inoculum and indicates that Tg–/– Treg cells effectively compete with WT cells during their production within the thymus. In marked contrast, there was a strikingly selective 20.9 ± 1.9-fold and 11.0 ± 0.9-fold preference for WT Treg cells in spleen and LN, respectively, while the ratio of conventional CD4+Foxp3– cells showed a slight preference for WT cells, in line with what was observed within the thymus (Fig. 6, E and F). There was also a significant fraction of peripheral CD4+Foxp3+ T cells that were both Thy-1.2– and CD45.1– (Fig. 6E). These Treg cells were recipient derived because 98% stained positively for Thy-1.1+ (not shown), suggesting that Treg cells may be somewhat radioresistant when compared with other T cells. Thus, although there were some recipient-derived Treg cells, the failure of donor-derived Tg–/– Treg cells to effectively compete with donor-derived WT Treg cells indicates that the pathway used by Tg–/– Treg cells in a noncompetitive setting is not normally a prominent mechanism for the homeostasis of peripheral Treg cells or effectively driven by weak IL-2R signals.


Figure 6
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 6. Competition between WT and Tg–/– T cells in the thymus and peripheral lymph tissue. Bone marrow chimeras were generated by infusing i.v. a T cell-depleted bone marrow into lethally irradiated recipients. Mice were sacrificed 8 wk later for FACS analysis. A, Representative dot plots for CD4 and CD8 expression in the thymus and LN. B, Percentage of CD25- or Foxp3-expressing CD4 T cells in the spleen and LN. C, Representative dot plot for CD4 and Foxp3 expression in the thymus with corresponding dot plots for the CD45.1 and Thy1.2 staining of gated CD4+Foxp3+ or CD4+Foxp3– cells. D, Ratio of WT to Tg–/– cells for the indicated thymocyte populations. E, Representative dot plot for CD4 and Foxp3 expression in the LN with corresponding dot plots for CD45.1 and Thy1.2 staining of gated CD4+Foxp3+ or CD4+Foxp3– cells. F, Ratio of WT to Tg–/– cells for the indicated populations. Data are means ± SEM for 10 individual mice.

 
Comparison of the growth and phenotypic properties of peripheral WT and Tg–/– Treg cells

The ineffective competition of Tg–/– Treg cells in the mixed thymocyte and bone marrow experiments raised the possibility that the properties of WT and Tg–/– Treg cells might not be identical. First, their proliferative properties were assessed by BrdU incorporation into cellular DNA in vivo. For this analysis, Treg cells were evaluated by enumerating BrdU incorporation into CD4+CD25high cells, as nearly all of these cells coexpress Foxp3+ (see Fig. 3A and Refs. 24 and 28). In comparison with WT Treg cells, peripheral Tg–/– CD4+CD25+ Treg cells showed a decreased fraction of cells that were BrdU+ (Fig. 7, A and B). This lower incorporation of BrdU by Tg–/– Treg cells appears to be specific for this T cell subset, because CD4+ CD25– T cells and all thymic CD4/CD8 subsets from normal and Tg–/– mice similarly incorporated BrdU (Fig. 7B and data not shown).


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 7. Homeostasis proliferation by mature WT and Tg–/– Treg cells. C57BL/6 and Tg–/– mice (8–12 wk of age) received BrdU in their drinking water (0.8 mg/ml) for 2, 4, 7, or 10 days. A, Representative FACS analysis for BrdU incorporation by CD4+ T cell subsets. B, BrdU incorporation was determined for the indicated T cell subsets by FACS. C, Drinking water contained BrdU for 10 days followed by water without BrdU for 5, 10, or 20 days. At these times BrdU incorporation into CD4+CD25+ T cells was measured. Data are means ± SEM for 3–6 animals per group per day.

