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Genetically Targeted MiceSpecific Ablation of Regulatory T Cells inLeads to Induction of Autoimmunity by

Cells+Cutting Edge: Depletion of Foxp3

Alexander Rudensky and Tim SparwasserLoddenkemper, Ashutosh Chaudhry, Paul deRoos, Jeong Kim, Katharina Lahl, Shohei Hori, Christoph

http://www.jimmunol.org/content/183/12/7631doi: 10.4049/jimmunol.0804308November 2009;

2009; 183:7631-7634; Prepublished online 18J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Cutting EdgeCutting Edge

Cutting Edge: Depletion of Foxp3� Cells Leads toInduction of Autoimmunity by Specific Ablation ofRegulatory T Cells in Genetically Targeted Mice1

Jeong Kim,2,3* Katharina Lahl,2† Shohei Hori,‡ Christoph Loddenkemper,§

Ashutosh Chaudhry,*¶ Paul deRoos,4*¶ Alexander Rudensky,*¶ and Tim Sparwasser5�

We have recently described two independent mouse mod-els in which the administration of diphtheria toxin (DT)leads to specific depletion of regulatory T cells (Tregs) dueto expression of DT receptor-enhanced GFP under thecontrol of the Foxp3 promoter. Both mouse models de-velop severe autoimmune disorders when Foxp3� Tregsare depleted. Those findings were challenged in a recentstudy published in this journal suggesting the expres-sion of Foxp3 in epithelial cells as the cause for diseasedevelopment. By using genetic, cellular, and immuno-histochemical approaches, we do not find evidence forFoxp3-expression in nonhematopoietic cells. DT injec-tion does not lead to a loss of epithelial integrity in ourFoxp3-DTR models. Instead, Foxp3 expression is Treg-specific and ablation of Foxp3� Tregs leads to the in-duction of fatal autoimmune disorders. Autoimmunitycan be reversed by the adoptive transfer of Tregs intodepleted hosts, and the transfer of Foxp3-deficient bonemarrow into T cell-deficient irradiated recipients leadsto full-blown disease development. The Journal of Im-munology, 2009, 183: 7631–7634.

T he existence of a dedicated population of suppressor Tcells has long been surmised. However, the immunol-ogy community en large treated this notion with con-

tempt because the markers and other molecular features for thepresumed suppressor T cell population were not known. Theidentification of CD25�CD4� T cells followed by the discov-ery of the transcription factor Foxp3 as a faithful marker forregulatory T cells (Tregs)6 allowed for a large body of publishedreports demonstrating the suppressive activity of Foxp3-ex-

pressing T cells (1–5). A fatal early onset autoimmune syn-drome in mice or humans harboring a nonfunctional Foxp3allele is manifest to the vital significance of Treg-mediated sup-pression. Collectively, the general consensus in the field is thatthe high level of Foxp3 expressed by Tregs is essential for theirsuppressive activity. In accordance with this idea, the loss ofFoxp3 expression in hematopoietic cells, more specifically in Tcells (1), or the loss of Tregs induces an autoimmune syndrome(6, 7) similar to that in mice with germline mutations in Foxp3(8). However, this model was recently challenged by Liu and col-leagues (9, 10). According to their studies, Foxp3 expression is notrestricted to Treg cells, let alone hematopoietic cells, but is wide-spread in variety of epithelial tissues including thymic, mammarygland, lung, and prostate epithelial cells (9–12). These investiga-tors argued that the loss of epithelial-specific Foxp3 expression isthe cause of life-threatening autoimmunity in immunodysregula-tion, polyendocrinopathy, enteropathy, X-linked syndrome(IPEX) patients, Foxp3-deficient mice, or mice engineered to elim-inate Foxp3-expressing cells, whereas Foxp3 expression in hema-topoietic cells, including Treg cells, is not essential (10). Therefore,we revisited the issue of expression of Foxp3 in nonlymphoid tis-sues using several strains of mice with targeted mutations of theFoxp3 locus and monoclonal and polyclonal Foxp3 Abs.

