Development of regulatory T cells requires IL-7R stimulation by IL-7 or TSLP

IMMUNOBIOLOGY

Development of regulatory T cells requires IL-7R� stimulation by IL-7 or TSLPRenata Mazzucchelli,1 Julie A. Hixon,1 Rosanne Spolski,2 Xin Chen,3 Wen Qing Li,1 Veronica L. Hall,4 Jami Willette-Brown,1

Arthur A. Hurwitz,1 Warren J. Leonard,2 and Scott K. Durum1

1Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute (NCI), National Institutesof Health (NIH), Frederick, MD; 2Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD; 3Basic Research Program,SAIC-Frederick, NCI-Frederick, MD; and 4Laboratory of Experimental Immunology, Cancer and Inflammation Program, Center for Cancer Research, NCI, NIH,Frederick, MD

Interleukin-7 (IL-7), a cytokine producedby stromal cells, is required for thymicdevelopment and peripheral homeosta-sis of most major subsets of T cells. Weexamined whether regulatory T (Treg)cells also required the IL-7 pathway byanalyzing IL-7R��/� mice. We observeda striking reduction in cells with theTreg surface phenotype (CD4, CD25,GITR (glucocorticoid-induced tumor ne-crosis factor [TNF]-like receptor),CD45RB, CD62L, CD103) or intracellular

markers (cytotoxic T-lymphocyte–associated antigen-4, CTLA-4, and fork-head box transcription factor 3, Foxp3).Foxp3 transcripts were virtually absentin IL-7R��/� lymphoid tissues, and noTreg cell suppressive activity could bedetected. There are 2 known ligands forIL-7R�: IL-7 itself and thymic stromallymphopoietin (TSLP). Surprisingly,mice deficient in IL-7 or the other chainof the TSLP receptor (TSLPR) devel-oped relatively normal numbers of Treg

cells. Combined deletion of IL-7 andTSLP receptor greatly reduced Treg celldevelopment in the thymus but was notrequired for survival of mature peri-pheral Treg cells. We conclude thatTreg cells, like other T cells, requiresignals from the IL-7 receptor, but un-like other T cells, do not require IL-7itself because of at least partially over-lapping actions of IL-7 and TSLP fordevelopment of Treg cells. (Blood. 2008;112:3283-3292)

Introduction

Regulatory T (Treg) cells have a critical suppressive function formaintenance of self-tolerance and prevention of autoimmunity,1,2

and their deficiency can predispose to gastritis, thyroiditis, diabetesand graft-versus-host disease. Initially, Treg cells were identified asa small percentage, approximately 10% to 15%, of mouse CD4�

T cells that expressed CD25, the � chain of IL-2R.3,4 Treg cellsalso are reported to express CD45RB,5 CD62L,6 cytotoxicT lymphocyte–associated antigen 4 (CTLA-4),7-9 glucocorticoid-induced tumor necrosis factor (TNF)–like receptor (GITR),10-12

CD103 (�E�7 integrin)13 and forkhead box transcription factor3 (Foxp3), an intracellular transcriptional regulator.14-16

In vitro, Treg cells can block proliferative responses of bothCD4� and CD8� CD25� cells by a mechanism that remains to beclearly defined but appears to be based on cell contact and to beindependent of cytokine production.17-21 A very recent papersuggests Tregs suppress target cells by interleukin-2 (IL-2) depriva-tion and subsequent apoptosis.22 In vivo, Treg cells suppressactivation and expansion of self-reactive T cells that have escapedthymic clonal deletion.1,3,23-25 In addition to cell contact, thesuppressive cytokines IL-10 and transforming growth factor (TGF)�have been implicated in vivo as mediators of inhibition.26-32

Several studies suggest that CD4�CD25� T cells mature in thethymus as a distinct T-cell population.5,7,10,15,29,33 High-affinity IL-2receptors are constitutively expressed on Treg cells, and IL-2 hasbeen implicated in the development, maintenance, and function ofthese cells.34 It has been reported that IL-2 may be required forperipheral expansion and homeostasis,35-38 and IL-2 appears to berequired for Treg cell function in the periphery.17,39-42 Mice

deficient in IL-2, IL-2R�, or IL-2R� lack regulatory T cells43,44 anddevelop severe autoimmune disease.42,45-49

IL-7 is a cytokine that is produced by stromal cells in lymphoidtissues and is required for development and homeostasis of mostsubsets of T cells.50-52 The IL-7 receptor is composed of IL-7R�and the common cytokine receptor � chain, �c

53,54. A relatedstromal factor, thymic stromal lymphopoietin (TSLP), also sharesIL-7R� but additionally has a distinctive receptor subunit,TSLPR.55,56 Naive and memory CD4� T cells require IL-7 forhomeostatic survival.57,58 In vitro it has been shown that IL-7 can,albeit less well than IL-2 or IL-4, promote the proliferation andsuppressor function of CD4�CD25� cells stimulated with anti-CD3.40 The Tr1 cell, another type of suppressor cell that acts bysecreting suppressive cytokines, has been shown to respond to IL-7in vitro.59 It has recently been reported that Treg cells develop,survive, and function normally in mice that lack IL-7,60 suggestingthe Treg cell lineage has requirements quite different from themajor lineages of T cells, all of which require IL-7. Moreover, arecent study in human Treg cells noted that IL-7R� chain isexpressed at much lower levels than on other T-cell subsets andsuggested that this low expression is a useful marker for distinguish-ing Treg cells.61 We independently evaluated the role of the IL-7pathway in the Treg cell lineage, and our results substantiate boththe independence of IL-7 and the low expression of IL-7R�.However, despite this, we find that IL-7R is absolutely required fornormal development of the Treg cell lineage. It had previously beenreported that Foxp3-positive cells required a �c stimulus inaddition to the IL-2 receptor62; the present study implicates IL-7R�

Submitted February 4, 2008; accepted July 4, 2008. Prepublished online asBlood First Edition paper, July 29, 2008; DOI:10.1182/blood-2008-02-137414.