 
The rate of increase in BrdU labeling was initially similar for both the control and the Tg–/– Treg cells (Fig. 7B). However, after day 7 the fraction of BrdU+ cells slightly decreased in normal C57BL/6 Treg cells while they continued to increase in Tg–/– Treg cells. This observation raised the possibility that there may be a difference in the survival of normal and Tg–/– BrdU-labeled Treg cells. To assess this possibility, we examined the rate of loss of BrdU of cells that were first labeled with BrdU for 10 days in vivo. Twenty days after BrdU uptake ceased, ~50% fewer WT Treg cells contained BrdU. Because BrdU incorporation by normal Treg cells reached a plateau at the start of chase period, the decrease of BrdU+ cells likely reflects lower survival. In marked contrast, there was essentially no decrease in BrdU-labeled Tg–/– Treg cells, indicating that all of the Treg cells initially labeled with BrdU survived throughout the entire chase period.

Even though Tg–/– Treg cells show distinct growth properties, their phenotypic characteristics are largely similar to those of WT Treg cells. Besides Foxp3 and CD25 (Fig. 1B), the expression of CD69, CD62L, and GITR by peripheral Tg–/– Treg cells was also similar to that of WT Treg cells (Fig. 8, A and B), even when analyzing the BrdU+ and BrdU– cells (Fig. 8C). One exception was CD103, which was detected on a lower fraction of Tg–/– Treg cells. Nevertheless, recently proliferating BrdU+ cells were more frequent for WT and Tg–/– Treg cells that expressed CD69 or CD103 or lacked CD62L. Thus, peripheral Tg–/– Treg cell homeostasis is maintained by relatively slow growth and an extended survival rate while still largely expressing characteristic key phenotypic markers in a normal fashion.


Figure 8
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 8. Phenotypes of peripheral WT and Tg–/– CD4+CD25+ Treg cells. LN cells from individual C57BL/6 (WT) or Tg–/– adult mice were subjected to FACS analysis for the indicated markers after gating on CD4+CD25high T cells. Shown are representative FACS histograms (A) and the percentages of Treg cells positive (B) for the indicated surface markers. Data are means ± SEM for 3–7 mice per group. C, LN cells from individual WT or Tg–/– mice were subjected to FACS analysis for the indicated markers after gating on the CD4+CD25high BrdU+ or BrdU– T cells. Data are means ± SEM for 3 or 4 mice per group.

 
Suppressor function by CD4+CD25+ Tg–/– Treg cells

Using a relatively large number of Treg cells, previous data from our laboratory demonstrated that Tg–/– Treg cells inhibited anti-CD3-induced proliferation of CD4+ T cells (11), suggesting that the Treg suppressor function does not depend upon IL-2. Recently, several studies have implied that IL-2 may play a role in generating the CD4+CD25+ Treg cell suppressor function in vitro (29, 30, 31). Based on this finding, we re-examined the capacity of purified Tg–/– CD4+CD25+ Treg cells to suppress proliferation by CD4+ T cells in vitro. For these experiments, we tested a broader ratio of CD4+ responding T cells to Treg cells and varied the stimulation by anti-CD3. When compared with normal mice, CD4+CD25+ Treg cells within the periphery of Tg–/– mice are usually represented at a somewhat higher fraction of the CD4+ T cell population (Fig. 9A) (11). When stimulated with a low concentration of anti-CD3 (0.1 µg/ml), Tg–/– Treg cells suppressed proliferation to nearly the level seen with Treg cells from normal mice (Fig. 9B). However, at a higher dose of anti-CD3 (0.25 µg/ml) the Tg–/– Treg cells were less potent suppressor cells at all ratios of responder to Treg cells (Fig. 9C). These data are consistent with Tg–/– mice containing Treg cells with somewhat lower suppressor activity, suggesting that optimal IL-2R signaling is normally required to fully maintain Treg cell suppressor function.


