Increased Foxp3+ CD4+ Regulatory T Cells with Intact Suppressive Activity but Altered Cellular Localization in Murine Lupus
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080314
ISSN1525-2191
AutoresJun Abe, Satoshi Ueha, Jun‐ichi Suzuki, Yoshiaki Tokano, Kouji Matsushima, Sho Ishikawa,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoFoxp3+ CD4+ regulatory T (Treg) cells play a pivotal role in the maintenance of dominant self tolerance. Understanding how the failures of immune control by Treg cells are involved in autoimmune diseases is important for the development of effective immunotherapies. In the present study, we analyzed the characteristics of endogenous Treg cells in (NZB × NZW) F1 (BWF1) mice, a murine model of systemic lupus erythematosus. Unexpectedly, Treg number and frequency in aged BWF1 mice with developing lupus nephritis were increased, not decreased, and in vitro suppressive activity in lymphoid organs was intact. In addition, Treg cells trafficked to target organs because cells were present in the kidney and lung. Treg cells of aged BWF1 mice exhibited altered localization within lymph organs, however, and an altered phenotype, with higher expression levels of chemokine receptors and activation markers, suggesting a highly activated cellular state. Notably, the expression levels of co-stimulatory molecules were also markedly enhanced in the Treg cells of aged BWF1 mice. Furthermore, Treg cells of BWF1 mice did not show any suppressive effects on antibody production in vitro. Taken together, we conclude that Treg cells in BWF1 mice are not predisposed to functional incompetence but rather are present in a highly activated state. Foxp3+ CD4+ regulatory T (Treg) cells play a pivotal role in the maintenance of dominant self tolerance. Understanding how the failures of immune control by Treg cells are involved in autoimmune diseases is important for the development of effective immunotherapies. In the present study, we analyzed the characteristics of endogenous Treg cells in (NZB × NZW) F1 (BWF1) mice, a murine model of systemic lupus erythematosus. Unexpectedly, Treg number and frequency in aged BWF1 mice with developing lupus nephritis were increased, not decreased, and in vitro suppressive activity in lymphoid organs was intact. In addition, Treg cells trafficked to target organs because cells were present in the kidney and lung. Treg cells of aged BWF1 mice exhibited altered localization within lymph organs, however, and an altered phenotype, with higher expression levels of chemokine receptors and activation markers, suggesting a highly activated cellular state. Notably, the expression levels of co-stimulatory molecules were also markedly enhanced in the Treg cells of aged BWF1 mice. Furthermore, Treg cells of BWF1 mice did not show any suppressive effects on antibody production in vitro. Taken together, we conclude that Treg cells in BWF1 mice are not predisposed to functional incompetence but rather are present in a highly activated state. Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology characterized by a massive production of autoantibodies against various nuclear antigens. The deposit of immune complexes in the target organs, ie, skin, kidney, lung, joints, and central nervous system, is thought to cause fatal dysfunction of the body system. (NZB × NZW) F1 (BWF1) is a mouse strain that has been widely used as a model for SLE since the 1960s. These mice spontaneously develop severe autoimmune disease highly resembling human SLE in terms of serological and hematological abnormalities, and severe nephritis accompanying massive production of anti-nuclear antibodies.1Andrews BS Eisenberg RA Theofilopoulos AN Izui S Wilson CB Mcconahey PJ Murphy ED Roths JB Dixon FJ Spontaneous murine lupus-like syndromes—clinical and immunopathological manifestations in several strains.J Exp Med. 1978; 148: 1198-1215Crossref PubMed Scopus (1337) Google Scholar Reconstitution of SCID (severe combined immunodeficiency) mice with cultured pre-B cells of BWF1 mice recapitulates many symptoms of the disease of BWF1 mice. Cultured pre-B cells alone, however, are