The publication costs of this article were defrayed in part by page charge

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as providing both a �c stimulus, and when paired with the TSLPR,a �c-independent stimulus.

Methods

Mice

IL-7R��/� (B6.129S7-Il7rtm1Imx/J) and Rag2�/� were originally purchasedfrom The Jackson Laboratory (Bar Harbor, ME) and maintained byhomozygous breeding at NCI-Frederick, MD. A different colony ofIL-7R��/� mice, also originally purchased from The Jackson Laboratory,was maintained at NCI-Bethesda, MD, and provided by C. L. Mackall.TSLPR�/� and TSLPR�/� mice were generated and maintained at theLaboratory of Molecular Immunology, National Institutes of Health(NIH), Bethesda, MD.63 C57BL/6 mice were purchased from the AnimalProduction Area, NCI-Frederick Cancer Research and Development Center(Frederick, MD). Rag�/�IL7�/� mice were obtained from R. Murray (EOSBiotechnology, San Francisco, CA). Animal care was provided in accor-dance with NIH Animal Use and Care guidelines. Experiments wereperformed following protocols approved by the NIH Committee. In ourexperiments, we refer to C57BL/6 as the wild-type control for IL-7�/� andIL-7R��/� mice, while the littermate TSLPR�/� mice were the control forTSLPR�/� mice. All untreated mice analyzed were 6 to 8 weeks old andusually sex-matched.

Flow cytometry

For analysis of thymic, splenic, or lymph node cells, single-cell suspensionswere prepared immediately after tissue harvesting. Erythrocytes were removedusing the ACK lysing buffer (Biosource, Rockville, MD). The followingantibodies were purchased from BD PharMingen (San Diego, CA): purifiedanti-CD16/CD32 (2.4G2); fluorescein isothiocyanate (FITC), peridinin chloro-phyll protein (PerCP), allophycocyanin (APC), or phycoerythrin-cyanine 5 (PE-Cy5)-anti–CD4 (RM4-5); FITC-anti–CD3 (� chain 145-2C11); PE, FITC, orAPC- anti-CD25 (PC61); biotinylated-anti–CD25 (7D4); FITC-anti-CD45RB(16A); FITC-anti–CD103 (integrin �E�7, M290); FITC-anti–CD62L (L-selectin, LECAM, Ly-22, clone MEL-14); PE-anti–CD152 (cytotoxicT-lymphocyte antigen 4 [CTLA-4, clone UC10-4F10-11)]); PE-anti–phospho-Stat5 (Stat5p, clone Y694); FITC-rat immunoglobulin IgG2a�; andPE-Ar Ham IgG1�. FITC-anti–goat IgG was purchased from Sigma-Aldrich (St Louis, MO). Polyclonal anti-glucocorticoid–induced tumornecrosis factor receptor (GITR) was obtained from R&D Systems (Minne-apolis, MN). PE-Cy5-anti–CD127 (IL-7R�, clone A7R34) and APC orPE-anti–Foxp3 (FJK-16s) antibodies were purchased from e-Bioscience(San Diego, CA).

For cell surface staining, cells were washed twice with FACS buffer(phosphate buffered saline [PBS], 0.5% bovine serum albumin [BSA]),treated with Fc-block (anti-CD16/CD32 antibodies) for 15 minutes at 4°Cand then incubated with the proper fluorochrome-conjugate antibodies for30 minutes at 4°C. Cells were then washed twice in FACS buffer andanalyzed. For CTLA-4 and Foxp3 intracellular staining, cells were fixedand labeled following the manufacturer’s protocols. Intracellular anti-Stat5p staining was performed following the protocol published by Van DeWiele and colleagues.64

Flow cytometric analysis was performed on FACScan using CELLQuestSoftware (BD Biosciences, San Jose, CA). Postacquisition FACS data wereanalyzed using FCS Express version 3 software (De Novo Software, LosAngeles, CA).

Isolation and reverse transcription of RNA

Spleens, lymph nodes, and thymi were harvested and immediately homoge-nized in 600 �L RNeasy lysis buffer (Qiagen, Valencia, CA) with �-mercaptoethanol (Sigma-Aldrich). Total RNA was isolated using theRNeasy Mini Kit (Qiagen). To avoid DNA contamination, in RNA isolatedfrom tissue eluted RNA was incubated with rDNase I for 30 minutes at37°C (DNA-free kit, Ambion, Austin, TX). An amount of total RNA equalto 500 ng or less was reverse transcribed using SuperScipt III First-Strand

Synthesis System (Invitrogen, Carlsbad, CA). Briefly, the RNA wasincubated at 65°C for 5 minutes in a volume of 20 �L containing 1 �L of50 �M oligo(dT)20 and 1 �L of 10 mM dNTPs mix, and then quicklychilled on ice. The cDNA synthesis was performed in a total volume of40 �L containing 20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 10 mMDTT, 40U RNaseOUT, and 100U of SuperScript III RT at 50°C for50 minutes followed by heat inactivation at 85°C for 5 minutes. The RNAtemplate was removed from the cDNA:RNA hybrid molecules by digestionwith 2 U of RNase H at 37°C for 20 minutes. At least 2 �L of this reactionmixture were used for real-time or conventional polymerase chainreaction (PCR) analysis.