Materials and MethodsMice

DEREG (depletion of regulatory T cells; Ref. 7), C57BL/6, and BALB/c micewere bred at the animal facility of the Institute for Medical Microbiology, Im-munology, and Hygiene (Technical University, Munich, Germany). Foxp3GFP

(13) reporter mice and Foxp3DTR (where DTR is diphtheria toxin receptor; Ref.6) mice were bred at the animal facility of the Department of Immunology,University of Washington (Seattle, WA). All animal experiments were per-formed under specific pathogen-free conditions and in accordance with insti-tutional, state, and federal guidelines.

*Department of Immunology, University of Washington, Seattle, WA 98195; †Laboratory ofImmunology and Vascular Biology, Department of Pathology, Stanford University School ofMedicine, Stanford, CA 94305; ‡Research Unit for Immune Homeostasis, RIKEN ResearchCenter for Allergy and Immunology, Yokohama, Kanagawa, Japan; §Institute of Pathology/Research Center ImmunoSciences, Charite Universitaetsmedizin Berlin, Campus BenjaminFranklin, Berlin, Germany; ¶Howard Hughes Medical Institute, University of Washington,Seattle, WA 98195; and �Institute of Infection Immunology, TWINCORE, Center for Exper-imental and Clinical Infection Research, Hannover, Germany, a joint venture between theMedical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI)

Received for publication December 23, 2008. Accepted for publication October 13, 2009.

The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB/TR22 and SFB 587).

2 J.K. and K.L. contributed equally to this work.3 Current address: Genentech, 1 DNA Way, South San Francisco, CA 94080.4 Current address: Immunology Program, Memorial Sloan Kettering Cancer Center, Box212, 1275 York Avenue, New York, NY 10065.5 Address correspondence and reprint requests to Dr. Tim Sparwasser, Institute ofInfection Immunology, TWINCORE, Center for Experimental and Clinical InfectionResearch, Feodor-Lynen-Straße 7, 30625 Hannover, Germany. E-mail address:tim.sparwasser@twincore.de6 Abbreviations used in this paper: Treg, regulatory T cell; DEREG, depletion of regula-tory T cells; DT, diphtheria toxin; DTR, DT receptor; IPEX, immunodysregulation, poly-endocrinopathy, enteropathy, X-linked syndrome; sf, scurfy.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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Immunohistochemistry

In brief, sections from formalin-fixed, paraffin-embedded samples were sub-jected to a heat-induced epitope retrieval step before incubation with primaryAbs for 30 min. For detection of Foxp3, the rat Ab clone FJK-16s (eBioscience)was applied at a dilution of 1/10 or 1/100 and slides were blocked using a com-mercial peroxidase-blocking reagent (DakoCytomation), followed by a second-ary rabbit anti-rat Ab (DakoCytomation) and the EnVision peroxidase kitagainst rabbit Abs (catalog no.K4003, DakoCytomation). In addition, a poly-clonal rabbit anti-mouse Foxp3 Ab (dilution 1/1000; catalog no. ab54501, Ab-cam) was used, followed by a secondary biotinylated donkey anti-rabbit IgG(Dianova) and the streptavidin peroxidase kit (catalog no. K5001, Dako-Cytomation). For Ki-67 (TEC-3 Ab) (dilution 1/500; DakoCytomation),cleaved caspase-3 (Asp175) (dilution 1/200; Cell Signaling), androgen receptor(dilution 1/100; catalog no. ab47563, Abcam), and estrogen receptor (dilution1/20; catalog no. ab21232, Abcam) biotinylated rabbit anti-rat (DakoCytomation)or donkey anti-rabbit (Dianova) secondary Abs were used followed by thestreptavidin alkaline phosphatase kit (catalog no. K5005, DakoCytomation).Alkaline phosphatase was revealed by Fast Red as chromogen and peroxidasewas developed with a highly sensitive diaminobenzidine chromogenic substratefor 10 min.