not sufficient to fully reproduce the disease.2Reininger L Radaszkiewicz T Kosco M Melchers F Rolink AG Development of autoimmune-disease in SCID mice populated with long-term in vitro proliferating (NZB × NZW)F1 pre-B cells.J Exp Med. 1992; 176: 1343-1353Crossref PubMed Scopus (108) Google Scholar These data suggest that cellular subset(s) in addition to B cells are necessary for the development of the lupus-like syndrome of BWF1 mice, although abnormalities of the immune system predominantly lie within B cells. One of the possible candidates is CD4+ T cells, because depletion of CD4+ T cells with anti-CD4 antibody from 5 months old, slightly before the disease onset, prevents the development of the disease.3Wofsy D Chiang NY Greenspan JS Ermak TH Treatment of murine lupus with monoclonal antibody to L3T4. I. Effects on the distribution and function of lymphocyte subsets and on the histopathology of autoimmune disease.J Autoimmun. 1988; 1: 415-431Crossref PubMed Scopus (23) Google Scholar, 4Ermak TH Steger HJ Wofsy D Treatment of murine lupus with monoclonal-antibody to L3T4. II. Effects on immunohistopathology of thymus, spleen, and lymph node.Lab Invest. 1989; 61: 447-456PubMed Google Scholar CD4+ T cells are, therefore, also required for the development of the disease in BWF1 mice, possibly by providing help for the production of high-affinity autoantibodies. Studies in this decade have clearly shown the key roles of naturally occurring regulatory T (Treg) cells in the maintenance of dominant self tolerance of the immune system.5Sakaguchi S Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2934) Google Scholar Treg cells in normal mice are mostly of thymic origin and are considered to be autoreactive T-cell clones that have bypassed negative selection by unknown mechanism(s).6Liston A Rudensky AY Thymic development and peripheral homeostasis of regulatory T cells.Curr Opin Immunol. 2007; 19: 176-185Crossref PubMed Scopus (127) Google Scholar There also exists Treg cells of extra-thymic origin induced from conventional T cells during immune responses,7Kretschmer K Apostolou I Hawiger D Khazaie K Nussenzweig MC von Boehmer H Inducing and expanding regulatory T cell populations by foreign antigen.Nat Immunol. 2005; 6: 1219-1227Crossref PubMed Scopus (1048) Google Scholar although the underlying mechanisms of this process are still unclear. Foxp3, a member of forkhead-box family of transcription factors, is specifically expressed in the whole life of Treg cells and programs their functional properties.8Gavin MA Rasmussen JP Fontenot JD Vasta V Manganiello VC Beavo JA Rudensky AY Foxp3-dependent programme of regulatory T-cell differentiation.Nature. 2007; 445: 771-775Crossref PubMed Scopus (932) Google Scholar, 9Williams LM Rudensky AY Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3.Nat Immunol. 2007; 8: 277-284Crossref PubMed Scopus (698) Google Scholar, 10Lin W Haribhai D Relland LM Truong N Carlson MR Williams CB Chatila TA Regulatory T cell development in the absence of functional Foxp3.Nat Immunol. 2007; 8: 359-368Crossref PubMed Scopus (389) Google Scholar In contrast to the previously used marker CD25 or combination of CD25 and CD62L, expression of Foxp3 is specific for Treg cells, and thus can be used for the definitive identification of these cells.11Fontenot JD Rasmussen JP Williams LM Dooley JL Farr AG Rudensky AY Regulatory T cell lineage specification by the forkhead transcription factor FoxP3.Immunity. 2005; 22: 329-341Abstract Full Text Full Text PDF PubMed Scopus (1941) Google Scholar Immunoregulatory function of Treg cells is dependent on Foxp3 and genetic deficiency of Foxp3 causes fatal organ-specific autoimmune disease because of the lack of functional Treg cells.12Fontenot JD Gavin MA Rudensky AY Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (6209) Google Scholar, 13Hori S Nomura T Sakaguchi S Control of regulatory T cell development by the transcription factor Foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (58) Google Scholar, 14Khattri R Cox T Yasayko SA Ramsdell F An essential role for Scurfin in CD4+CD25+ T regulatory cells.Nat Immunol. 