Sorted cells were pelleted and resuspended in 1 mL of Trizol (Invitro-gen, Carlsbad, CA). Total RNA was isolated using the standard Trizolmethod. Total RNA equal to 1 �g or less was reverse transcribed using thecDNA archive kit (Applied Biosystems, Foster City, CA). Briefly, RNA wasresuspended in a volume of 20 �L containg 2 �L of 10 RT buffer, 10 random hexamers, 0.4 uL 10 mM dNTPs, and 1 �L of reverse transcriptase.RNA was incubated at room temperature for 10 minutes and moved to 37°Cfor 2 hours. 10 ng of cDNA was used for each quantitative real-time PCR.

Foxp3 and IL-7R quantitative real-time PCR

Quantitative real-time PCR.was used to determine the mRNA expressionlevel of Foxp3 in spleens, lymph nodes, and thymus using the followingprimers for Foxp3 and the housekeeping gene Hprt: Foxp3 F: 5 CTG CCTACA GTG CCC CTA G 3, Foxp3 R: 5 CAT TTG CCA GCA GTG GGTAG 3,14 HPRT F: 5 TCC CAG CGT CGT GAT TAG CGA TGA 3, HPRTR: 5 AAT GTG ATG GCC TCC CAT CTC CTT CAT GAC AT 3.65

Expression was normalized to the level of the housekeeping gene Hprt ineach sample. Real-time PCR reactions were performed by an ABI Prism7900 instrument (Applied Biosystems), and the PCR scheme was thefollowing: 95°C for 15 minutes, then 40 cycles at 95°C for 15 seconds, and55°C degrees for 30 seconds, followed by the dissociation curve of 95°C for15 minutes, 55°C for 15 minutes, and 95°C for 15 minutes.

Gene expression assays (Applied Biosystems) for Foxp3 and IL-7receptor were used to determine the mRNA expression level of Foxp3 andIL-7 receptor in sorted cells. Briefly, 10 ng of cDNA was put in a finalvolume of 20 �L containing 10 �L of Taqman Univeral PCR mix, 1 �L ofprimer/probe gene expression assay. The quantitative PCR samples wererun on an ABI 7300. The PCR scheme was 50°C for 2 minutes, 95°C for10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute.

Samples were run in triplicate, and the results were analyzed using the��Ct method.66 Analysis was made using a proper data analysis workbook.67

Functional assays

Single-cell suspensions were obtained from mouse spleens. Erythrocyteswere removed using the ACK lysing buffer (Biosource). CD3�CD4�CD25�

and CD3�CD4�CD25� cells were sorted using MoFlo cytometer (Cytoma-tion, Fort Collins, CO), yielding a purity of both subsets more than 99%.CD3�CD4�CD25� T cells (5 104) were seeded in triplicate in 96-well,round-bottom plate in RPMI-1640 (Mediatech, Herndon, VA) with 10%fetal bovine serum (Hyclone, Logan, UT) containing 2 mM glutamine,100 IU/mL penicillin (Mediatech), 100 �g/mL streptomycin (Mediatech),10 mM Hepes (Gibco-BRL, Grand Island, NY), 1 mM sodium pyruvate(Gibco-BRL), and 50 �M �-mercaptoethanol (Sigma-Aldrich), plus0.5 �g/mL soluble anti-CD3 antibody (� chin, 145-2C11; BD PharMingen).T cell–depleted and irradiated spleen cells (2 105cell/well) were used asantigen-presenting cells (APCs) and were prepared depleting of CD90�

cells using CD90 (Thy 1.2) microbeads and an LD MACS column(Miltenyi Biotec, Auburn, CA). CD3�CD4�CD25� T cells were added tothe wells at a ratio (CD4�CD25� T cells:CD4�CD25� T cells) of 10:0,10:1, 5:1, 2:1, and 1:1. Cells were pulsed with 37 000 becquerels[3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) per well forthe last 15 hours of the 72-hour culture period. Cells were then harvestedonto filter membranes using a harvester (Inotech Biosystems International,Rockville, MD), and the amount of incorporated [3H]thymidine wasmeasured with a Wallac MicroBeta counter (PerkinElmer Life and Analyti-cal Sciences, Waltham, MA).

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In vitro analysis of IL-7R� expression on splenic CD4�CD25�

cells after IL-7 stimulation

A single-cell suspension was prepared from C57BL/6 spleens. Two millioncells were plated in wells of a 24-well plate at a concentration of 2 106

cells/mL in RPMI-1640 (Mediatech) with 10% fetal bovine serum (Hy-clone) containing 2 mM glutamine, 100 IU/mL penicillin (Mediatech),100 �g/mL streptomycin (Mediatech), and 55 �M �-mercaptoethanol(Invitrogen, Carlsbad, CA). Cells were treated with IL-7 or IL-2 (Pepro-Tech, Rocky Hill, NJ) at a final concentration of 50 ng/mL and 20 ng/mL,respectively, and cultured overnight at 37°C in a humidified atmospherewith 5% CO2. Fresh cells and overnight untreated cells were used as areference control.

Cell lines

The IL-7–dependent thymocyte cell line D168 was maintained in RPMI-1640 (Mediatech) supplemented with 10% FBS (Hyclone), 2 mML-glutamine, 100 IU/mL of penicillin (Mediatech),100 �g/mL of streptomy-cin (Mediatech), 50 �M �-mercaptoethanol (Invitrogen), and 50 ng/mLmurine-recombinant IL-7 (PeproTech). The retroviral package cell linephoenix-Eco69 was maintained in DMEM (Mediatech), supplemented with10% FBS (Hyclone) and 100 IU/mL of penicillin (Mediatech), and100 �g/mL of streptomycin (Mediatech).