Western blotting

Mice tissues were collected, weighed, resuspended in Tris buffer (20 mM Tris,5 mM magnesium acetate, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, and aprotease inhibitor cocktail from Roche), and homogenized for 10 s using a Poly-tron homogenizer at 60% power followed by the addition of lysis buffer (50mM Tris-Cl, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate,0.1% SDS, and 20 mM MOPS). Tissue extracts were incubated at 4°C for 30min followed by centrifugation at 13,000 rpm for 20 min. Supernatants werecollected, quantified for protein levels (Coomassie protein assay reagent;Thermo Fisher), boiled in 2� SDS loading buffer, and separated in 4–12%Tris-glycine polyacrylamide gels followed by Western blotting using an affinity-purified rabbit Foxp3 Ab previously generated in the laboratory (no.1586) andanti-rabbit IgG-HRP as secondary Ab (Amersham/GE Healthcare). Blottingfor �-actin using a mouse mAb of corresponding specificity (catalog no. A2228, Sigma-Aldrich) served as a loading control.

DT treatment and Treg cell transfer

For immunohistochemistry, Tregs were depleted from DEREG (7) and controlmice by i.p. injection of DT (50 mg/kg body weight; Merck) on two consecu-tive days, day �2 and day �1 before tissue collection. For Treg transfer, Tregcells were eliminated in 5- to 6-wk-old Foxp3DTR recipient mice (6). In brief, 50mg/kg DT (Sigma-Aldrich or Calbiochem) was injected i.p. into Foxp3DTR

mice on two consecutive days, day 1 and day 2. CD4� Foxp3GFP� cells (5 �105) isolated from Foxp3GFP reporter mice were injected i.v. on day 3 of theexperiment (13). DT was injected on days 4, 6, and 8, and mice were eutha-nized on day 9 of the experiment.

Results and DiscussionCentral to their model of Foxp3 function in epithelial cells, Liuand colleagues reported Foxp3 protein expression in epithelialcells by immunohistochemical staining (10). According to theirarguments, eliminating Foxp3-expressing cells in Foxp3DTR

mice is expected to dramatically destroy the epithelial tissue ar-chitecture (10). To investigate whether tissue destruction was aconsequence of DT-mediated depletion of Foxp3� cells, we in-jected Foxp3DTR (DEREG) mice with DT twice before theanalysis of several organs by H&E staining. No tissue destruc-tion could be observed in any of the analyzed organs (Fig. 1). Insharp contrast to the aforementioned observations, high levelsof Foxp3 protein were detected in cells with lymphoid but notepithelial morphology (Fig. 1) in all tissues where epithelialFoxp3 expression was reported. By using the commerciallyavailable mAb against Foxp3 (clone FJK-16s; eBioscience), wedid not detect any Foxp3 protein expression in epithelial cells ofthe lung, prostate, or thymic cortex (Fig. 1). The latter was infull agreement with a previous report (14), and staining was alsonot detectable even when Abs were used at a 10-fold higher con-centration (data not shown). Similar results were obtained us-ing a polyclonal, affinity-purified, rabbit anti-mouse Foxp3 Ab

(supplemental Fig. 1A).7 In addition to immunohistochemicalstaining of tissue sections, we also prepared whole tissue extractsfrom colon, lung, prostate, and spleen of wild-type, Rag1�/�,and Foxp3�/� mice for Western blot analysis of Foxp3 expres-sion. We failed to detect Foxp3 protein in lung and prostatetissues isolated from Rag-deficient mice, further corroboratingour findings through an independent method of Foxp3 detec-tion (Fig. 2). Furthermore, eliminating Foxp3-expressing cellsin Foxp3DTR (DEREG) (7) mice resulted in the loss of Foxp3signal among lymphoid cells, whereas very weak backgroundlevels of nonspecific cytoplasmic Foxp3 staining in epithelialcells remained unchanged (Fig. 1). Importantly, the overall tis-sue architecture supported by epithelial cells was unperturbedin the DT-treated Foxp3DTR (DEREG) mice. DT-treated ani-mals were followed closely in various experimental settings, andwe never observed tissue destruction. Furthermore, DEREGmice have been used by different groups (see for example Refs.15–19) with no reports of epithelial tissue damage.