2003; 4: 337-342Crossref PubMed Scopus (2414) Google Scholar Furthermore, many groups have reported the reduced number and/or function of Treg cells in both organ-specific and systemic autoimmune diseases.15Valencia X Lipsky PE CD4+CD25+FoxP3+ regulatory T cells in autoimmune diseases.Nat Clin Pract Rheumatol. 2007; 3: 619-626Crossref PubMed Scopus (132) Google Scholar A recent study has shown that the decreased frequency of Treg cells in the peripheral blood was associated with disease activity in SLE patients.16Valencia X Yarboro C Illei G Lipsky PE Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus.J Immunol. 2007; 178: 2579-2588PubMed Google Scholar Frequency of Treg cells identified as CD25+ CD62Lhi CD4+ T cells in the spleen was also decreased in aged BWF1 mice.17Scalapino KJ Tang QZ Bluestone JA Bonyhadi ML Daikh DI Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells.J Immunol. 2006; 177: 1451-1459Crossref PubMed Scopus (210) Google Scholar Accordingly, adoptive transfer of in vitro-expanded Treg cells, or administration of histone-derived peptides or peptides derived from the complementarity-determining region 1 of anti-double-strand DNA immunoglobulin has been shown to ameliorate the disease in BWF1 mice by a mechanism involving Treg cells.17Scalapino KJ Tang QZ Bluestone JA Bonyhadi ML Daikh DI Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells.J Immunol. 2006; 177: 1451-1459Crossref PubMed Scopus (210) Google Scholar, 18Sharabi A Zinger H Zborowsky M Sthoeger ZM Mozes E A peptide based on the complementarity-determining region 1 of an autoantibody ameliorates lupus by up-regulating CD4+CD25+ cells and TGF-β.Proc Natl Acad Sci USA. 2006; 103: 8810-8815Crossref PubMed Scopus (82) Google Scholar, 19La Cava A Ebling FM Hahn BH Ig-reactive CD4+ CD25+ T cells from tolerized (New Zealand Black × New Zealand White)F-1 mice suppress in vitro production of antibodies to DNA.J Immunol. 2004; 173: 3542-3548PubMed Google Scholar, 20Kang HK Michaels MA Berner BR Datta SK Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets.J Immunol. 2005; 174: 3247-3255PubMed Google Scholar These studies suggest that the function of endogenous Treg cells is, at least partially, abrogated by unidentified mechanisms in BWF1 mice. Despite the effort to develop therapeutic methods involving Treg cells, their nature in BWF1 mice remains unclear. Here we performed a detailed characterization of Treg cells in BWF1 mice using Foxp3 as their marker. Our results demonstrated that aged BWF1 mice had increased frequency and number of Treg cells with apparently normal function, but with an activated phenotype including enhanced expression of co-stimulatory molecules and altered localization. Female 6- to 8-week-old BWF1 and BALB/c mice were purchased from Japan SLC (Shizuoka, Japan) and were kept under specific pathogen-free conditions in the animal facility of our laboratory until analysis. Mice were used at 6 to 10 or 32 to 40 weeks of age as young or aged, respectively. All animal experiments were approved by the animal care committee of The University of Tokyo. Monoclonal anti-mouse CD4 (clone RM4-5), CD5 (55-7.3), CD8α (53-6.7), CD11b (M1/70), CD16/32 (2.4G2), CD19 (1D3), CD23 (B3B4), CD25 (7D4), CD43 (S7), CD44 (IM7), CD45 (30-F11), CD45R (RA3-6B2), CD62L (MEL-14), CD69 (H1.2F3), CD90.2 (53-2.1), CD103 (M290), OX40 (OX-86), CXCR4 (2B11/CXCR4), CCR5 (C34-3448), NK1.1 (PK136), TER-119 (TER-119), and streptavidin were purchased from BD Biosciences (San Diego, CA); monoclonal anti-mouse 4-1BB (17B5), ICOS (7E.17G9), F4/80 (BM8), CCR7 (4B12), and Foxp3 (FJK-16s) were purchased from eBioscience (San Diego, CA); monoclonal anti-mouse CXCR3 (220803) was purchased from R&D Systems (Minneapolis, MN). Antiserum raised against mouse type IV collagen was purchased from LSL (Tokyo, Japan). Details of monoclonal anti-mouse CCR4 antibody (clone 2G11) will be described elsewhere by Nagakubo and colleagues.21Tsunemi Y Saeki H Nakamura K Nagakubo D Nakayama T Yoshie O Kagami S Shimazu K Kadono T Sugaya M Komine M Matsushima K Tamaki K CCL17 transgenic mice show an enhanced Th2-type response to both allergic and non-allergic stimuli.Eur J Immunol. 