Retroviral infection

Retroviral constructs pMIGR1-Foxp3 and the empty vector pMIGR1 (akind gift from Dr S. Sakaguchi14) were transfected into the phoenix-Ecopackage cell line using FuGENE 6 reagent.69 The retrovirus-containingsupernatant was collected and loaded onto a Retro-Nectin (Takara, SantaAna, CA)–coated plate, and then the D1 cells were added and infectedovernight. Green fluorescent protein–positive cells were analyzed forIL-7R� expression 24 hours after infection.

IL-7 response of Treg cells

Splenic CD4�CD25�, CD4�CD25�, and CD4� cells were purified by cell

sorting (MoFlo cytometer, Cytomation) or MACS columns (MiltenyiBiotec). Cells (0.5-2 106) were resuspended in 500 �L of RPMI-1640(Mediatech), incubated at 37°C for 5 minutes and then stimulated with IL-7(PeproTech) at a final concentration of 50 ng/mL for 20 minutes. Afterstimulation, cells were stained for intracellular Stat5p and then analyzed by flowcytometry.

Adoptive transfer of T cells

IL-7R��/� and Rag�/� mice were irradiated with 300 rad of whole bodyirradiation. Four hours later, the mice were injected in the tail vein with asingle-cell suspension of 10 106 bone marrow cells obtained fromC57BL/6 donor mice and maintained on SMZ antibiotic until the day ofsacrifice. Spleens, thymi, and lymph nodes of host mice were harvestedafter 8 weeks and analyzed by flow cytometry.

C57BL/6 and IL-7�/� mice were irradiated with 300 rad of whole bodyirradiation. Four hours later, the mice were injected in the tail vein with asingle cell suspension of 10 106 bone marrow cells obtained fromTSLPR�/� or TSLPR�/� donor mice and maintained on SMZ antibioticuntil the day of sacrifice. Spleens and lymph nodes of host mice wereharvested after 8 weeks and analyzed by flow cytometry.

IL-7�/�, Rag�/�, IL-7�/�Rag�/�, and C57BL/6 mice were irradiatedwith 300 rad of whole body irradiation. Four hours later, the mice wereinjected in the tail vein with a single cell suspension of purified CD4� cells(15 106) obtained from TSLPR�/� or TSLPR�/� donor mice. Cells werelabeled with carboxyfluorescein succinimidyl ester (CFSE). Recipientswere maintained on SMZ antibiotic until the day of sacrifice. Spleens and lymphnodes of host mice were harvested after 6 days and analyzed by flow cytometry.

Treatment with anti–IL-7 neutralizing mAb

Eight-week-old TSLPR�/� and TSLPR�/� mice were intraperitoneallyinjected 3 times a week for 4 weeks with 1 mg M25, a mouse IgG2b

anti–human IL-7 neutralizing monoclonal antibody or M1, anti-FLAG, amouse IgG2b isotype-matched control (Amgen, Seattle, WA).

Results

Foxp3� cells are reduced in tissues of IL7R��/� mice

To determine whether Treg cells require the IL-7 system like mostsubsets of T cells, we first examined the expression of Foxp3, anuclear protein that is primarily expressed by Treg cells and isessential for their development and function. RNA was extractedfrom thymus, spleen, lymph nodes, and bone marrow fromC57BL/6, IL-7R��/�, IL-7�/�, or TSLPR�/� mice. Real-time PCRwas used to quantify Foxp3 transcripts in cells pooled from at least4 mice of each strain. The results shown in Figure 1A indicate thatexpression was clearly lower in thymus, lymph nodes, and spleenof IL-7R��/� mice as compared with TSLPR�/� and IL-7�/� mice.Since the IL-7�/� thymus is deficient in most thymocytes subsets,except Tregs, this could explain the relative enrichment of Foxp3transcripts compared with C57BL/6. That explanation would notaccount for the TSLPR�/� result, since there are relatively normalthymocyte numbers in these mice. The other panels of Figure 1Ashow that in the periphery, there are similar values for C57BL/6,IL-7�/�, and TSLPR�/� mice.

To verify that our real-time PCR reaction for Foxp3 reactedselectively with Treg cells, we compared thymic Treg cells(CD4�CD25�) with other subsets of thymocytes. Thymocytesfrom C57BL/6 mice were sorted into fractions of double-positive(DP), double-negative (DN), and 3 different single-positive (SP)populations: CD8�CD4�CD25�, CD8�CD4�CD25�, andCD8�CD4�. RNA was amplified and as expected, the expressionof Foxp3 in C57BL/6 was abundant in CD4�CD25� cells andvirtually absent in DP, DN, and CD8�CD4� subsets (Figure 1B).Similar results were obtained using sorted splenocytes fromIL-7�/�, TSLPR�/�, and TSLPR�/� mice (Figure 1C), confirmingthe specific expression of Foxp3 in CD4�CD25� cells, whereas thefew spleen cells obtained from IL-7R��/� mice that expressedCD4 and CD25 did not express Foxp3. Similar results wereobtained in thymocytes and lymph nodes (data not shown). Thenumber of cells with the phenotypic markers of Tregs (CD4�CD25�

and CD4�Foxp3�) were greatly diminished in spleens of IL-7R��/� mice, but present in IL-7�/� or TSLPR�/� mice (Figure1D). In IL-7R��/� mice, the reduction in CD4�Foxp3� cells (22.7-fold) was similar to the reduction in CD4�Foxp3� cells (30.3-fold),suggesting similar dependency on this receptor for their generationand/or maintenance.