7 The online version of this article contains supplemental material.

FIGURE 1. Lack of Foxp3 expression in epithelial cells. Prostate, lung, andthymus from DEREG mice with or without DT administration (1 �g twice ondays �2 and �1 before analysis) on either BALB/c or C57/Bl6 backgroundshowed no expression of Foxp3 in epithelial cells of prostatic glands, respiratoryepithelium of the bronchi or alveolar lining cells (intraparenchymal lymph nodein the left column with scattered Foxp3� Tregs; arrow) or in the thymic cortex(Foxp3� Tregs in the medulla serve as positive intrinsic control). The rate ofproliferation (Ki-67) and apoptosis (cleaved (cl.) caspase-3) in epithelial cellswas unaltered by DT injection (insets, third row from top; germinal center in alymph node used as positive control). Ki-67 labeling also served as a positivecontrol for nuclear Ags, similar to the expression of the androgen receptor (AR)in the prostate epithelial cells (inset, second row from top) representative for allmouse groups depicted). HE represents H&E staining.

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Because all studies by Liu et al. were performed in BALB/cmice, we crossed our C57BL/6 Foxp3DTR mice onto a BALB/cbackground for 10 generations to exclude strain-specific differ-ences (9–12). Identical to our results on the C57BL/6 back-ground, nonhematopoietic tissues were not affected by DT ad-ministration in BALB/c Foxp3DTR (DEREG) mice (Fig. 1).This was further confirmed by cleaved caspase-3 labeling ofprostate tissue showing, according to Ki-67 staining, very littleongoing apoptosis and a low rate of regular epithelial prolifer-ation, both of which remained unchanged in the differentmouse genotypes with or without DT injection (Fig. 1).

Additionally, in contrast to another study that reportedbroad Foxp3 expression by breast epithelial cells and its role asa cancer suppressor (12), we did not detect Foxp3 expression innormal breast tissue using the monoclonal (Fig. 3) or polyclonalAb (supplemental Fig. 1B). Again, tissue morphology was notaltered following DT treatment in this organ.

Altogether, we have been unable to independently confirmthe assertion that Foxp3 is highly expressed by epithelial cells.Therefore, we conclude that autoimmunity induced by elimi-nation of Foxp3-expressing cells does not represent the loss ofepithelial cells or its integrity (or architecture). To account forthese discordant results, we speculate that the polyclonal rabbit

anti-Foxp3 Ab used in previous studies of Foxp3 expression inepithelial cells nonspecifically cross-reacts with an unidentifiedprotein Ag in addition to or instead of Foxp3.

The strongest argument for the contention that Foxp3 ex-pression in epithelial cells is biologically important is derivedfrom earlier observation that the transfer of Foxp3-deficientbone marrow into Foxp3-sufficient, Rag-deficient recipientmice does not induce autoimmunity (11). This result suggestedthat Foxp3 deficiency in bone marrow-derived cells was not pri-mary to autoimmune disease instigation, challenging the cur-rent dogma. In a stark contrast to these findings, we and othergroups have reported that bone marrow transfer from Foxp3-deficient mice into Rag�/� recipients leads to autoimmunelymphoproliferative disease (2, 20, 21). Additionally, wild-typebone marrow transferred into Foxp3sf (where sf is scurfy)Rag�/� mice does not cause the disease (22). The disease wasnot caused by mature pathogenic T cells possibly contaminatedwithin the Foxp3sf donor bone marrow cells because the transferof fetal liver cells from Foxp3sf mice or bone marrow cells fromFoxp3sf nude mice, both of which do not contain any maturepathogenic T cells, also caused identical disease in irradiatedRag�/� recipients (14, 22). The usage of recipient mice with agenetic deficiency in T cell generation is essential for assessingthe contribution of Foxp3 expression in hematopoietic cells(22). As we demonstrated, the use of T lymphocyte-deficientrecipients is necessary because radiation-resistant Treg cells inwild-type recipients rapidly reconstitute the Treg cell compart-ment after irradiation (22). Thus, expanded host-derived Tregcells spare irradiated recipient mice from lethal autoimmunity,irrespective of the donor bone marrow genotype. Notably, anearly bone marrow transfer study cited by Liu and colleagues toindependently support their model for Foxp3 action in nonhe-matopoietic tissues used irradiated wild-type mice as recipientsof Foxp3-deficient bone marrow (23). Therefore, the result ob-tained by Liu and colleagues has not, to date, been indepen-dently reproduced. It should also be pointed out that the bonemarrow transfers in a study that championed a role for Foxp3expression in nonhematopoietic cells lacked a positive control,i.e., bone marrow transfer from Foxp3� mice into Foxp3�/�

Rag�/� mice, obfuscating the interpretation of the key finding(11). In contrast, our studies tested all combinations of donorand host Foxp3 genotypes and demonstrated no contribution ofFoxp3 deficiency in nonhematopoietic tissues to disease devel-opment (22). Finally, bone marrow transplantation serves as aneffective treatment for IPEX patients in agreement with ourbone marrow chimera studies in mice (24).