2006; 36: 2116-2127Crossref PubMed Scopus (43) Google Scholar, 22Ueha S Yoneyama H Hontsu S Kurachi M Kitabatake M Abe J Yoshie O Shibayama S Sugiyama T Matsushima K CCR7 mediates the migration of Foxp3+ regulatory T cells to the paracortical areas of peripheral lymph nodes through high endothelial venule.J Leukoc Biol. 2007; 82: 1230-1238Crossref PubMed Scopus (39) Google Scholar Single cell suspensions of the thymus, spleen, and lymph nodes were prepared by passing the tissue through a cell strainer (BD Bioscience). Single cell suspension of the kidney and lung were prepared by dissociating the tissue with collagenase D (Roche, Basel, Switzerland). Mononuclear cells in the kidney and lung were isolated from the single cell suspension by Percoll (Invitrogen, Carlsbad, CA) gradient centrifugation. CD25+ CD4+ T cells were isolated from the single cell suspension of various organs by magnetic enrichment of CD25+ cells followed by fluorescence-activated cell sorting with the Epics Altra cell sorter (Beckman Coulter, Fullerton, CA). CD25− CD4+ T cells were isolated from the single cell suspension of spleen by magnetic depletion of the cells positive for CD8α, CD11b, CD25, B220, CD138, NK1.1, or TER-119. B1 cells were isolated from peritoneal lavage cells by magnetic depletion of the cells positive for CD23, Thy-1.2, or F4/80. B2 cells were isolated from spleen by magnetic depletion of the cells positive for CD43, Thy-1.2, or TER-119. All procedures involving magnetic isolation were performed with an autoMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were incubated with fluorochrome- or biotin-labeled antibodies for 20 minutes at 4°C, following the blockade of FcγRII/III with unlabeled anti-CD16/32 for 10 minutes at 4°C; for the staining with biotin-labeled anti-CCR7, incubation after the blockade of Fc receptors was performed at 37°C. Biotin-labeled antibodies were visualized by further incubating with phycoerythrin-conjugated streptavidin for 15 minutes at 4°C. Staining of Foxp3 was performed according to the manufacturer's instructions. Data were collected using BD LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR). Explanted tissues embedded in OCT compound were snap-frozen in liquid nitrogen and stored at −80°C until use. Six-μm-thick sections of frozen tissues were fixed with cold acetone for 10 minutes and rehydrated with phosphate-buffered saline (PBS) for 10 minutes at room temperature. Rehydrated sections were blocked for nonspecific binding of proteins with Blocking One (Nacalai Tesque, Kyoto, Japan) for 20 minutes at room temperature and incubated with unlabeled or biotinylated antibodies, or antisera for 60 minutes at room temperature. Sections were then incubated with Alexa Fluor-labeled anti-Ig secondary antibodies or streptavidin (Invitrogen) for 30 minutes at room temperature. After the staining, sections were fixed with phosphate-buffered 4% paraformaldehyde for 10 minutes at room temperature and were mounted with Prolong Gold Antifade Reagent (Invitrogen). Specimens were observed under IX70 confocal laser-scanning microscopy (Olympus, Tokyo, Japan). Images obtained from confocal microscopic observation were processed with Win ROOF software (Mitani Corporation, Fukui, Japan), and the number of the signals was counted manually or automatically using Win ROOF software. 2 × 104 cells/well of purified CD25− CD4+ T cells were stimulated with 2 μg/ml of concanavalin A (Sigma-Aldrich, St. Louis, MO) and 8 × 104 cells/well of mitomycin-C (Sigma-Aldrich)-treated Thy1.2− splenocytes with or without titrated number of CD25+ CD4+ T cells were incubated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mmol/L HEPES, 55 μmol/L 2-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin in a round-bottom 96-well plate for 72 hours at 37°C. CD25+ CD4+ T cells were cultured under the same conditions to measure their proliferative capacity in the absence of CD25− CD4+ T cells. Cells were pulsed with 1 μCi/well [3H-methyl]-thymidine (GE Health Care, Buckinghamshire, UK) for the last 6 to 8 hours of the culture, and proliferation was measured by cpm value of the harvested cells. Suppressive activity of CD25+ CD4+ T cells was expressed as percent suppression23Itoh M Takahashi T Sakaguchi N Kuniyasu Y Shimizu J Otsuka F Sakaguchi S Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance.J Immunol. 