The number of cells with the Treg cell surface markers CD4,CD25, and Foxp3 were determined at different ages in IL-7R��/�

mice. Throughout life, these cells were greatly reduced in numberin both thymus and spleen, compared with WT mice (not shown).

It was surprising from data in Figure 1 that, in contrast to thestriking deficiency in IL-7R��/� mice, IL-7�/� or TSLPR�/� micehad relatively normal numbers of cells with the Treg cell markersCD4, CD25, and Foxp3. We therefore examined a larger panel ofTreg cell markers on CD4�CD25� cells to determine whether thesewere also relatively normal in IL-7�/� and TSLPR�/� spleen cells,but deficient in IL-7R��/� spleen cells. The frequency and level ofexpression was relatively normal for CD45RB, CD103, CD62L,GITR and intracellular CTLA-4 (not shown). Thus, the number andphenotype of Treg cells was relatively normal in IL-7�/� orTSLPR�/� mice. However, these markers also confirmed thedeficiency in Treg cells in IL-7R��/� mice.

TREGS, IL-7, AND TSLP 3285BLOOD, 15 OCTOBER 2008 � VOLUME 112, NUMBER 8




Figure 1. Expression of Foxp3 mRNA in tissues ofdifferent strains of mice. (A) Relative expression ofFoxp3 mRNA by real-time PCR. RNA was extracted fromhomogenized suspensions of thymus, spleen, or lymphnodes. Symbols represent the fold increase or decreaseexpression in individual mice, and the bar indicates themean. (B) To verify that detection of Foxp3 by real-timePCR was selective for Treg cells, thymocytes fromC57BL/6 mice were sorted for Treg cell markers. Expres-sion was highest in the CD4�CD25� subset that includesTreg cells. (C) Expression of Foxp3 mRNA by real-timePCR in sorted CD4�CD25� or CD4�CD25� cells fromspleens of C57BL/6, IL-7�/�, IL-7R��/�, TSLPR�/�, andTSLPR�/� mice. Cells for each strain were pooled from atleast 2 mice. (D) Quantification of Treg cells (CD4�CD25�

and CD4�Foxp3�) in spleen of IL-7R��/� mice wascompared with C57BL/6, IL-7�/�, or TSLPR�/�. Cellswere gated on CD3. Representative individual mice fromgroups of at least 4 mice are shown.





It was unexpected to find such a striking difference betweenIL-7R��/� and IL-7�/� mice, since other features of phenotypes inthese mice are virtually indistinguishable. To verify that this wasnot unique to our colony of IL-7R��/� mice (NCI-FCRDC), weevaluated a second colony (NCI-Bethesda). Both colonies ofIL-7R��/� mice showed the same Foxp3 deficiency, the 2 colonieshaving been founded years apart from breeders purchased fromJackson Laboratories. Thus, development of CD4 cells expressingFoxp3 and CD25, which are essential for function of Treg cells, isdependent on signals from IL-7R. Given this requirement forIL-7R, it was surprising that neither IL-7�/� nor TSLPR�/� miceshowed a deficiency in Foxp3, since the only ligands known forIL-7R� are IL-7 itself and TSLP. One explanation is that, whereasall other subsets of T cells require IL-7 (but not TSLP), Treg cellsmay use either IL-7 or TSLP interchangeably, and this hypothesiswill be supported by findings described later in this report.

IL-7R��/� mice lack functional Treg cells

We compared the suppressive function of CD4�CD25� spleno-cytes from IL-7R��/�, IL-7�/�, TSLPR�/� and wild-type controlmice (Figure 2). IL-7R��/� cells, despite enrichment for theinfrequent CD4�CD25� cells, showed no significant inhibition ofproliferation of wild-type responders. IL-7�/� and TSLPR�/� cellsshowed suppressive function that was comparable to that ofwild-type Treg cells when assayed either on wild-type respondersor on responders of their own knockout strain. Thus, Treg cells,defined by the criteria of surface markers and suppressive function, areabsent in IL-7R��/� mice but present in IL-7�/� and TSLPR�/� mice.

IL-7R� expression and signaling in Treg cells

Having shown that Foxp3� cells required IL-7R� for development,we examined the expression of IL-7R� on Foxp3� cells fromC57BL/6 mice. Thymus, spleen, and lymph node cells were stainedfor surface expression of IL-7R� versus intracellular content ofFoxp3 protein. IL-7R� was only detectable by flow cytometry on aminority of the Foxp3� cells from thymus and spleen and lymph node(Figure 3A). This expression on a subset of murine Treg cells thereforediffers from human peripheral blood Treg cells, which lack IL-7R�.61

We then considered that the low expression on freshly isolatedFoxp3� could reflect a recent encounter with IL-2 or IL-7 in vivo,which down-regulates IL-7R� on other T-cell subsets.70-72 Cellswere placed in culture overnight, which indeed raised expression ofIL-7R� on other T-cell subsets in thymus, spleen, and lymph node.

However, only thymic Treg cells up-regulated IL-7R�, whereassplenic or lymph node Treg cells did not up-regulate IL-7R�(Figure 3B). Culture with IL-7 down-regulated IL-7R� expressionon Treg cells from thymus, spleen, or lymph node. Taken together,these results suggest that the low level of IL-7R� on the majority offreshly isolated Treg cells from spleen or lymph node does notnecessarily result from recent encounters with IL-7 or IL-2,although thymic Treg cells may have had such a recent encounter.

