A Function for IL-7R for CD4?CD25?Foxp3?T Regulatory
Allison L. Bayer,2*†Joon Youb Lee,‡Anabel de la Barrera,†Charles D. Surh,‡
and Thomas R. Malek*†
The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-
deficient mice are not completely devoid of Foxp3?cells, but rather lack population of mature CD4?CD25?Foxp3highTregcells and
contain few immature CD4?CD25?Foxp3lowT cells. Interestingly, common ? chain (?c) knockout mice have been shown to have a near
important during thymic development of CD4?CD25?Foxp3?Tregcells apart from the IL-2. The present study was undertaken to
determine whether the ?c-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Tregcell production. These studies
revealed that mice double deficient in IL-2R? and IL-7R? contained a striking lack in the CD4?Foxp3?population and the Tregcell
defect recapitulated the ?c knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production
of CD4?CD25?Foxp3?Tregcells, indicating that normal thymic Tregcell production likely depends on signaling through both IL-2 and
IL-7 receptors. Selective thymic reconstitution of IL-2R? in mice double deficient in IL-2R? and IL-7R? established that IL-2R? is
dominant and sufficient to restore production of Tregcells. Furthermore, the survival of peripheral CD4?Foxp3lowcells in IL-2R??/?
mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Tregcell development
and peripheral homeostasis. The Journal of Immunology, 2008, 181: 225–234.
ery to actively suppress autoreactive T cells that escaped thymic neg-
of the better-characterized molecules of Tregcells. Furthermore, the
IL-2R is functionally important for Tregcells as IL-2- and IL-2R-
duction of these cells (1). The IL-2/IL-2R interaction, primarily
homeostasis in peripheral immune tissues (2–5). An important con-
sequence of the IL-2/IL-2R interaction in Tregcells is to maintain
optimal expression of Foxp3 and CD25. The Foxp3 gene contains
consensus-binding sequences for STAT5 that are required for tran-
scription, indicating an important role STAT5 in Foxp3 expression (6,
mouse and human CD4?CD25?regulatory cells through STAT5-
aturally occurring CD4?CD25?Foxp3?regulatory T
cells (Treg)3develop in the thymus as a fully functional
distinct subset of CD4?T cells that migrate to the periph-
dependent signals revealing the critical link between IL-2 and STAT5
8). Mice deficient in IL-2R?, IL-2R?, or IL-2 are not devoid of
Foxp3?-expressing CD4 T cells, but rather contains a reduced num-
ber of immature CD4?CD25?Foxp3lowT cells. Although these im-
mature Foxp3lowCD4 T cells in IL-2?/?mice exhibit some suppres-
sor function in vitro, they do not suppress autoreactive T cells in vivo
and are found at very low numbers in the periphery of IL-2R??/?
mice that were rendered autoimmune-free through adoptive transfer
of a low number of wild-type (WT) Tregcells (2, 9). Furthermore,
when endogenous Foxp3 is attenuated in Tregcells, mice succumb to
lethal autoimmunity (10). These results taken together are consistent
with an intrinsic defect in IL-2/IL-2R-deficient Foxp3lowCD4?T
Induction of Foxp3 is a hallmark of commitment to Tregcell lin-
eage (11). Therefore, the signals leading to Foxp3 expression in the
thymus represent key steps in development of this T cell subset. In
this regard, common ? chain (?c)?/?mice exhibit a much more strik-
ing defect in the development of Foxp3?cells than IL-2- or IL-2R-
deficient mice, with a near complete absence of these cells in both the
thymus and periphery (3, 5). Therefore, another ?c-cytokine along
with IL-2 functions during thymic development of Tregcells. Because
the development of natural Tregcells depends on signaling through
STAT5 (3, 6, 12, 13), in the current study we assessed whether IL-7
or IL-15, which like IL-2 also activates STAT5 (14, 15), contributes
to expression of Foxp3 and Tregcell production. By evaluating mice
that do not respond to IL-2, we uncovered a role for IL-7R signaling
for Tregcell development and peripheral homeostasis that usually is
dominated by IL-2.
Materials and Methods
C57BL/6, B6129SF2/S, IL-15R??/?, and ?c?/?mice were obtained from
The Jackson Laboratory. The thymic-targeted transgenic WT IL-2R? ex-
pressed in IL-2R??/?mice (designated 2R?TGin this study; thymic
*Department of Microbiology and Immunology and†Diabetes Research Institute,
Miller School of Medicine, University of Miami, FL 33136; and‡Department of
Immunology, Scripps Research Institute, La Jolla, CA 92037
Received for publication December 17, 2007. Accepted for publication May 2, 2008.