1999; 162: 5317-5326PubMed Google Scholar calculated as following: 100 × [cpm (responder) − cpm(CD25+ + CD25−)]/cpm(responder). In vitro antibody production by B cells was analyzed as previously described24Sekigawa I Ishida Y Hirose S Sato H Shirai T Cellular basis of in vitro anti-DNA antibody-production—evidence for T-cell dependence of IgG-class anti-DNA antibody-synthesis in the (NZB × NZW)F1 hybrid.J Immunol. 1986; 136: 1247-1252PubMed Google Scholar with several modifications. Briefly, 2 × 105 B1 or B2 cells isolated from young or aged BWF1 mice and equal numbers of CD25− CD4+ T cells isolated from the spleen of young or aged BWF1 mice were cultured with or without 1 × 105 CD25+ CD4+ T cells in supplemented RPMI 1640 medium for 5 days at 37°C. The concentration of IgG antibody in the culture supernatant was measured by enzyme-linked immunosorbent assay using a Mouse IgG quantitation kit (Bethyl, Montgomery, TX). Mice were perfused with 30 mL of PBS, and spleen, lymph nodes, kidney, and lung were excised. Tissues were homogenated with TRIzol reagent (Invitrogen), and purified total RNA was reverse-transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with an ABI Prism 7500 (Applied Biosystems) using primers and Taq Man probes listed in Table 1.Table 1Primers and Probes for Real-Time PCRGeneSenseProbeAntisenseCCL195′-GAAAGCCTTCCGCTACCTTCT-3′5′-CCCATCCCGGCAATCCTGTTCTTA-3′5′-CCCTTAGTGTGGTGAACACAACA-3′CCL215′-GGCTATAGGAAGCAAGAACCAAGT-3′5′-TTACTTCTACCGACGTCCCACGGA-3′5′-TCAGGCTTAGAGTGCTTCCG-3′CXCL95′-TGATAAGGAATGCACGATGCTC-3′5′-AGCCGAGGCACGATCCACTACAAATC-3′5′-TTCCTTGAACGACGACGACTTT-3′CXCL105′-CGTCATTTTCTGCCTCATCCT-3′5′-AAGCTTGAAATCATCCCTGCGAGCC-3′5′-TGGTCTTAGATTCCGGATTCAG-3′CXCL125′-GCTCTGCATCAGTGACGGTAA-3′5′-ATCGCCAGAGCCAACGTCAAGCAT CT-3′5′-AGCCGTGCAACAATCTGAAG-3′GAPDH5′-AGTATGACTCCACTCACGGCAA-3′5′-AACGGCACAGTCAAGGCCGAGAAT-3′5′-TCTCGCTCCTGGAAGATGGT-3′ Open table in a new tab Statistical significance of the difference between data sets was analyzed by Welch's unpaired t-test for the comparison of two groups or by one-way analysis of variance with Bonferroni's multiple comparison test for more than three groups. P < 0.05 was considered to be statistically significant. Suppressive activity of Treg cells is strongly correlated with the expression of Foxp3.11Fontenot JD Rasmussen JP Williams LM Dooley JL Farr AG Rudensky AY Regulatory T cell lineage specification by the forkhead transcription factor FoxP3.Immunity. 2005; 22: 329-341Abstract Full Text Full Text PDF PubMed Scopus (1941) Google Scholar To clarify whether an increase or decrease in the frequency and/or number of Treg cells exists, we analyzed the population of Foxp3+ CD4+ T cells by flow cytometry. We found that aged BWF1 mice had substantially increased frequency (Figure 1A) and number (Figure 1B) of Foxp3+ CD4+ T cells in the lymphoid organs compared with young BWF1 mice. A recent study has shown an age-dependent increase in CD25− Foxp3+ CD4+ T cells in 24-month-old normal mice,25Nishioka T Shimizu J Iida R Yamazaki S Sakaguchi S CD4+CD25+Foxp3+ T cells and CD4+CD25−Foxp3+ T cells in aged mice.J Immunol. 2006; 176: 6586-6593PubMed Google Scholar but increased Foxp3+ CD4+ T cells in aged BWF1 mice was not merely an age-dependent event because age-matched BALB/c mice did not show a marked increase in Foxp3+ CD4+ T cells (Figure 1, C and D). Valencia and colleagues16Valencia X Yarboro C Illei G Lipsky PE Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus.J Immunol. 