In addition to bone marrow transfer studies, a large body ofgenetic evidence has established that Foxp3 expression in T cellsand more specifically in Treg cells is required to prevent fatalautoimmunity. Mice with a Foxp3 deficiency restricted to the Tcell lineage through CD4-Cre-mediated deletion of a condi-tional Foxp3 allele are indistinguishable from mice with thegermline ablation of the Foxp3 gene (1, 14). In contrast, nearcomplete deletion of Foxp3 in thymic epithelial cells was incon-sequential, i.e., no changes in thymocyte development and nosigns of autoimmunity were observed (14). To accommodatethese findings, Liu and colleagues suggested that CD4-Cre isexpressed and mediates Foxp3 deletion in thymic epithelial cellsand that the latter, not Foxp3 deficiency in T cells, causes theautoimmune syndrome in these mice (11). However, extensivegenetic analyses of Cre-mediated recombination in CD4-Cre

FIGURE 2. Western blot analysis fails to detect Foxp3 protein expression inlung and prostate tissues in T cell-deficient mice. Lung, prostate, and spleenwere analyzed for Foxp3 expression by Western blotting of whole tissue ex-tracts. As negative controls, organs from Rag1�/� and Foxp3�/� mice were in-cluded, and nuclear (nucl.) extracts from CD4�GFP� Treg cells FACS purifiedfrom LN and spleens of Foxp3GFP mice served as positive control. Blots shownhere are representative of three separate experiments. wt, Wild type.

FIGURE 3. Breast tissue stains negative for Foxp3 in immunohistochemis-try. Absence of Foxp3 expression in normal epithelial cells of mammary glandsin breast tissue from DT-treated BALB/c and DEREG BALB/c mice; splenicparenchyma served as positive control. Expression of the estrogen receptor (ER)in the epithelium of mammary glands served as a control for the labeling ofnuclear Ags (inset).

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transgenic mice using a highly sensitive reporter allele failed todetect Cre expression in thymic epithelial cells, and geneticallycontrolled immunhistochemical analysis failed to detect Foxp3protein expression in thymic epithelial cells (14). Lastly, wehave demonstrated that Treg cell-specific Foxp3 deletion viaretroviral Cre delivery to purified Treg cells isolated from con-ditional Foxp3-knockout mice abrogates Treg cell suppressiveactivity, causing fulminating autoimmunity when these cellswere transferred into lymphopenic recipients either alone or to-gether with T cells from Foxp3�/� mice (25). Hence, fatal au-toimmunity in mice and humans with Foxp3 mutations or inmice engineered to inducibly eliminate Foxp3-expressing cellsis ascribed to the essential role of Foxp3 in Treg cells.

If autoimmunity in Foxp3-deficient mice and humans is indeedcaused by the lack of Treg cells, restoration of the Treg cell com-partment is predicted to cure disease. In agreement with this no-tion, we and five other groups have demonstrated that injectingpurified Treg cells into Foxp3-deficient mice is sufficient to pre-vent life-threatening autoimmunity (1, 21, 22, 26–28). Addition-ally, the transfer of Treg cells into Foxp3DTR mice treated with DTto eliminate Foxp3-expressing cells inhibits tissue pathology in theliver, lung, and skin (Fig. 4). In contrast to these results, Liu andcolleagues recently demonstrated that injecting sorted Treg cellsinto scurfy mice did not alleviate morbidity (9). Thus, our resultsand the published data from a number of other groups are at oddswith those generated in the Liu laboratory.