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.
1This work was supported by National Institutes of Health R01 CA45957 and
AI055815 and Juvenile Diabetes Research Foundation International 4-2004-361, Ju-
venile Diabetes Research Foundation Center at the University of Miami-Diabetes
2Address correspondence and reprint requests to Dr. Allison L. Bayer, Miller School
of Medicine University of Miami, Departments of Microbiology and Immunology and
Diabetes Research Institute, 1450 Northwest 10th Avenue, Miami, FL 33136. E-mail
3Abbreviations used in this paper: Treg, T regulatory cell; DKO, double knockout;
SP, single positive; DP, double positive; TG, thymic transgene; TSLP, thymic stromal
lymphopoietin; WT, wild type; ?c, common ? chain.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
transgene (TG)) and the thymic-targeted WT IL-7R? expressed in IL-
7R??/?mice (designated 7R?TGin this study) on the C57BL/6 genetic
background have been previously described (16, 17). These mice, along
with IL-7R??/?mice, were maintained in the breeding colony at the
University of Miami. Double deficient IL-7 and IL-15 (double knockout
(DKO)IL-7/IL-15) mice on the C57BL/6 genetic background have been
previously described (18) and were bred and maintained at The Scripps
Research Institute. Thymic transgenic mice expressing WT IL-2R?
and/or IL-7R? on the double deficient IL-2R??/?and IL-7R??/?
(DKO2R?/7R?) were generated by intercrossing 2R?TGand 7R?TG. The
progeny were then crossed, resulting in 2R?TG7R?KOand IL-2R??/?
7R?TGmice that were used as breeding pairs to generate and maintain
the following lines of mice: DKO2R?/7R?, 2R?TG7R?TGDKO2R?/7R?,
2R?TGDKO2R?/7R?, and 7R?TGDKO2R?/7R?. Protocols were approved
by the Institutional Animal and Use Committee at the University of
Miami and at The Scripps Research Institute.
Pacific Blue-conjugated CD4, PerCP-conjugated CD4 and CD8, and PE-
Cy7-conjugated CD25 were purchased from BD Biosciences. Biotin-con-
jugated anti-CD3? and anti-CD127, FITC-conjugated anti-CD25, Cy5-
conjugated anti-CD8, and the Foxp3 staining kit were purchased from
eBioscience. Pacific Blue-conjugated streptavidin was purchased from Mo-
C57BL/6, DKO2R?/7R?, 2R?TG7R?TGDKO2R?/7R?, 2R?TGDKO2R?/7R?,
and 7R?TGDKO2R?/7R?mice were sacrificed 3–15 wk of age. The
B6126SF2/S and IL-15R??/?or ?c?/?mice were sacrificed at 7–12 or 6 wk
of age, respectively. Inguinal, axillary, brachial, and cervical lymph
nodes were collected at the time of sacrifice along with the spleen and
thymus. Tissues were made into single-cell suspensions and subjected
to FACS analysis.
For four-color analysis, cells were incubated for 20 min at 4°C with Pacific
Blue-CD4, PerCP-CD8, and PE-Cy7-CD25 followed by two washes with
HBSS containing 0.2% BSA and 150 mM NaN3. The PE-Foxp3 staining
was performed according to the manufacturer’s kit instructions (eBioscience).
FACS analysis was performed as previously described (19) using a Becton
Dickinson LSRII and Diva software. The total number of events collected for
analysis was between 500,000 and 1,000,000.
FACS dot plots for Foxp3 and CD25 expression after gating on CD4?CD8?SP thymocytes (A) or CD4?splenocytes (B) from the indicated mice. C,
Percentage of Foxp3 and CD25 cells among the gated SP CD4 thymocytes (left panels) or CD4 splenocytes (right panels) and (D) number of CD4?Foxp3?
cells per 1 ? 106thymocytes (left panel) or splenocytes (right panels) in B6129SF2/S and IL-15R??/?mice (top panels) or B6 and IL-7R??/?(bottom
panels). Data are means ? SEM for five to eight mice per group.
Individual contribution of IL-15R or IL-7R signaling in Tregcell thymic development and peripheral homeostasis. Shown are representative
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