2007; 178: 2579-2588PubMed Google Scholar reported a decreased suppressive activity of CD25+ CD4+ T cells in SLE patients, possibly because of the lower proportion of Foxp3+ cells among CD25+ CD4+ T cells. This result, however, does not exclude the possibility that a functional defect intrinsic to Treg cells exists as well. To test the functional competency of Treg cells of BWF1 mice, we performed an in vitro suppression assay. Because Foxp3 expression could be detected only in permeabilized cells, we used CD25+ CD4+ T cells as a surrogate for Foxp3+ CD4+ T cells. Concurrent with the high proportion of Foxp3+ cells among CD25+ CD4+ T cells even after disease onset (Figure 2A), CD25+ CD4+ T cells isolated from the spleen and lymph nodes of both young and aged BWF1 mice did not proliferate on stimulation (Figure 2B) and showed suppressive activity (Figure 2C). Furthermore, CD25+ CD4+ T cells isolated from the kidney (Figure 2C) and lung (data not shown), ie, the target organs, of aged BWF1 mice also showed suppressive activity comparable to those from the spleen and lymph nodes. CD25+ CD4+ T cells of thymus or lymph nodes showed similar suppressive activity (data not shown). We did not note a significant difference in the suppressive activity between young and aged, or lymphoid and nonlymphoid CD25+ CD4+ T cells in BWF1 mice at any dose of CD25+ CD4+ T cells. Taken together, our data suggest that aged BWF1 mice have an expanded pool size of Treg cells with intact suppressive activity. Defective migration into the site of inflammation is known to impair the in vivo suppressive activity of Treg cells even if they were functionally competent in vitro.26Tang QZ Adams JY Tooley AJ Bi MY Fife BT Serra P Santamaria P Locksley RM Krummel MF Bluestone JA Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice.Nat Immunol. 2006; 7: 83-92Crossref PubMed Scopus (664) Google Scholar Because our data indicated that Treg cells of BWF1 mice have intact suppressive activity in vitro, we asked whether Treg cells in aged BWF1 mice infiltrated into the target organs, ie, kidney and lung. Flow cytometric analysis of mononuclear cells within the target organs revealed that Foxp3+ as well as Foxp3− CD4+ T cells infiltrated into these organs, and that the frequency of Foxp3+ cells in CD4+ T cells was comparable to that in the lymph nodes of normal mice11Fontenot JD Rasmussen JP Williams LM Dooley JL Farr AG Rudensky AY Regulatory T cell lineage specification by the forkhead transcription factor FoxP3.Immunity. 2005; 22: 329-341Abstract Full Text Full Text PDF PubMed Scopus (1941) Google Scholar (18.76 ± 3.79% in the kidney and 14.08 ± 2.50% in the lung). Foxp3+ CD4+ T cells infiltrated into the glomeruli, interstitium, and perivascular region of the kidney along with Foxp3− CD4+ T cells (Figure 3B). Young BWF1 mice and nonautoimmune control mice did not show the infiltration of inflammatory cells (data not shown). Moreover, both Foxp3+ and Foxp3− CD4+ T cells were apparently distributed evenly within the infiltrating site of the target organs (Figure 3, A and C), indicating that clustering of these cells that were essential for Treg cell-mediated suppression26Tang QZ Adams JY Tooley AJ Bi MY Fife BT Serra P Santamaria P Locksley RM Krummel MF Bluestone JA Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice.Nat Immunol. 2006; 7: 83-92Crossref PubMed Scopus (664) Google Scholar, 27Tadokoro CE Shakhar G Shen SQ Ding Y Lino AC Maraver A Lafaille JJ Dustin ML Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.J Exp Med. 2006; 203: 505-511Crossref PubMed Scopus (412) Google Scholar would take place in the target organs as well as in the lymphoid organs. The thymus, another target organ of the disease in BWF1 mice, is the major site of the development of Treg cells.6Liston A Rudensky AY Thymic development and peripheral homeostasis of regulatory T cells.Curr Opin Immunol. 2007; 19: 176-185Crossref PubMed Scopus (127) Google Scholar Disruption of the architecture of the thymic medulla where development of Treg cells occurs is known to impair that process.28Fontenot JD Dooley JL Farr AG Rudensky AY Developmental regulation of Foxp3 expression during ontogeny.J Exp Med. 2005; 202: 901-906Crossref PubMed Scopus (340) Google Scholar To determine whether Treg cells are properly located within the thymus, we analyzed thymic sections by immunofluorescent staining. In BWF1 mice, thymic architecture is strongly affected by the disease,4Ermak TH Steger HJ Wofsy D Treatment of murine lupus with monoclonal-antibody to L3T4. II. Effects on immunohistopathology of thymus, spleen, and lymph node.Lab Invest. 