In summary, a significant body of genetic, immunohisto-chemical, and genetically controlled functional studies by usand others is irreconcilable with the view that Foxp3 function inepithelial cells, including the thymic epithelium, is a key factorin the prevention of severe autoimmune lesions associated withFoxp3 mutations.

DisclosuresThe authors have no financial conflict of interest.

References1. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the devel-

opment and function of CD4�CD25� regulatory T cells. Nat. Immunol. 4:330–336.

2. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell develop-ment by the transcription factor Foxp3. Science 299: 1057–1061.

3. Khattri, R., D. Kasprowicz, T. Cox, M. Mortrud, M. W. Appleby, M. E. Brunkow,S. F. Ziegler, and F. Ramsdell. 2001. The amount of scurfin protein determines pe-ripheral T cell number and responsiveness. J. Immunol. 167: 6312–6320.

4. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immunologicself-tolerance maintained by activated T cells expressing IL-2 receptor �-chains(CD25): breakdown of a single mechanism of self-tolerance causes various autoim-mune diseases. J. Immunol. 155: 1151–1164.

5. Sakaguchi, S., T. Yamaguchi, T. Nomura, and M. Ono. 2008. Regulatory T cells andimmune tolerance. Cell 133: 775–787.

6. Kim, J. M., J. P. Rasmussen, and A. Y. Rudensky. 2007. Regulatory T cells preventcatastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8:191–197.

7. Lahl, K., C. Loddenkemper, C. Drouin, J. Freyer, J. Arnason, G. Eberl, A. Hamann,H. Wagner, J. Huehn, and T. Sparwasser. 2007. Selective depletion of Foxp3� reg-ulatory T cells induces a scurfy-like disease. J. Exp. Med. 204: 57–63.

8. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko,J. E. Wilkinson, D. Galas, S. F. Ziegler, and F. Ramsdell. 2001. Disruption of a newForkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disor-der of the scurfy mouse. Nat. Genet. 27: 68–73.

9. Chang, X., P. Zheng, and Y. Liu. 2008. Homeostatic proliferation in the mice withgermline FoxP3 mutation and its contribution to fatal autoimmunity. J. Immunol.181: 2399–2406.

10. Chen, G. Y., C. Chen, L. Wang, X. Chang, P. Zheng, and Y. Liu. 2008. Cutting edge:broad expression of the FoxP3 locus in epithelial cells: a caution against early inter-pretation of fatal inflammatory diseases following in vivo depletion of FoxP3-express-ing cells. J. Immunol. 180: 5163–5166.

11. Chang, X., J. X. Gao, Q. Jiang, J. Wen, N. Seifers, L. Su, V. L. Godfrey, T. Zuo,P. Zheng, and Y. Liu. 2005. The Scurfy mutation of FoxP3 in the thymus stroma leadsto defective thymopoiesis. J. Exp. Med. 202: 1141–1151.

12. Zuo, T., L. Wang, C. Morrison, X. Chang, H. Zhang, W. Li, Y. Liu, Y. Wang, X. Liu,M. W. Chan, et al. FOXP3 is an X-linked breast cancer suppressor gene and an im-portant repressor of the HER-2/ErbB2 oncogene. Cell 129: 1275–1286.

13. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, andA. Y. Rudensky. 2005. Regulatory T cell lineage specification by the Forkhead tran-scription factor Foxp3. Immunity 22: 329–341.

14. Liston, A., A. G. Farr, Z. Chen, C. Benoist, D. Mathis, N. R. Manley, andA. Y. Rudensky. 2007. Lack of Foxp3 function and expression in the thymic epithe-lium. J. Exp. Med. 204: 475–480.

15. Osorio, F., S. LeibundGut-Landmann, M. Lochner, K. Lahl, T. Sparwasser, G. Eberl,and C. Reis e Sousa. 2008. DC activated via dectin-1 convert Treg into IL-17 pro-ducers. Eur. J. Immunol. 38: 3274–3281.

16. Pellegrini, M., T. Calzascia, A. R. Elford, A. Shahinian, A. E. Lin, D. Dissanayake,S. Dhanji, L. T. Nguyen, M. A. Gronski, M. Morre, et al. 2009. Adjuvant IL-7 an-tagonizes multiple cellular and molecular inhibitory networks to enhance immuno-therapies. Nat. Med. 15: 528–536.