Figure 9
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 9. Functional activity by peripheral WT and Tg–/– CD4+CD25+ Treg cells. A, Percentage of CD25+ cells after gating on CD4+ T cells for the Treg cells used to evaluate suppressor function. The indicated numbers of purified CD4+CD25+ Treg cells from either C57BL/6 (WT) or Tg–/– mice were cultured with 0.1 (B) and 0.25 µg/ml (C) anti-CD3, purified CD4+ T cells (5 x 104), and mitomycin C-treated, T cell-depleted spleen cells (5 x 104) from WT mice for 72 h. [3H]thymidine was added during the last 4–6 h of culture. The percentage of inhibition is based the response by CD4+ T cell cultured in the absence of CD4+CD25+ Treg cells. Data are means ± SEM for 4–6 separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although there are substantial data showing that IL-2 is essential for the production of an effective population of Treg cells, the extent to which IL-2 contributes to the thymic development vs the peripheral homeostasis of these cells is somewhat controversial. Despite the detection of CD4+Foxp3+ T cells within the thymus of mice deficient in IL-2, IL-2R{alpha}, or IL-2Rbeta (Refs. 19 and 20 and this report), our findings show that the IL-2/IL-2R interaction is active within the thymus because the expression of WT IL-2Rbeta in the thymus of IL-2Rbeta–/– (Tg–/–) mice increased the number of thymic Treg cells ~2-fold, and these cells expressed heightened levels of Foxp3 and CD25. These data suggest that IL-2 functions as a growth and differentiation factor during thymic Treg cell development.

There are two major subsets of thymic Foxp3+ Treg cells. One subset comprising ~20–25% of the cells represents immature CD24 (HSA)+ CD4+CD8+ DP Treg cells. These cells likely give rise to the mature CD24 (HSA)– CD4 SP subset that represents the large majority of thymic Treg cells. However, the coincident appearance of Foxp3+ DP and CD4 SP cells in thymic ontogeny raises the possibility that DP Foxp3+ cells are not obligatory precursors to all SP thymic Treg cells (24). When IL-2Rbeta expression was evaluated for Tg–/– Foxp3+ cells, only the most immature DP subset expressed normal levels of this protein, whereas IL-2Rbeta was down-regulated to nearly undetectable levels on the mature CD4 SP cells. These data indicate that IL-2R expression at the most immature stage of Treg cell development is sufficient for driving the normal development of thymic Treg cells or that the very low expression of IL-2Rbeta on the CD4 SP cells is functionally relevant. In either case, the competitive bone marrow chimeric mice demonstrate that this level of transgenic IL-2Rbeta expression on Tg–/– thymocytes is sufficient to effectively compete with WT thymocytes. When compared with DP Foxp3+ cells in nontransgenic IL-2Rbeta–/– mice, the thymus of WT and Tg–/– mice contained twice as many DP cells that already expressed high levels of Foxp3 and CD25. Although the thymus of IL-2Rbeta-deficient mice contained a lower number of DP Foxp3low CD25– cells, these cells readily increased by ~4-fold into SP CD4 Foxp3low CD25– cells. This relative increase is the same for the Foxp3high CD25+ subsets in thymus of WT and Tg–/– mice. Thus, this phase of cell growth was not impaired and IL-2 independent in the thymus of IL-2Rbeta-deficient mice. Collectively, these findings raise the possibility that IL-2R delivers an essential signal at the DP stage that functions as an important checkpoint in Treg cell development.

Most other studies favor the view that IL-2 is critical for peripheral Treg cell homeostasis (19, 20). Several recent studies concluded that the primary role of IL-2 lies in peripheral Treg cell expansion and survival rather than in thymic production. The main conclusion for this result was based on the presence of Foxp3+ cells in the thymus and the peripheral immune tissues of IL-2/IL-2R-deficient mice, with a much more striking deficit in the proportion of these cells in the periphery. These studies suggest that these CD4+ Foxp3low CD25– cells were functionally active but the low proportion of these cells failed to keep up and suppress autoreactive cells. However, there are no direct data that the Foxp3+ T cells in IL-2/IL-2R-deficient mice are functionally active in vivo, although they showed variable capacity to suppress T cells responses in vitro. One argument for the suppressive activity of the CD4+ Foxp3+ cells in IL-2-deficient mice is that the autoimmunity in Foxp3-decieint mice is somewhat more severe than that seen in IL-2/IL-2R-deficient animals. It is equally possible, however, that the T effector pool in Foxp3-deficient mice, which is responsive to IL-2, more rapidly causes disease than the T effector cells that cannot respond to IL-2, as immune responses in vivo without IL-2R signals are effective but somewhat less robust (11, 23, 32, 33).