Because Treg cells expressed lower levels of IL-7R� than otherT-cell subsets, we examined whether Foxp3 could influence theexpression of IL-7R�. Transduction of Foxp3 into an IL-7–dependent murine thymocyte line induced a significant decline inIL-7R� expression (Figure 3C). Thus, the low levels of IL-7R� onmature Treg cells may alternatively reflect down-regulation byFoxp3, rather than a recent encounter with IL-7 or IL-2. Inthymocytes, if Foxp3 inhibited IL7-R� expression, different sub-sets might reflect this relationship. Thymocytes were sorted intodifferent subpopulations and indeed, CD4� cells bearing CD25expressed much higher levels of Foxp3, and correspondingly lowerlevels of IL-7R�, than their CD25� counterparts (Figure 3D). Thisrelationship is consistent with Foxp3 inhibiting IL-7R� expression.The lack of IL-7R� in CD4�CD8� cells is well known72 and, fromthese results, is unrelated to Foxp3 expression.

Based on the level of receptor expression, the response of Tregcells to IL-7 was expected to occur in only a minority of cells.However, a vigorous phosphorylation of Stat5 was observed in themajority of Treg cells as shown in Figure 4 and was comparable toother CD4 cells. This indicates that despite the low expression ofIL-7 receptors on the majority of mature murine Treg cells, therewere sufficient numbers of receptors to signal a response. Thisraises the possibility that a requirement for signals from IL-7receptor could occur both in thymic development and peripheralsurvival of Treg cells, as in other T-cell subsets; however, as we willshow, other data support a developmental but not a survival role.

Anti–IL-7 antibody blocks the development of Treg cells inTSLPR�/� mice

Since Treg cells were deficient in IL-7R��/� mice but not in eitherIL-7�/� or TSLPR�/� mice, it suggested that the 2 ligands ofIL-7R� might have a redundant activity. To test this, we injectedanti–IL-7 monoclonal antibody (M25) into either TSLPR�/� orTSLPR�/� littermates, as a control. The depletion of splenic Tregcells by anti–IL-7 was more complete in TSLPR�/� mice than in

Figure 2. Lack of Treg cell activity from IL-7R��/�

mice. Suppressor populations were sorted for CD3,CD4, and CD25� or CD25�. For each individual experi-ment, spleen cells were pooled from 2 wild-type mice and10, 6, or 4 spleens from IL-7R��/�, IL-7�/�, or TSLPR�/�

mice, respectively. The top panel shows CD25� Tregcells ( ) from various knockout mice mixed withCD25� responders from wild-type mice, whereas thebottom panel shows both CD25� Treg cells and CD25�

responders from the same knockout mice. WT Treg cells( ) are shown as a control at each mixture ratio.





TSLPR�/� littermates (Figure 5), supporting a redundant functionfor IL-7 and TSLP in inducing the development of Treg cellsthrough an IL-7 receptor signal. It should be noted that there wasalso a depleting effect of anti–IL-7 in TSLPR�/� mice, suggestingthat of the 2 ligands, Treg cell progenitors may somewhat preferIL-7 over TSLP.

IL-7 or TSLP are required for thymic development, but notperipheral survival, of Treg cells

The results using antibody injections showed a block of thymicdevelopment of Treg cells, and it remained possible that peripheral

survival of these cells also depended on an IL-7 receptor signal,analogous to other subsets of T cells. We therefore performedchimerism and adoptive transfer experiments that would distin-guish whether the IL-7 receptor signal was required for bothdevelopment and survival. Bone marrow chimeras verified theredundant requirement for IL-7 or TSLP in thymic development ofTreg cells (Figure 6A). Thus, normal numbers of thymic Treg cellsdeveloped in mice lacking either IL-7 or TSLPR, but not both.

To determine whether an IL-7 receptor signal was required forperipheral survival of Treg cells, spleen cells were labeled withCFSE, and then transferred into recipient mice. Analysis of spleen

Figure 3. Expression of IL-7R� on Treg cells. (A) Surface IL-7R� versus intracellular Foxp3 were evaluated in cells from thymus, spleen, and lymph nodes of normalC57BL/6 mice. IL-7R� was variably expressed on Foxp3-positive cells. (B) Effect of overnight culture on expression of IL-7R�. The level of IL-7R� increased on some cells fromthymus spleen and lymph node of normal C57BL/6 mice. On Treg cells (CD4�CD25�) from thymus, the IL-7R� level increased, but it did not rise on Treg cells from spleen orlymph node. Culture with IL-7 down-regulated IL-7R� expression on Treg cells as well as other T cells. (C) Foxp3 down-regulates IL-7R� expression. The IL-7–dependentT-cell line D1 was infected with the retrovirus MigR1 expressing Foxp3, or with an empty MigR1 vector. After 24 hours, cells were analyzed for expression of IL-7R�. The resultis representative of 3 separate experiments. (D) Subsets of thymocytes were analyzed for expression of Foxp3 versus IL-7R. Cells were sorted according to expression of CD4,CD8, and CD25, then assayed by real-time PCR.





cells 6 days later showed no apparent decrease in survival if Tregcells were deprived of both IL-7 and TSLPR (Figure 6B). Weconclude that Treg cells require an IL-7 receptor signal fordevelopment, but unlike other T cell subsets, do not require thissignal for survival.