17. Zelinskyy, G., K. Dietze, T. Sparwasser, and U. Dittmer. 2009. Regulatory T cellssuppress antiviral immune responses and increase viral loads during acute infectionwith a lymphotropic retrovirus. PLoS Pathog. 5: e1000406.

18. Zelinskyy, G., K. K. Dietze, Y. P. Husecken, S. Schimmer, S. Nair, T. Werner,K. Gibbert, O. Kershaw, A. D. Gruber, T. Sparwasser, and U. Dittmer. 2009. Theregulatory T cell response during acute retroviral infection is locally defined and con-trols the magnitude and duration of the virus-specific cytotoxic T cell response. Blood114: 3199–3207.

19. von Allmen, C. E., N. Schmitz, M. Bauer, H. J. Hinton, M. O. Kurrer, R. B. Buser,M. Gwerder, S. Muntwiler, T. Sparwasser, R. R. Beerli, and M. F. Bachmann. 2009.Secretory phospholipase A2-IID is an effector molecule of CD4�CD25� regulatoryT cells. Proc. Natl. Acad. Sci. USA 106: 11673–11678.

20. Chikuma, S., and J. A. Bluestone. 2007. Expression of CTLA-4 and FOXP3 in cisprotects from lethal lymphoproliferative disease. Eur. J. Immunol. 37: 1285–1289.

21. Sather, B. D., P. Treuting, N. Perdue, M. Miazgowicz, J. D. Fontenot,A. Y. Rudensky, and D. J. Campbell. 2007. Altering the distribution of Foxp3� reg-ulatory T cells results in tissue-specific inflammatory disease. J. Exp. Med. 204:1335–1347.

22. Komatsu, N., and S. Hori. 2007. Full restoration of peripheral Foxp3� regulatory Tcell pool by radioresistant host cells in scurfy bone marrow chimeras. Proc. Natl. Acad.Sci. USA 104: 8959–8964.

23. Godfrey, V. L., J. E. Wilkinson, E. M. Rinchik, and L. B. Russell. 1991. Fatal lym-phoreticular disease in the scurfy (sf) mouse requires T cells that mature in a sf thymicenvironment: potential model for thymic education. Proc. Natl. Acad. Sci. USA 88:5528–5532.

24. Torgerson, T. R., and H. D. Ochs. 2007. Immune dysregulation, polyendocrinopa-thy, enteropathy, X-linked: Forkhead box protein 3 mutations and lack of regulatoryT cells. J. Allergy Clin. Immunol. 120: 744–750, quiz 751–742.

25. Williams, L. M., and A. Y. Rudensky. 2007. Maintenance of the Foxp3-dependentdevelopmental program in mature regulatory T cells requires continued expression ofFoxp3. Nat. Immunol. 8: 277–284.

26. Dudda, J. C., N. Perdue, E. Bachtanian, and D. J. Campbell. 2008. Foxp3� regula-tory T cells maintain immune homeostasis in the skin. J. Exp. Med. 205: 1559–1565.

27. Huter, E. N., G. A. Punkosdy, D. D. Glass, L. I. Cheng, J. M. Ward, andE. M. Shevach. 2008. TGF-�-induced Foxp3� regulatory T cells rescue scurfy mice.Eur. J. Immunol. 38: 1814–1821.

28. Smyk-Pearson, S. K., A. C. Bakke, P. K. Held, and R. S. Wildin. 2003. Rescue of theautoimmune scurfy mouse by partial bone marrow transplantation or by injectionwith T-enriched splenocytes. Clin. Exp. Immunol. 133: 193–199.

FIGURE 4. Reconstitution of Foxp3DTR mice with DT-insensitive Tregcells rescues autoimmunity. Treg cell-depleted Foxp3DTR mice were either un-treated (n � 3) or reconstituted (n � 3) with DT-insensitive Treg cells. Rep-resentative H&E staining of skin (ear), liver, and lung sections is shown. Micewere euthanized on day 9 after Treg cell depletion. Data are representative oftwo independent experiments.

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