The activity of transgenic IL-2Rbeta within the thymus as discussed above and the capacity of anti-IL-2 Ab to inhibit thymic Treg cell production (16) provides direct evidence of an important role for IL-2/IL-2R in thymic Treg cell development. This finding by itself does not preclude a role for IL-2R in peripheral Treg cell homeostasis. However, CD4+ Foxp3high CD25+ Treg cells were readily found in the periphery of Tg–/– mice even though they barely expressed IL-2Rbeta and correspondingly showed substantially impaired IL-2R signaling. It is likely that thymic Tg–/– CD4 SP Treg cells are also poorly responsive to IL-2 upon exiting the thymus and seeding peripheral LN, because they also expressed minimal IL-2Rbeta and did not favorably compete when mixed with WT thymic Treg cells during IL-2-dependent expansion after adoptive transfer into IL-2Rbeta-deficient neonates. One potential trivial explanation for peripheral Tg–/– cells in adults is that they were primarily due to recent thymic output. However, Treg cells persisted in the spleen and LN of autoimmune-free Tg–/– mice 8 wk after adult thymectomy, indicating that constant thymic output does not compensate for the lack of a fully functional IL-2R. Collectively, these findings suggest that peripheral Treg cell homeostasis and function are either IL-2R independent or influenced by minimal IL-2R signaling.

Further characterization of Tg–/– Treg cells provides some initial information concerning the mechanism(s) for these peripheral Treg cells. With respect to major phenotypic markers of Treg cells such as CD25, Foxp3, CD62L, CD69, and GITR, the phenotypes of Treg cells from Tg–/– and WT mice are largely comparable within the thymus and periphery, arguing against the notion that peripheral Tg–/– Treg cells represent a distinct subpopulation of cells. When compared with Treg cells from normal C57BL/6 mice, BrdU labeling studies demonstrate that there is a reduced fraction of Tg–/– Treg cells that undergo DNA synthesis. This comparison also revealed that Tg–/– Treg cells did not readily lose the BrdU label when assessed in pulse-chase type experiments. These data are consistent with a population of Treg cells in the periphery of Tg–/– mice with lower proliferative activity but a longer life span when IL-2Rbeta expression and signaling is greatly diminished. These properties, especially the slow turnover, may in part explain why Treg cells with impaired IL-2R signaling still readily populate the periphery of Tg–/– mice and then persist in the mature peripheral immune compartment with a normal complement of Treg cells even without newly produced Treg cells from the thymus. This explanation is not at odds with the finding that Treg cells from the periphery of adult Tg–/– or WT mice cells cannot engraft and prevent autoimmunity when transferred to IL-2Rbeta–/– or IL-2–/– neonatal mice, respectively, as this model requires extensive IL-2-driven proliferation by the few donor Treg cells that initially engraft the LN (11, 16).

C57BL/6 IL-2Rbeta–/– mice contain a high proportion of T cells with an activated phenotype and exhibit severe lymphoproliferation that is first evident by 2 wk of age and rapidly progresses to severe multiorgan autoimmunity accompanied by a wasting syndrome that leads to death by 8- to 12-wk of age. Tg–/– mice do not exhibit any of these severe symptoms and are vigorous breeders with a normal life span, establishing that the Treg cells in these mice are effective in controlling autoimmunity (11). However, when the suppressor activity of Tg–/– Treg cells was tested in a standard in vitro assay, these cells exhibited somewhat less efficient inhibition when compared with WT Treg cells. This finding is comparable to that reported for the suppressive activity of TCR-transgenic Treg cells from IL-2R{alpha}-deficient mice (19) and is in line with several other studies that suggest that IL-2R function is important for Treg cell suppressive activity (30, 34). When compared with WT mice, several characteristics of Tg–/– mice, i.e., somewhat larger LN due to increased CD4 T cell cellularity, minimally elevated serum IgG1 levels, an occasionally high titer of autoanti-DNA Abs (Ref. 22 and see Fig. 3C), and mild inflammatory infiltrates in the liver of some older (>6 mo) animals (T. Malek, unpublished data and S. Ziegler, Benaroya Research Institute, Seattle, WA, personal communication), are consistent with mice containing Treg cells that at not fully functional. The increased proportion of Treg cells relative to the total pool of CD4+ T cells in the periphery of Tg–/– mice might represent a compensatory mechanism for this somewhat lower suppressor cell activity. However, the lower turnover of peripheral Tg–/– Treg cells, as discussed above, may simply account for the increased number in Treg cells in these mice. Alternatively, the lower activity of the Tg–/– Treg cells might reflect impaired IL-2R signals by the transgenic Treg cells during the in vitro culture. We do not favor this interpretation, because if IL-2 was mandatory in the in vitro assay we would expect that the Tg–/– Treg cells would exhibit an impaired suppressor activity that was independent of the dose of anti-CD3 or the number of Treg cells.