Discussion

Treg cells have attracted much attention in recent years due to theirpotency in suppressing immune responses, suggesting applicationsof enhancing their activity to treat autoimmune diseases andinhibiting them to promoting immune responses. It was previouslyreported that Treg cells are independent of IL-7,60 and human Tregcells express low levels of IL-7R�,61 distinguishing them frommost other major subsets of T cells that require IL-7 during thymicdevelopment and in the periphery. Here we show that in IL-7R��/�

mice, Treg cells do not develop in the thymus and are absent in

peripheral lymphoid organs. It was previously reported that Tregcell development requires a �c signal in addition to that from theIL-2 receptor.62 The present study implicates the IL-7R� chain asproviding both a �c stimulus, and when paired with the TSLPR, a�c-independent stimulus. The identification of a key role for TSLP

Figure 4. IL-7 stimulation of Treg cells induces phosphorylation of Stat5.Spleen cells freshly isolated from C57BL/6 mice were sorted into differentpopulations, stimulated for 20 minutes with IL-7, then stained for intracellularphospho-Stat5. Most Treg cells responded to IL-7 despite low expression ofIL-7R� on the majority of cells.

Figure 5. Depletion of Treg cells in TSLPR�/� mice by injection of anti–IL-7 Ab.Anti–IL-7 (M25) or control antibody (M1) were injected 3 times per week for 4 weeksinto either TSLPR�/� or TSLPR�/� littermates. The number of Treg cells in thymusand spleen was determined by staining for intracellular Foxp3. Values shown are for3 individual mice per group, and the bar indicates the mean in each group.

Figure 6. Treg cells require an IL-7R� signal for thymic development but not forperipheral survival. (A) Treg cells require IL-7 or TSLP for thymic development.TSLPR�/� or TSLPR�/� bone marrow was used to reconstitute irradiated IL-7�/� orC57BL/6 recipients. Eleven weeks later, the thymus was analyzed for CD4�Foxp3�

cells. Representative data from 3 separate experiments is shown, and the totalnumber of Treg cells per thymus is indicated in the upper right quadrant. (B) MatureTreg cells do not require IL-7 or TSLP for survival. Purified CD4� spleen cells fromTSLPR�/� or TSLPR�/� mice were labeled with CFSE and transferred into irradiatedrecipients that expressed IL-7 or lacked it, as indicated. Six days later, spleen cellswere analyzed for CD4 and Foxp3 expression. TSLPR�/� Treg cells survived equallywell in hosts with or without IL-7.





in Treg development indicates another important role for TSLPbeyond its importance in mediating inflammatory processes such asallergic inflammation in the lung and atopic skin diseases.73-77

We verified the report60 that, unlike other subsets of T cells,Treg cells developed relatively normally in IL-7�/� mice, suggest-ing that the IL-7 receptor is activated by more than one ligandduring Treg cell development and implicating TSLP. Of the2 ligands, Treg cell progenitors may somewhat favor IL-7 overTSLP, since anti–IL-7 treatment reduces Treg cells in mice thathave TSLPR (Figure 5), although as we have emphasized, thiseffect is stronger in mice lacking TSLPR. A recent study78 usingcultures of human thymocytes reported that TSLP can indirectlyinduce Treg cell development; TSLP (but not IL-7) promoteddendritic cell development, which in turn supported Treg celldevelopment. However, it is now clear that both mouse and humanTSLP can act directly on CD4� T cells as well as on DCs.63,79 Ourstudy did not find that TSLPR was obligatory for Treg celldevelopment in the mouse thymus.

Our interpretation that TSLP has a redundant function (withIL-7) is actually based on studies with TSLPR-, rather thanTSLP-deficient cells. It remains a formal possibility that TSLP isnot the only ligand for TSLPR, although none have yet beenidentified. Thus, an alternative ligand for TSLPR paired withIL-7R� would also explain our results.

The IL-2 pathway was shown to be required for thymicdevelopment of Treg cells in some studies,48,49,80,81 but not others.35

Other reports show a requirement for IL-2 in persistence of Tregcells in peripheral organs37 or in peripheral development fromnonsuppressive T cells.35 Thus, mice deficient in IL-2 itself,IL-2R�, or IL-2R� all show a deficiency in Treg cells, excessiveimmune responses, and colitis. From our study, IL-7R� deficiencyleads to a Treg cell defect but, unlike other Treg cell deficiencymodels, there has been no colitis reported; this suggests thatIL-7R��/� mice also lack, in addition to Treg cells, the effectorT cells that are capable of excessive immune responses.

A recent study82 investigated whether the required regions ofIL-2R� in Treg cell development could be satisfied by regions ofIL-7R�. They concluded that, although the intracellular domain ofIL-7R� was capable of delivering signals for Treg cell develop-ment, apparently only IL-2, and not IL-7, was present in themicroenvironment in which Treg cells developed Foxp3 expres-sion. Taken together with our findings, this suggests that in thymicdevelopment, the IL-7R� requirement could occur early, at thepro–T-cell stage, as for other T-cell lineages. Later in thymicdevelopment, IL-2 may be required for induction of Foxp3 and thedifferentiation to Treg cells.

In other types of lymphocytes, the IL-7 receptor delivers signalsfor survival, proliferation, and differentiation. In pro-T cells, themost important response to IL-7 receptor signals appears to besurvival, since IL-7R� deficiency can be largely reversed byoverexpressing the survival protein Bcl-283,84 or deleting the deathprotein Bax.85 In CD8 T-cell development, IL-7 delivers a differen-tiation signal,86 and in �� T-cell development it induces rearrange-ment of the TCR� locus.87 In peripheral T cells, IL-7 receptorsignals also induce proliferation88,89 mediated by cell cycle regula-tors as well as survival based on protection from the death proteinBim.90 The requirement for IL-7R� in Treg cell development couldbe an effect on survival, proliferation, or differentiation.