1989; 61: 447-456PubMed Google Scholar, 29Sato T Ishikawa S Akadegawa K Ito T Yurino H Kitabatake M Yoneyama H Matsushima K Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus.Eur J Immunol. 2004; 34: 3346-3358Crossref PubMed Scopus (67) Google Scholar but medullary localization of Treg cells remained virtually unchanged even after the manifestation of severe nephritis (Figure 3, D and E). Localization of Treg cells within the thymus is also confined to the medulla in BALB/c mice irrespective of their age (Supplemental Figure 1, A and B, see http://ajp.amjpathol.org). Treg cells have to be located in the site of antigen presentation within the secondary lymphoid organs to make contacts with their target cells.26Tang QZ Adams JY Tooley AJ Bi MY Fife BT Serra P Santamaria P Locksley RM Krummel MF Bluestone JA Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice.Nat Immunol. 2006; 7: 83-92Crossref PubMed Scopus (664) Google Scholar, 27Tadokoro CE Shakhar G Shen SQ Ding Y Lino AC Maraver A Lafaille JJ Dustin ML Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.J Exp Med. 2006; 203: 505-511Crossref PubMed Scopus (412) Google Scholar Because our analyses on the number, function, and site of the development of Treg cells could not find any obvious defect, we examined the localization of Treg cells within the secondary lymphoid organs of BWF1 mice. Treg cells in aged BWF1 mice were located in the follicles and red pulp as well as periaortic lymphoid sheath in the spleen, whereas Treg cells in young BWF1 mice were mostly located in the periaortic lymphoid sheath (Figure 3, F and G; Supplemental Figure 2, see http://ajp.amjpathol.org). Similar localization of Treg cells were observed in the renal lymph node where Treg cells were located in the follicles and medulla as well as paracortex in aged BWF1 mice, whereas the localization of Treg cells in young BWF1 mice was relatively confined to paracortex (Figure 3, H and I; Supplemental Figure 2, see http://ajp.amjpathol.org). Such altered localization was not limited to Treg cells, but was also seen in Foxp3− conventional T cells. In contrast, localization of Treg cells in BALB/c mice was not altered with age and was similar to that of young BWF1 mice (Supplemental Figures 1, C–F, and 2, see http://ajp.amjpathol.org). Localization of T cells is tightly regulated by various chemokines and their receptors to achieve efficient induction of immune response or tolerance.30Cyster JG Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs.Annu Rev Immunol. 2005; 23: 127-159Crossref PubMed Scopus (727) Google Scholar To elucidate the molecule(s) responsible for the altered localization of Treg cells, we next analyzed the expression of chemokine receptors involved in the function of Treg cells31Sather BD Treuting P Perdue N Miazgowicz M Fontenot JD Rudensky AY Campbell DJ Altering the distribution of Foxp3+ regulatory T cells results in tissue-specific inflammatory disease.J Exp Med. 2007; 204: 1335-1347Crossref PubMed Scopus (330) Google Scholar, 32Yuan Q Bromley SK Means TK Jones KJ Hayashi F Bhan AK Luster AD CCR4-dependent regulatory T cell function in inflammatory bowel disease.J Exp Med. 2007; 204: 1327-1334Crossref PubMed Scopus (106) Google Scholar, 33Wysocki CA Jiang Q Panoskaltsis-Mortari A Taylor PA McKinnon KP Su LS Blazar BR Serody JS Critical role for CCR5 in the function of donor CD4+CD25+ regulatory T cells during acute graft-versus-host disease.Blood. 2005; 106: 3300-3307Crossref PubMed Scopus (212) Google Scholar, 34Müller M Carter SL Hofer MJ Manders P Getts DR Getts MT Dreykluft A Lu B Gerard C King NJC Campbell IL CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system.J Immunol. 2007; 179: 2774-2786PubMed Google Scholar by flow cytometry. Both Foxp3+ and Foxp3− CD4+ T cells in the spleen showed decreased CCR7 expression (Figure 4C) and enhanced CXCR4 expression (Figure 4E) in aged BWF1 mice. On th
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