The detection of suboptimal and transient STAT5 activation by peripheral Tg–/– Treg cells raises the possibility that this low IL-2R signaling on its own is sufficient for peripheral Treg cells in vivo. This notion seems unlikely, because in contrast to WT Treg cells, Tg–/– Treg cells failed to engraft and persist when adoptively transferred to IL-2-sufficient IL-2Rbeta–/– mice (11). Furthermore, IL-2 did not promote the survival of these cells in vitro. Impaired IL-2Rbeta signaling by peripheral Tg–/– Treg cells is also evident by their distinctive in vivo homeostasis as revealed by BrdU studies and their slightly impaired functional activity. Importantly, when lethally irradiated recipient mice were reconstituted with a mixture of T cell-depleted bone marrow from WT and Tg–/– mice, Tg–/– cells effectively competed with WT cells in the reconstitution of the Treg cell pool in the thymus but not in the peripheral immune compartment. This result strongly suggests that normally there is a critical IL-2-dependent step for peripheral Treg cells. Thus, the persistence of Tg–/– Treg cells in the periphery in a noncompetitive setting with minimal to no effective IL-2R signaling likely represents a minor mechanism in maintaining Treg cells in situations in which a fully functional IL-2R pathway is active. This minor mechanism is either IL-2 independent or weak IL-2 signals cooperate with signals through other surface receptors. Although we cannot distinguish between these possibilities, the largely normal expression of CD25 by Tg–/– Treg cells when directly assessed ex vivo and the inability of IL-2 to up-regulate CD25 expression in vitro point to some IL-2-independent signaling for Tg–/– Treg cells in vivo.

The mixed chimera approach cannot discriminate whether the normal dominance for IL-2 in the periphery for Treg cells is at the level of their early expansion in LN during the initial stages of their production or their subsequent homeostasis in the steady state. However, our experience is that blockade of Treg cell production is most effective when anti-IL-2 is administered to neonatal normal mice (16). IL-2 blockade during the neonatal period also resulted in autoimmune gastritis in BALB/c mice and early diabetes, peripheral neuritis, gastritis, and thyroiditis in NOD mice (17). Thus, it appears that early developmental steps in the periphery may typically depend upon IL-2. However, the observation that Treg cells from adult WT C57BL/6 mice exhibited greater uptake and subsequent loss of BrdU when compared with the CD4+ CD25– T cell subset or Tg–/– Treg cells indicates that IL-2 may normally function as a growth and death factor for Treg cells and that WT Treg cells are not anergic in vivo, as noted by others (11, 35, 36, 37).

We favor a model in which the essential role for IL-2 resides at the earliest stages of the production of Treg cells in the thymus. If IL-2-signals are delivered to immature thymic Treg cells, continued thymic production, peripheral expansion, and homeostasis of mature Treg cells can occur without a requirement for sustained IL-2R signaling and may essentially be IL-2 independent. TCR and costimulatory interactions through CD28, CD40L, 4-1BB, CD7, or IL-4 each have been implicated in controlling aspects of Treg cell growth or function (38, 39, 40, 41, 42, 43). Therefore, signaling through these molecules may compensate for poor IL-2R signaling to maintain Treg cells. This notion may be of practical relevance, as this might mean that Treg cells may persist in therapeutic protocols that target IL-2 action on effector cells. Nevertheless, it seems likely that IL-2R signaling remains the dominant means for Treg cell expansion and homeostasis in the periphery.