Mature peripheral Treg cells did not require IL-7 or TSLP forsurvival, and only a minority of freshly isolated Foxp3� cellsexpresses enough IL-7R� to fall above control levels detected byflow cytometry (Figure 3A). Despite the low receptor expression,

all Treg cells were capable of responding to IL-7 by phosphorylat-ing Stat5 (Figure 4); this indicates that a very low level of receptordoes not preclude responses to IL-7, and also suggests that IL-7could have important actions on mature Treg cells in vivo, but notbe absolutely required. This effect on Treg cells is consistent withdirect actions of TSLP on both mouse63 and human79 CD4� T cells.We initially thought that the low receptor expression might havebeen explained by a recent encounter with IL-2 or IL-7, whichdown-regulate IL-7R�70,71; but other subsets of CD4 or CD8 cells,once removed from their IL-7–containing environment, spontane-ously up-regulate IL-7R�. However, peripheral Treg cells, afterovernight culture in the absence of IL-7, did not up-regulateIL-7R�, unlike other subsets of T cells. The low expression ofIL-7R� on Treg cells could be partly due to an effect of Foxp3,which we show down-regulated IL-7R� expression (Figure 3B).

It should be noted that treatment of TSLPR�/� mice withanti–IL-7 antibody for a month showed modest splenic depletion ofTregs (approximately a third in Figure 5), whereas transfer of Tregsinto IL-7�/� mice showed no depletion in 6 days (Figure 6B). Thecontrol antibody injections did not deplete Tregs (Figure 5), thus acompletely nonspecific antibody effect is unlikely, although im-mune complexes of IL-7/�IL-7 might be a factor. One explanationis that a month of anti–IL-7 treatment partially inhibits thymic Treggeneration in the adult mouse thymus (Figure 5), which in turninhibits seeding of peripheral Tregs. Although IL-7�/� mice hadsubstantial numbers of Treg cells in our experiments (Figure 1D),the study of de Latour60 reported an overall reduction in Tregs inIL-7�/� mice, although the reduction was less severe than otherT-cell subsets.

Although a direct effect of IL-7 receptor on Treg cells is thesimplest explanation of our findings, an indirect effect cannot beruled out as yet; for example, it may induce production of othercytokines that could directly act on Treg cells. Other cytokineshave been reported to promote Treg cells, including IL-2, TGF�,IL-10, IL-4, and IL-13 91), although only IL-2 has been implicatedin the naturally occurring Treg cells that are addressed in ourstudies. Since IL-2 is required for Treg cells, it is possible that theIL-7R� requirement is for production of IL-2. However, this seemsunlikely given the difference in IL-7R�– versus IL-7–deficientmice: the former lack Treg cells but have a general T-cell deficiencyidentical to the latter, presumably including an identical degree ofIL-2 deficiency.

If the requirement for IL-7R� stimulation is through directaction on a thymic progenitor, our findings suggest a distinctionbetween the progenitor for Treg cells and that of other ��T cells.The latter depend exclusively on the ligand IL-7, whereas weobserve that Treg cells depend on IL-7R� but not exclusively onIL-7. Pro-T2 and -T3 cells depend on IL-7 for survival.92 Thus,deletion of IL-7 (or IL-7R�) results in loss of pro-T2 and -T3cells93,94 and the majority of ��T cells that descend from them. Ourresults show that Treg cell development is relatively normal in theabsence of IL-7 itself, verifying the findings of Peffault deLatour60) and suggesting that unlike the majority of ��T cells, Tregcells may not develop from typical IL-7–dependent pro-T2 and -T3cells. This adds to the previously noted distinctive characteristics ofthymic Treg cells.48,80,81

Acknowledgments

We thank C. Willis (Amgen) for providing anti–IL-7 antibody; M.Guimond and C. L. Mackall for providing a verifying source of





IL-7R��/� mice; R. Wyles and S. Stull for animal technicalassistance; K. Noer, R. Matthai, S. Bauchiero, and M. Anderson forflow cytometry assistance; and J. J. Oppenheim for comments onthe manuscript.

This work was supported by the Intramural Research Programof the NIH, NCI, and with federal funds from the NCI, NIH, undercontract N01-CO-12400.

The content of this publication does not necessarily reflectthe views or policies of the Department of Health and HumanServices, nor does mention of trade names, commercial prod-ucts, or organizations imply endorsement by the United Statesgovernment.

Authorship

Contribution: R.M. designed and performed research, analyzeddata, and wrote the paper; J.A.H., R.S., X.C., W.L., V.L.H., andJ.W.B. performed research; A.A.H. and W.J.L. designed research,and S.K.D. designed research, analyzed data, and wrote the paper.

Conflict-of-interest disclosure: R.S. and W.J.L. are listed as coinven-tors on applications for and/or issued patents related to TSLP. Theremaining authors declare no competing financial interests.

Correspondence: Scott K. Durum, NCI-FCRDC, Building 560,Room 31-71, Frederick, MD 21702-1201; e-mail: [email protected].

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online July 29, 2008 originally publisheddoi:10.1182/blood-2008-02-137414

2008 112: 3283-3292

Willette-Brown, Arthur A. Hurwitz, Warren J. Leonard and Scott K. DurumRenata Mazzucchelli, Julie A. Hixon, Rosanne Spolski, Xin Chen, Wen Qing Li, Veronica L. Hall, Jami TSLP

stimulation by IL-7 orαDevelopment of regulatory T cells requires IL-7R

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Development of regulatory T cells requires IL-7R stimulation by IL-7 or TSLP