   Acknowledgments
 
We thank Linjian Zhu for technical assistance and Larry Boise for helpful discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 National Institutes of Health Grants CA45957 and AI055815. Back

2 Address correspondence and reprint requests to Dr. Thomas R. Malek, University of Miami Miller School of Medicine, Department of Microbiology and Immunology, R138, 1600 Northwest 10th Avenue, Miami, FL 33146. E-mail address: tmalek{at}med.miami.edu Back

3 Abbreviations used in this paper: Treg, T regulatory cell; 7-AAD, 7-aminoactinomycin; DP, double positive; Foxp3, Forkhead box P3 transcription factor; GITR, glucocorticoid-induced TNFR; Gy, gray; LN, lymph node; HSA, heat-stable Ag; MFI, mean fluorescence intensity; LTbeta, lymphotoxin-beta; SP, single positive; Tg–/–, thymic-specific expression of IL-2Rbeta in IL-2Rbeta–/– mice; WT, wild type. Back

Received for publication June 13, 2006. Accepted for publication July 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Apostolou, I., A. Sarukhan, L. Klein, H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3: 756-763. [Medline]
  2. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2: 301-306. [Medline]
  3. Sakaguchi, S.. 2000. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101: 455-458. [Medline]
  4. Shevach, E. M.. 2000. Regulatory T cells in autoimmmunity. Annu. Rev. Immunol. 18: 423-449. [Medline]
  5. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  6. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  7. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337-342. [Medline]
  8. Malek, T. R., A. L. Bayer. 2004. Tolerance, not immunity, crucially depends on IL-2. Nat. Rev. Immunol. 4: 665-674. [Medline]
  9. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  10. Shevach, E. M.. 2000. Suppressor T cells: Rebirth, function and homeostasis. Curr. Biol. 10: R572-R575. [Medline]
  11. Malek, T. R., A. Yu, V. Vincek, P. Scibelli, L. Kong. 2002. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice: implications for the nonredundant function of IL-2. Immunity 17: 167-178. [Medline]
  12. Papiernik, M., M. L. de Moraes, C. Pontoux, F. Vasseur, C. Penit. 1998. Regulatory CD4 T cells: expression of IL-2R{alpha} chain, resistance to clonal deletion and IL-2 dependency. Int. Immunol. 10: 371-378. [Abstract/Free Full Text]
  13. Almeida, A. R., N. Legrand, M. Papiernik, A. A. Freitas. 2002. Homeostasis of peripheral CD4+ T cells: IL-2R{alpha} and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J. Immunol. 169: 4850-4860. [Abstract/Free Full Text]
  14. Furtado, G. C., M. A. Curotto de Lafaille, N. Kutchukhidze, J. J. Lafaille. 2002. Interleukin 2 signaling is required for CD4+ regulatory T cell function. J. Exp. Med. 196: 851-857. [Abstract/Free Full Text]
  15. Wolf, M., A. Schimpl, T. Hunig. 2001. Control of T cell hyperactivation in IL-2-deficient mice by CD4+CD25– and CD4+CD25+ T cells: evidence for two distinct regulatory mechanisms. Eur. J. Immunol. 31: 1637-1645. [Medline]
  16. Bayer, A. L., A. Yu, D. Adeegbe, T. R. Malek. 2005. Essential role for interleukin-2 for CD4+CD25+ T regulatory cell development during the neonatal period. J. Exp. Med. 201: 769-777. [Abstract/Free Full Text]
  17. Setoguchi, R., S. Hori, T. Takahashi, S. Sakaguchi. 2005. Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 201: 723-735. [Abstract/Free Full Text]
  18. Murakami, M., A. Sakamoto, J. Bender, J. Kappler, P. Marrack. 2002. CD25+CD4+ T cells contribute to the control of memory CD8+ T cells. Proc. Natl. Acad. Sci. USA 99: 8832-8837. [Abstract/Free Full Text]
  19. D’Cruz, L. M., L. Klein. 2005. Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nat. Immunol. 6: 1152-1159. [Medline]
  20. Fontenot, J. D., J. P. Rasmussen, M. A. Gavin, A. Y. Rudensky. 2005. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6: 1142-1151. [Medline]
  21. Suzuki, H., T. M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J. J. Simard, P. S. Ohashi, H. Griesser, et al 1995. Deregulated T cell activation and autoimmunity in mice lacking interleulin 2 receptor beta. Science 268: 1472-1476. [Abstract/Free Full Text]
  22. Malek, T. R., B. O. Porter, E. K. Codias, P. Scibelli, A. Yu. 2000. Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors. J. Immunol. 164: 2905-2914. [Abstract/Free Full Text]
  23. Malek, T. R., A. Yu, P. Scibelli, M. G. Lichtenheld, E. K. Codias. 2001. Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells. J. Immunol. 166: 1675-1683. [Abstract/Free Full Text]
  24. Fontenot, J. D., J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Developmental regulation of Foxp3 expression during ontogeny. J. Exp. Med. 202: 901-906. [Abstract/Free Full Text]
  25. Bensinger, S. J., P. T. Walsh, J. Zhang, M. Carroll, R. Parsons, J. C. Rathmell, C. B. Thompson, M. A. Burchill, M. A. Farrar, L. A. Turka. 2004. Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells. J. Immunol. 172: 5287-5296. [Abstract/Free Full Text]
  26. Xu, W., M. Yan, L. Lu, L. Sun, J. Theze, Z. Zheng, X. Liu. 2001. The p38 MAPK pathway is involved in the IL-2 induction of TNFbeta gene via the EBS element. Biochem. Biophys. Res. Commun. 289: 979-986. [Medline]
  27. Luther, S. A., A. Bidgol, D. C. Hargreaves, A. Schmidt, Y. Xu, J. Paniyadi, M. Matloubian, J. G. Cyster. 2002. Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J. Immunol. 169: 424-433. [Abstract/Free Full Text]
  28. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329-341. [Medline]
  29. Tang, Q., K. J. Henriksen, M. Bi, E. B. Finger, G. L. Szot, J. Ye, E. Masteller, H. McDevitt, M. Bonyhadi, J. A. Bluestone. 2004. In vitro expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 199: 1455-1465. [Abstract/Free Full Text]
  30. Thornton, A. M., E. E. Donovan, C. A. Piccirillo, E. M. Shevach. 2004. Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J. Immunol. 172: 6519-6523. [Abstract/Free Full Text]
  31. Thornton, A. M., C. A. Piccirillo, E. M. Shevach. 2004. Activation requirements for the induction of CD4+CD25+ T cell suppressor function. Eur. J. Immunol. 34: 366-376. [Medline]
  32. Nelson, B. H.. 2004. IL-2, regulatory T cells, and tolerance. J. Immunol. 172: 3983-3988. [Abstract/Free Full Text]
  33. Yu, A., J. Zhou, N. Marten, C. C. Bergmann, M. Mammolenti, R. B. Levy, T. R. Malek. 2003. Efficient induction of primary and secondary T cell-dependent immune responses in vivo in the absence of functional IL-2 and IL-15 receptors. J. Immunol. 170: 236-242. [Abstract/Free Full Text]
  34. de la Rosa, M., S. Rutz, H. Dorninger, A. Scheffold. 2004. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 34: 2480-2488. [Medline]
  35. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, A. Bandeira. 2001. CD25+CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166: 3008-3018. [Abstract/Free Full Text]
  36. Fisson, S., G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, B. L. Salomon. 2003. Continuous activation of autoreactive CD4+CD25+ regulatory T cells in the steady state. J. Exp. Med. 198: 737-746. [Abstract/Free Full Text]
  37. Klein, L., K. Khazaie, H. von Boehmer. 2003. In vivo dynamics of antigen-specific regulatory T cells not predicted from behavior in vitro. Proc. Natl. Acad. Sci. USA 100: 8886-8891. [Abstract/Free Full Text]
  38. Cozzo, C., J. Larkin, 3rd, A. J. Caton. 2003. Cutting edge: self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T cells. J. Immunol. 171: 5678-5682. [Abstract/Free Full Text]
  39. Guiducci, C., B. Valzasina, H. Dislich, M. P. Colombo. 2005. CD40/CD40L interaction regulates CD4+CD25+ T reg homeostasis through dendritic cell-produced IL-2. Eur. J. Immunol. 35: 557-567. [Medline]
  40. Sempowski, G. D., S. J. Cross, C. S. Heinly, R. M. Scearce, B. F. Haynes. 2004. CD7 and CD28 are required for murine CD4+CD25+ regulatory T cell homeostasis and prevention of thyroiditis. J. Immunol. 172: 787-794. [Abstract/Free Full Text]
  41. Tang, Q., K. J. Henriksen, E. K. Boden, A. J. Tooley, J. Ye, S. K. Subudhi, X. X. Zheng, T. B. Strom, J. A. Bluestone. 2003. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J. Immunol. 171: 3348-3352. [Abstract/Free Full Text]
  42. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, R. M. Steinman. 2003. Direct expansion of functional CD25+CD4+ regulatory T cells by antigen-processing dendritic cells. J. Exp. Med. 198: 235-247. [Abstract/Free Full Text]
  43. Zheng, G., B. Wang, A. Chen. 2004. The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells. J. Immunol. 173: 2428-2434. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Kuczma, R. Podolsky, N. Garge, D. Daniely, R. Pacholczyk, L. Ignatowicz, and P. Kraj
Foxp3-Deficient Regulatory T Cells Do Not Revert into Conventional Effector CD4+ T Cells but Constitute a Unique Cell Subset
J. Immunol., September 15, 2009; 183(6): 3731 - 3741.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. L. Bayer, M. Jones, J. Chirinos, L. de Armas, T. H. Schreiber, T. R. Malek, and R. B. Levy
Host CD4+CD25+ T cells can expand and comprise a major component of the Treg compartment after experimental HCT
Blood, January 15, 2009; 113(3): 733 - 743.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. Sgouroudis, A. Albanese, and C. A. Piccirillo
Impact of Protective IL-2 Allelic Variants on CD4+Foxp3+ Regulatory T Cell Function In Situ and Resistance to Autoimmune Diabetes in NOD Mice
J. Immunol., November 1, 2008; 181(9): 6283 - 6292.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
E. C. Lewis, M. Mizrahi, M. Toledano, N. DeFelice, J. L. Wright, A. Churg, L. Shapiro, and C. A. Dinarello
{alpha}1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice
PNAS, October 21, 2008; 105(42): 16236 - 16241.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Inaba, M. Steeves, P. Nguyen, and T. L. Geiger
In Vivo Suppression of Naive CD4 T Cell Responses by IL-2- and Antigen-Stimulated T Lymphocytes in the Absence of APC Competition
J. Immunol., September 1, 2008; 181(5): 3323 - 3335.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Laronne-Bar-On, D. Zipori, and N. Haran-Ghera
Increased Regulatory versus Effector T Cell Development Is Associated with Thymus Atrophy in Mouse Models of Multiple Myeloma
J. Immunol., September 1, 2008; 181(5): 3714 - 3724.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. L. Bayer, J. Y. Lee, A. de la Barrera, C. D. Surh, and T. R. Malek
A Function for IL-7R for CD4+CD25+Foxp3+ T Regulatory Cells
J. Immunol., July 1, 2008; 181(1): 225 - 234.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M.-E. Blais, S. Brochu, M. Giroux, M.-P. Belanger, G. Dulude, R.-P. Sekaly, and C. Perreault
Why T Cells of Thymic Versus Extrathymic Origin Are Functionally Different
J. Immunol., February 15, 2008; 180(4): 2299 - 2312.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
P. J. Spence and E. A. Green
Foxp3+ regulatory T cells promiscuously accept thymic signals critical for their development
PNAS, January 22, 2008; 105(3): 973 - 978.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. Li, E. J. Gowans, C. Chougnet, M. Plebanski, and U. Dittmer
Natural Regulatory T Cells and Persistent Viral Infection
J. Virol., January 1, 2008; 82(1): 21 - 30.
[Full Text] [PDF]

Home page
 NEJMHome page
The International Multiple Sclerosis Genetics Cons
Risk Alleles for Multiple Sclerosis Identified by a Genomewide Study
N. Engl. J. Med., August 30, 2007; 357(9): 851 - 862.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bayer, A. L.
Right arrow Articles by Malek, T. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bayer, A. L.
Right arrow Articles by Malek, T. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS