Cells Intestinal Lamina Propria As Regulatory T Cells in Human Bright CD25 +CD4

The Journal of Immunology

CD4؉CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells1

Shin Makita,* Takanori Kanai,2* Shigeru Oshima,* Koji Uraushihara,* Teruji Totsuka,* Taisuke Sawada,* Tetsuya Nakamura,* Kazutaka Koganei,† Tsuneo Fukushima,† and Mamoru Watanabe*

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4؉CD25bright T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4؉ T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4؉CD25؊ T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4؉CD25؉ T cells, however, cells expressing CD25 at high levels (CD4؉CD25bright) suppressed the proliferation and various cytokine productions of CD4؉CD25؊ T cells. LP CD4؉CD25bright T cells by themselves produced fewer amounts of IL-2, IFN-␥, and IL-10. Interestingly, LP CD4؉CD25bright T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4؉CD25bright T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis. The Journal of Immunology, 2004, 173: 3119–3130.

he gastrointestinal tract is home to the largest number of of an intact immune system for the intestinal homeostasis is re- leukocytes in the body as well as being the site where vealed by the fact that a number of immune manipulations, includ- these cells encounter abundant exogenous stimuli. De- ing deletion of cytokine genes and alterations in T subsets, lead T R spite this potential immune stimulus, it is well known that immune to the development of an animal model of inflammatory bowel responses in the intestine remain in a state of controlled inflam- disease (IBD) (10Ð12). Evidence emerging from these studies sug- mation (1). Regulation of the immune response here is a balance gests that pathogenic responses in the intestine are derived by res- between the need to mount protective immunity toward pathogens ident bacteria and are controlled by a functionally specialized pop- while not activating damaging inflammatory responses to the ulation of T cells in the GALT (13).

plethora of harmless Ags present, including those derived from A variety of TR cells that display regulatory function in vitro or resident bacteria (2Ð4). To maintain the intestinal homeostasis, in vivo have been described. These can be subdivided into different including immunological tolerance, functionally distinct subsets subsets based on the expression of cell surface markers, production have been clearly deﬁned in T cells (5, 6). Among these subsets, of cytokines, and mechanisms of action. Recent studies focused on the regulatory T (T )3 cell subset down-regulates immune re- ϩ R CD25 as the best marker for CD4 TR cells in mice and humans sponses for both foreign and self-Ags and effectively participates (14Ð16). CD4ϩCD25ϩ T cells, which constitute ϳ10% of periph- in the suppression of autoimmune disorders (7Ð9). The importance eral murine and human CD4ϩ T cells, express CTLA-4 (17Ð20), glucocorticoid-induced TNFR family-related protein (GITR) (21,

http://classic.jimmunol.org 22), and the forkhead/winged helix transcription factor Foxp3 (23Ð *Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; and †Department of Surgery, Yokohama City 25). Although autoimmune diseases, including IBD, can be in- Hospital, Yokohama, Japan duced by reconstituting immunodeﬁcient mice with peripheral ϩ ϩ ϩ Received for publication December 10, 2003. Accepted for publication June 23, 2004. CD4 T cells, which have been depleted of CD4 CD25 (26) or ϩ ϩ The costs of publication of this article were defrayed in part by the payment of page CD4 GITR T cells (27), not only the existence of human intes- charges. This article must therefore be hereby marked advertisement in accordance tinal CD4ϩCD25ϩ T cells in normal or diseased condition but with 18 U.S.C. Section 1734 solely to indicate this fact. R

Downloaded from also their role in the pathogenesis of IBD are largely unknown. 1 This study was supported in part by Grants-in-Aid for Scientific Research, Scientific Research on Priority Areas, Exploratory Research and Creative Scientific Research Conversely, several findings, for example. the evidence of the ac- ϩ ϩ ϩ from the Japanese Ministry of Education, Culture, Sports, Science and Technology; cumulated CD25 , CD69 , and CD71 (transferrin receptor) the Japanese Ministry of Health, Labor and Welfare; the Japan Medical Association; cells, indicate an increased state of activation of the T cell system Foundation for Advancement of International Science; Terumo Life Science Foun- dation; Ohyama Health Foundation; Yakult Bio-Science Foundation; and Research in IBD (28). Although intestinal lamina propria (LP) T cells are Fund of Mitsukoshi Health and Welfare Foundation. already preactivated, the activation is further increased in both the 2 Address correspondence and reprint requests to Dr. Takanori Kanai, Department of circulation and mucosa from IBD patients as determined by sev- Gastroenterology and Hepatology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail ad- eral activation markers as compared with those from normal indi- dress: [email protected] viduals (29). Furthermore, this activated phenotype is increased in 3 Abbreviations used in this paper: TR, regulatory T; IBD, inflammatory bowel dis- involved compared with uninvolved areas or control tissue (29), ease; GITR, glucocorticoid-induced TNFR family-related protein; LP, lamina propria; being so far believed that these activated CD4ϩCD25ϩ cells LPMC, lamina propria mononuclear cell; MMC, mitomycin C; CD, Crohn’s disease; UC, ulcerative colitis; rh, recombinant human; CT, critical threshold; PB, peripheral should be pathogenic for the development of IBD. These conflict- blood; MFI, mean fluorescence intensity. ing findings prompted us to determine whether or not the LP

CD4ϩCD25ϩ T cells in IBD patients were pathogenic (effector) or BNI3, PE-conjugated anti-CTLA-4; MQ1-17, PE-conjugated

protective (TR) in this study. anti-IL-2; JES3-19F1, PE-conjugated anti-IL-10; 4S.B3, PE- and Furthermore, there is much other evidence showing that 1) the FITC-conjugated anti-IFN-␥ (BD Pharmingen, San Diego, CA); peripheral CD4ϩCD25Ϫ T cell population also possesses some 110416, PE-conjugated anti-human GITR (R&D Systems, regulatory activity (26, 30Ð34), 2) anergic/memory T cells have Minneapolis, MN). regulatory properties (35Ð38), and 3) LP CD4ϩ T lymphocytes, Purified recombinant human IL-2 (rhIL-2; kindly provided by regardless of CD25 expression, are generally memory T cells, and Shionogi Pharmaceutical, Osaka, Japan), purified anti-human CD3 hyporesponsive to TCR-mediated proliferative signals (39, 40), mAb (HIT3a; BD Pharmingen), Con A (Sigma-Aldrich), PHA indicating these cells could be highly differentiated effector or ef- (Sigma-Aldrich), and mitomycin C (MMC, Sigma-Aldrich) were fector memory T cells (41, 42) with a raised threshold of activation used for in vitro coculture assay. that prevents immune responses to harmless intraluminal Ags. In the present study, we investigate the existence and the role of Isolation of LP mononuclear cells (LPMCs) from intestinal the human LP CD4ϩCD25bright as well as CD4ϩCD25ϩ and mucosa CD4ϩCD25Ϫ T cells. We demonstrate here that human LP CD4ϩ LPMCs were isolated from surgically resected intestinal speci- T cells from the normal intestine, regardless of CD25 expression, mens using enzymatic techniques as previously described (43). express other regulatory markers, CTLA-4, GITR, and Foxp3, and Briefly, the dissected mucosa was incubated in calcium and mag- show anergic and memory features, but only CD4ϩCD25bright LP nesium-free HBSS containing 2.5% FBS and 1 mM DTT (Sigma-

T cells retain TR function, which can mediate potent suppression Aldrich) to remove mucus. The mucosa was then incubated in of autologous T cell proliferation and various cytokine produc- medium containing 0.75 mM EDTA (Sigma-Aldrich) for 60 min at tions, such as IFN-␥ and IL-2. Furthermore, we show here that 37¡C. During this treatment, intraepithelial lymphocytes and epi-

these TR cells are significantly increased in patients with thelial cells were released from the tissue and tissues containing active IBD. LPMCs were collected and incubated in medium containing 0.02% collagenase (Worthington Biochemical, Freehold, NJ). The frac- Materials and Methods tion was pelleted twice through a 40% isotonic percoll solution and Patients and samples the cells were then centrifuged over a Ficoll-Hypaque density gra- dient (40% and 60%). The purity of resulting LPMCs was ana- Normal mucosal samples were obtained from macroscopically and lyzed by flow cytometry. microscopically unaffected areas of 54 colonic specimens from The CD4ϩ T cells from LPMCs and PBMCs were purified by patients with colon cancer who underwent surgery and were ob- positive selection with the CD4ϩ MultiSort kit (Miltenyi Biotec, tained from the surgical resected samples of intestinal inflamed Auburn, CA) (27). The MMC-treated CD4Ϫ fraction was used as mucosa of 24 patients with colonic type Crohn’s disease (CD) and APCs in the following experiments. The CD4ϩCD25Ϫ, 25 patients with ulcerative colitis (UC). The mucosa was prepared CD4ϩCD25ϩ, and CD4ϩCD25bright populations were isolated immediately after stripping away the underlying submucosa by from LPMCs and PBMCs by sorting using a FACSVantage (BD blunt dissection. All experiments were approved by the Committee Biosciences, San Jose, CA) in accordance with the method by on Human Subjects of both Tokyo Medical and Dental University by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. Baecher-Allan et al. (44), who demonstrated that the regulatory Hospital (Tokyo, Japan) and Yokohama City Hospital (Yokohama, CD4ϩ T cells in PBMCs of healthy human subjects preferentially Japan). Informed consent was obtained from all patients before the reside within the CD4ϩCD25bright T cell population. Cells were study. Disease activity in each patient with CD was analyzed ac- incubated with FITC-conjugated anti-CD4 and PE-conjugated anti- cording to the Crohn’s Disease Activity Index and endoscopic and CD25 mAbs. The analysis and sort gates were restricted to the histopathological data. The disease had been present from 6 mo to population of lymphocytes by means of their forward and side 15 years. When the experimental study was performed in patients scatter properties. Large, activated T cells were excluded. On re- with CD, 4 patients were taking only steroids, 4 were taking both analysis, the forward and side scatter properties of the steroids and sulfasalazine, 3 were receiving both azathiopurine and CD4ϩCD25bright cells were not appreciably different from those of ϩ Ϫ http://classic.jimmunol.org sulfasalazine, 11 were taking only sulfasalazine, and 2 had been the CD4 CD25 population, indicating that these cell populations undergoing nonspecific therapy for the previous 3 mo. In the UC are similar in size. In another set of experiments, LPMCs were group, disease activity was defined by the Truelove-Witts criteria stained with CyChrome-anti-CD4, FITC-anti-CD25, and PE-anti- and endoscopic (Matts grade) and histopathological data. When CD45RO or PE-anti-CD71 mAbs, and the various subsets of cells the experimental study was performed, 1 patient was receiving were purified by a FACSVantage. For polyclonal activation, cells only steroids, 14 were receiving both steroids and sulfasalazine, 6 were cultured with 5 ␮g/ml plate-bound anti-CD3 (UCHT1; BD were taking only sulfasalazine, 3 were receiving azathiopurine, Downloaded from Pharmingen) and 5 ␮g/ml soluble anti-CD28 (CD28.2; BD Pharm- steroids and sulfasalazine, and 1 patient had been undergoing non- ingen) for 48 h and used for FACS and RT-PCR analyses. specific treatment for at least 3 mo. FACS analysis of surface and intracellular Ags Culture media, reagents, and Abs For two-color flow cytometry analysis, freshly isolated LPMCs In all in vitro assays, cells were cultured in RPMI 1640 medium and PBMCs were stained with FITC-conjugated anti-CD4 and PE- supplemented with 2 mM L-glutamine, 100 IU/ml penicillin/100 conjugated anti-CD25 (27). For three-color flow cytometry anal- ␮g/ml streptomycin (Sigma-Aldrich, St. Louis, MO), 2 ϫ 10Ϫ5 M ysis, the samples stained with CyChrome-conjugated anti-CD4 and 2-ME, and 10% FCS (Invitrogen, Carlsbad, CA). FITC-conjugated anti-CD25 were also stained with control PE- The following mAbs were used for purification of cell popula- IgG1, or PE-conjugated CD45RO, PE-conjugated anti-CD45RA, tions and flow cytometry analysis: RPA-T4, FITC- and Cy- PE-conjugated anti-HLA-DR, or PE-conjugated anti-GITR mAb. Chrome-conjugatedanti-humanCD4;M-A251,PE-conjugatedanti- Before staining with PE-conjugated anti-CTLA-4 mAb, the cells human CD25; HI100, PE-conjugated anti-human CD45RA; were fixed and permeabilized with Cytofix/Cytoperm (BD Pharm- UCHL1, PE-conjugated anti-human CD45RO; G46-6, PE-conju- ingen) at 4¡C for 30 min. Staining and washing were performed gated anti-HLA-DR; M-A712, PE-conjugated anti-human CD71; in Perm/Wash Buffer (BD Pharmingen), and cells were washed The Journal of Immunology 3121

once in PBS before analysis. For GITR staining, the sorted Results ϩ Ϫ ϩ bright CD4 CD25 and CD4 CD25 cells stimulated with anti- Phenotypical properties of human CD4ϩCD25ϩ and CD3/CD28 mAbs were also used. CD4ϩCD25Ϫ LP T cells Cell stimulation assays Paired samples of PBMCs and LPMCs from 15 individuals were ϩ ϩ ϩ Ϫ analyzed by ﬂow cytometry for the presence of the CD4 CD25 The CD4 CD25 cells were plated at 1.0 ϫ 104/well, while the ϩ T cells. Consistent with previous reports describing human natu- CD4 CD25bright cells were plated at 0, 0.5 ϫ 104,or1.0ϫ 104/ rally occurring CD4ϩCD25ϩ T cells (44, 46-49), a total of 6.0 Ϯ well (27). Thus, upon coculture, the cells were combined at various R 0.5% of the peripheral blood (PB) CD4ϩ T cells were CD25ϩ (Fig. ratios of T responder in 96-well round-bottom plates (Corning R 1, A and B). Similarly, 6.4 Ϯ 0.6% of the LP CD4ϩ T cells was Costar, Cambridge, MA) in the presence or absence of Con A (5 also CD25ϩ (Fig. 1, A and B). In addition, the mean ﬂuorescence ␮g/ml), PHA (5 ␮g/ml), or soluble anti-CD3 mAb (1 ␮g/ml). To intensity (MFI) of LP CD4ϩCD25ϩ T cells is similar to that of the evaluate the effect of IL-2 on the break of anergy, cells were cul- PB CD4ϩCD25ϩ T cells (data not shown). Since it has been re- tured with soluble anti-CD3 (1 ␮g/ml) in the presence or absence cently reported that both naturally occurring CD4ϩCD25ϩ T of rhIL-2 (100 U/ml). To assess a role of suppressive activity via R

competition between TR cells and responder cells for IL-2, various concentrations of rhIL-2 (0, 2, 5, 10, or 100 U/ml) were added. All wells received 5 ϫ 104 MMC-treated CD4Ϫ cells as APCs. To determine proliferation, 50 ␮l of the culture supernatant was removed from each before 1 ␮Ci of [3H]thymidine (NEN, Boston, MA) was added on day 3, the final9hofculture before harvesting. Percent proliferation was determined as (cpm incorporated in the coculture)/(cpm of responder population alone) ϫ 100%. Cytokine assays Intracytofluorimetric analysis of IL-2, IL-10, and IFN-␥ synthesis at the single-cell level was performed. Briefly, 1 ϫ 106 cells were stimulated with 5 ng/ml PMA plus 500 nM ionomycin for 6 h, the last 4 of which was in the presence of 5 ␮g/ml brefeldin A (Gol- giPlug; BD Pharmingen). Cells were collected, washed, fixed, and saponin permeabilized (Cytofix/Cytoperm; BD Pharmingen) and stained with cytokine-specific mAbs or isotype. In another set of experiments, the supernatants, which were removed before addition of [3H]thymidine at proliferation assays, were diluted and analyzed by a cytometric bead array (Th1/Th2 Cytokine CBA 1; BD

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. Pharmingen) according to the manufacturer’s instructions. RT-PCR for the detection of Foxp3 Total cellular RNA was extracted from 5 ϫ 105 freshly sorted CD4ϩCD25Ϫ and CD4ϩCD25bright cells and those stimulated with anti-CD3/CD28 mAbs using the RNeasy Mini kit (Qiagen, Valen- cia, CA), and yield was estimated spectrophotometrically. One hundred ﬁfty nanograms of the total RNA was reverse transcribed using the Superscript First-Strand Synthesis System (Invitrogen). http://classic.jimmunol.org Foxp3 levels were measured by dual-labeled probe RT-PCR using Smart Cycler (Cepheid, Sunnyvale, CA). The PCR contained 0.3

mM of each primer, 0.2 mM probe, 3 mM MgCL2 and 0.75 U of Platinum Taq polymerase (Invitrogen). The primer sequences were designed to bracket an intron to avoid ampliﬁcation of genomic DNA (45). Their sequences were as follows; Foxp3 primers: 5Ј-

Downloaded from TTCATGCACCAGCTCTCAACGG-3Ј and 5Ј-TCGTCCATCC TCCTTTCCTTGATC-3Ј (Sawader, Tokyo, Japan). PCR cycling conditions consisted of 95¡C for 6 min, followed by 45 cycles of 95¡C for 15 s, 60¡C for 30 s, and 72¡C for 30 s. Cycle threshold (CT) values were compared against a standard curve to estimate starting amounts of mRNA, and the relative expression of Foxp3 mRNA between samples was estimated by normalizing these val- ϩ ϩ ues against 18S rRNA CT values were generated using a preopti- FIGURE 1. Identiﬁcation of the PB and LP CD4 CD25 and ϩ bright mized 18S rRNA primer and probe set (Applied Biosystems, Fos- CD4 CD25 T cells. A, Freshly isolated human PBMCs and LPMCs were assessed by a FACSCalibur. Representative sorting gates of the three ter City, CA). Ϫ ϩ cell populations, CD25 , CD25 (CD25int plus CD25bright), and CD25bright ϩ ϩ Statistical analysis are shown. B, The percentages of the PB and LP whole CD4 CD25 T cells in total CD4ϩ cells isolated from normal individuals (n ϭ 15) was The results were expressed as the mean Ϯ SD. Groups of data were determined by a FACSCalibur. C, The percentages of the PB and LP compared by Mann-Whitney U test. Differences were considered CD4ϩCD25bright T cells in total CD4ϩ cells isolated from normal individ- to be statistically signiﬁcant when p Ͻ 0.05. uals (n ϭ 15) was determined by a FACSCalibur. ϩ bright 3122 LP CD4 CD25 TR CELLS

ϩ ϩ ϩ bright cells (44, 50, 51) and anergic memory TR cells (52) preferentially not the LP CD4 CD25 and CD4 CD25 T cells also express ϩ bright resided in the CD4 CD25 T cell population in humans, we well-known TR markers, such as CTLA-4 and GITR, although subdivided the CD4ϩCD25ϩ T cells into CD4ϩCD25bright and these are also known as T cell activation markers. Expectedly, both CD4ϩCD25int T cells. Also, 1.06 Ϯ 0.23% and 0.90 Ϯ 0.16% of intracellular CTLA-4 and surface GITR were signiﬁcantly in- the PB and the LP CD4ϩ T cells, respectively, were creased in/on the LP CD4ϩCD25ϩ and CD4ϩCD25bright T cells as CD4ϩCD25bright (Fig. 1C), indicating that the LP CD4ϩ T cells compared with the LP CD4ϩCD25Ϫ T cells (Fig. 3A). Interest-

also may contain TR cells as well. ingly, these markers were significantly up-regulated in/on the The PB and the LP CD4ϩCD25Ϫ, CD4ϩCD25ϩ, and CD4ϩCD25bright T cells as compared with the CD4ϩCD25ϩ T CD4ϩCD25bright T cells were next analyzed for expression of cells (Fig. 3A). However, unexpectedly, the LP CD4ϩCD25Ϫ T surface Ags to gain insight into their mechanism of action and cells did also express CTLA-4 intracellularly, albeit the PB ϩ Ϫ to more fully characterize the TR population in human intestinal CD4 CD25 T cells did not (Fig. 3A). In turn, GITR on the LP LP. Consistent with previous reports (53), CD45RO, which can CD4ϩCD25Ϫ T cells was also up-regulated as compared with that be associated with proliferative responses to recall Ags (mem- on the PB CD4ϩCD25Ϫ T cells (Fig. 3A). These data were also ory T cells), was expressed at significantly higher levels by the confirmed by the differences of the MFI Ϯ SD from each popu- PB CD4ϩCD25ϩ and CD4ϩCD25bright populations as com- lation (Fig. 3A). pared with the CD4ϩCD25Ϫ (Fig. 2). In contrast to the PB Recently, Foxp3, which encodes a forkhead/winged-helix tran- CD4ϩ T cells, all groups, the LP CD4ϩCD25ϩ and scription factor known as scurfin (45), was found to be expressed ϩ bright ϩ Ϫ ϩ ϩ CD4 CD25 , and CD4 CD25 T cells preferentially ex- specifically by naturally occurring CD4 CD25 TR cells, but not pressed CD45RO. In contrast, the expression of CD45RA, by previously activated CD4ϩCD25ϩ T cells in mice. Thus, we considered a marker for naive T cells, showed the opposite next questioned whether or not the LP CD4ϩCD25Ϫ and expression profile. Consistent with the evidence that activated CD4ϩCD25bright T cells expressed Foxp3. Consistent with previ- human T cells express HLA-DR molecules on their surface ous mice studies (23Ð25), the PB CD4ϩCD25bright T population, (54), the PB and LP CD4ϩCD25ϩ and CD4ϩCD25bright T cells predominantly transcribed Foxp3, whereas the PB CD4ϩCD25Ϫ T expressed significantly higher HLA-DR, as compared with the cells did not (Fig. 3B). Somewhat surprising, the LP CD4ϩCD25Ϫ PB CD4ϩCD25Ϫ T cells. In addition, the LP CD4ϩCD25Ϫ T T cells as well as the LP CD4ϩCD25bright T cells did transcribe the cells did express HLA-DR as compared with the PB gene (Fig. 3B), although the semiquantitative RT-PCR confirmed CD4ϩCD25Ϫ T cells (Fig. 2). Taken together, the LP CD4ϩ T that Foxp3 in the LP CD4ϩCD25Ϫ T cells was clearly expressed, cells, regardless of CD25 expression, showed the phenotype but to a significantly smaller extent than that in the LP ϩ bright characterized as activated memory T cells. CD4 CD25 T cells (Fig. 3C). These results indicate that TR cells may reside within the LP CD4ϩCD25Ϫ T cell population as ϩ bright LP CD4 CD25 T cells express CTLA-4, GITR, and Foxp3 well as the LP CD4ϩCD25bright T cell population, which expressed Since CD4ϩCD8ϪCD25ϩ T cells in the PB and thymus from nor- CTLA-4, GITR, and Foxp3. mal individuals are T cells (44, 46Ð49), we assessed whether or R LP CD4ϩ T cells regardless of CD25 expression are anergic ϩ ϩ by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. Since the CD4 CD25 TR cells show a partially anergic phenotype, we then questioned whether the LP CD4ϩCD25bright/ CD4ϩCD25Ϫ T cells proliferate with or without PHA, Con A, or soluble anti-CD3 mAb stimulation in the presence of MMC- treated APCs. In this proliferation assay, we first separated the CD4ϩCD25bright T cells and CD4ϩCD25Ϫ T cells from paired PBMCs and LPMCs, respectively, using a FACSVantage. In both the PB and LP CD4ϩ T cells, the CD4ϩCD25bright subsets were found to be hyporesponsive to PHA, Con A, or anti-CD3 mAb ϩ Ϫ http://classic.jimmunol.org stimulation as compared with the CD4 CD25 T cells (Fig. 4), indicating that the PB and the LP CD4ϩCD25bright were anergic. Similarly, the PB and the LP CD4ϩCD25ϩ subsets were also hyporesponsive to PHA, Con A, or anti-CD3 mAb stimulation as compared with the PB and the LP CD4ϩCD25Ϫ T cells, respectively (data not shown). Furthermore, the LP CD4ϩCD25Ϫ T cells

Downloaded from did proliferate to a signiﬁcantly smaller extent as compared with the PB CD4ϩCD25Ϫ T cells, indicating that the LP CD4ϩCD25Ϫ cells were also anergic as compared with the paired PB CD4ϩCD25Ϫ cells. However, in the presence of exogenously added rhIL-2 at the concentration of 100 U/ml, stimulation with anti-CD3 mAb elicited proliferation not only in the PB and the LP CD4ϩCD25bright T cells but also in the LP CD4ϩCD25Ϫ T cells (Fig. 4).

LP CD4ϩCD25bright T cells are regulatory FIGURE 2. Human PB CD4ϩCD25ϩ (CD4ϩCD25bright), LP ϩ ϩ ϩ bright ϩ Ϫ ϩ We next investigated the regulatory properties of the LP CD4 CD25 (CD4 CD25 ), and CD4 CD25 T cells are CD45RO , ϩ bright ϩ Ϫ CD45RAϪ, and HLA-DRϩ activated memory T cells. The cells were CD4 CD25 /CD4 CD25 T cells by testing their ability to stained for the cell surface markers as indicated in the upper part of the suppress the proliferative responses of the PB and LP ϩ Ϫ histograms. Gates were set as described in Fig. 1. Shown is a representative CD4 CD25 T cells obtained from the same individuals. In a ϩ experiment of a total of seven performed independently. series of experiments, we used the CD4 CD25bright T cells for The Journal of Immunology 3123

assessing their regulatory activity, because our preliminary data showed that the PB and LP CD4ϩCD25ϩ T cells, including CD25bright and CD25int subpopulations, did not suppress the proliferation of the PB CD4ϩCD25Ϫ responder cells at a high ratio of

1TR:1 responder (data not shown). In contrast, the PB CD4ϩCD25bright T cells were able to suppress the proliferation of ϩ Ϫ the PB CD4 CD25 cells when cocultured at a ratio of 0.5 TR:1 responder or 1 TR:1 responder in the presence of MMC-treated APCs and PHA (percent proliferation compared with that at cul- Ϯ turing with responder alone, 0.5 TR:1 responder, 19.5 4.4% Ͻ Ϯ Ͻ ϭ ( p 0.05); 1 TR:1 responder; 12.6 2.6% ( p 0.05), n 7) (Fig. 5A), indicating that the PB CD4ϩCD25bright T cells were regulatory. As a control, it was shown that titration of the same dose of the PB CD4ϩCD25Ϫ cells into the cultures did not affect the degree of proliferation, thereby excluding the possibility that an increase in total responder cell number was responsible for the suppressive effect (Fig. 5A). However, the LP CD4ϩCD25bright T cells could not suppress the proliferation of the LP CD4ϩCD25Ϫ

responder cells even at a high ratio of 1 TR:1 responder (percent proliferation, 88.5 Ϯ 4.6% ( p ϭ 0.32), n ϭ 7; Fig. 5B), probably affecting the original feature of the anergic LP CD4ϩCD25Ϫ cells as shown in Fig. 4. To re-estimate their regulatory properties, we next used the PB CD4ϩCD25Ϫ T cells from the same individuals as responders for the proliferation assay. Expectedly, the LP CD4ϩCD25bright T cells were able to suppress the proliferation of the PB CD4ϩCD25Ϫ cells when cocultured at a ratio of both 0.5

TR:1 responder and 1 TR:1 responder (percent proliferation, 0.5 Ϯ Ͻ TR:1 responder, 22.7 4.4% ( p 0.05); 1 TR:1 responder, 12.8 Ϯ 2.2% ( p Ͻ 0.05), n ϭ 7; Fig. 5C), indicating that the LP CD4ϩCD25bright T cells were also regulatory. In contrast, the LP CD4ϩCD25Ϫ T cells could not suppress the proliferation of the PB CD4ϩCD25Ϫ cells (Fig. 5C), although these cells expressed CTLA-4, GITR, and Foxp3 and were anergic (Figs. 3 and 4).

ϩ bright

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. LP CD4 CD25 T cells suppress cytokine productions from the CD4ϩCD25Ϫ T cells The cytokine proﬁle of the LP CD4ϩCD25bright T cells and the effect of these cells on cytokines produced by the CD4ϩCD25Ϫ cells were examined. After stimulation with PMA and calcium ionophore, no detectable levels of IL-2, IFN- ␥, and IL-10 could be measured in both the PB and LP CD4ϩCD25bright T cells (Fig. 6A). In contrast, the PB and LP CD4ϩCD25Ϫ T cells produced IL-2 and IFN-␥, but not IL-10 (Fig. 6A). The striking difference http://classic.jimmunol.org between the LP CD4ϩCD25bright and CD4ϩCD25Ϫ T cell populations was that CD25bright cells failed to secrete IL-2, indicating

CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.01, LPMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.05; CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.01; Downloaded from percent positive CTLA-4; PBMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.01; CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.005, LPMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.05; CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.01) (MFI of GITR; PBMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.05; CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.05, LPMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.05; CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.01; MFI of CTLA-4; PBMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.05; FIGURE 3. Expression of CTLA4, GITR, and Foxp3 in/on the PB and LP CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.005, LPMC, CD4ϩCD25Ϫ vs CD4ϩCD25Ϫ/CD4ϩCD25ϩ/CD4ϩCD25bright T cells. A, The PBMCs and LP- CD4ϩCD25ϩ, p Ͻ 0.05; CD4ϩCD25Ϫ vs CD4ϩCD25bright, p Ͻ 0.01).B, MCs from normal individuals were collected, stained, and analyzed by ﬂow Expression of Foxp3 in a subpopulation of PB and LP cells. The PBMCs cytometry as described in Materials and Methods. Histograms are gated on and LPMCs were sorted into the indicated compartments using a FACS- CD4ϩCD25Ϫ, CD4ϩCD25ϩ, and CD4ϩCD25bright cells. CTLA-4 staining Vantage and nonsaturating RT-PCR analyses were conducted. C, Quanti- was performed after cell permeabilization; the isotype control is shown as a ﬁcation of relative Foxp3 mRNA levels in indicated CD4ϩ T cell subsets. light line. The percent positive cells Ϯ SD and the MFI Ϯ SD from each cDNA samples were subjected to real-time semiquantitative PCR analyses, population of four independent analyses is presented in each histogram (per- and the relative quantity of Foxp3 in each sample was normalized to the .p Ͻ 0.05 ,ء .cent positive of GITR; PBMC, CD4ϩCD25Ϫ vs CD4ϩCD25ϩ, p Ͻ 0.05; relative quantity of G3PDH ϩ bright 3124 LP CD4 CD25 TR CELLS

FIGURE 4. Proliferation of the PB and LP CD4ϩCD25Ϫ/CD4ϩCD25bright T cells in response to mitogen stimulation. Cells (1 ϫ 104) were stimulated for 72 h with or without 5 ␮g/ml PHA or 5 ␮g/ml Con A in the presence of APCs (5 ϫ 104, MMC-treated CD4-de- .p Ͻ 0.05 ,ء .(pleted cells

ϩ Ϫ

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. that these cells have a speciﬁc defect in production of IL-2 (Fig. Since stimulation of peripheral CD4 CD25 T cells through 6A). To next determine whether the LP CD4ϩCD25bright T cells TCR and CD28 leads to several outcomes, including the prolifer- suppress cytokine secretion by CD4ϩCD25Ϫ responder T cells, ation and induction of CD25 expression, it is possible that some or 3 ϩ bright the supernatants were collected at 63 h before addition of [ H]thy- all of the LP CD4 CD25 TR cells are the result of recent midine at proliferation assays and analyzed by a cytometric bead activation in the periphery. As shown in Fig. 7D, both CD25 and array, which allows multiparameter analysis in a single sample. As GITR were up-regulated after the stimulation by anti-CD3/CD28 shown in Fig. 6B, although the PB CD4ϩCD25Ϫ T cells (1 ϫ 104 mAbs, although their expression was relatively low when com- and 2 ϫ 104 cells) in the absence of the LP CD4ϩCD25bright T pared with that of CD4ϩCD25bright cells. To next determine the cells produced a large amount of IL-2, IL-5, TNF-␣, and IFN-␥, nature of the Foxp3 expression in previous activation of LP T cells, ϩ bright ϩ Ϫ ϩ Ϫ http://classic.jimmunol.org the LP CD4 CD25 , but not the LP CD4 CD25 T cells, did we demonstrated Foxp3 expression in both sorted CD4 CD25 clearly suppress these cytokine productions, indicating that the LP and CD4ϩCD25bright cells stimulated with anti-CD3/CD28 mAbs. CD4ϩCD25bright T cell did affect not only the proliferation, but As shown in Fig. 7, E and F, anti-CD3/CD28-stimulated LP also the cytokine productions of the surrounding responder cells. CD4ϩCD25Ϫ cells expressed the Foxp3 transcripts to an extent similar to that in anti-CD3/CD28-stimulated LP CD4ϩCD25bright GITR/Foxp3 expressions in LP T cells after TCR stimulation cells. However, the expression of Foxp3 tended to be increased

Downloaded from We next assessed the expression of GITR and FoxP3 on/in LP T after the stimulation as compared with that in the starting freshly ϩ Ϫ cells in association with activation and memory markers, because isolated population of CD4 CD25 T cells, but was not signiﬁcant.

it has been reported that some anergic activated/memory T cells ϩ bright have regulatory properties, indicating the peripheral development Origin of LP CD4 CD25 T cells ϩ ϩ ϩ of TR cells. To do so, we first divided the LP CD4 cells into Although human naturally arising CD4 CD25 TR cells are CD71ϩ (activated) and CD71Ϫ or CD45ROϩ (memory) and thought to be mainly derived from thymus (46), a recent study by Ϫ ϩ ϩ ϩ CD45RO cells, respectively, thereafter we examined the expres- Walker et al. (55) suggests that Foxp3 CD4 CD25 TR can be sion of CD25/GITR and Foxp3 using FACS and RT-PCR analysis. also developed from peripheral Foxp3ϪCD4ϩCD25Ϫ T cells by in As shown in Fig. 7, A and B, both CD25/GITR and Foxp3 were vitro anti-CD3/CD28 mAb stimulation. Furthermore, it is well preferentially expressed on/in LP CD71ϩ and CD45ROϩ T cells known that anergic human CD4ϩ T cells that are accompanied by Ϫ Ϫ as compared with LP CD71 and CD45RO T cells, respectively. an increased level of CD25 expression possess TR activity (52). Furthermore, the semiquantitative RT-PCR confirmed that the Thus, it was important to determine whether our human LP ϩ ϩ ϩ bright Foxp3 expression in the LP CD71 and CD45RO cells was sig- CD4 CD25 cells were naturally arising TR cells from the nificantly higher than that in the LP CD71Ϫ and CD45ROϪ cells, thymus or anergic cells that were developed in the periphery. As ϩ ϩ respectively (Fig. 7C). one of the differences between naturally arising CD4 CD25 TR The Journal of Immunology 3125 by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd.

FIGURE 6. The CD4ϩCD25bright do not secrete cytokines but can suppress the production of IL-2, IL-5, TNF-␣, and IFN-␥ by cocultured ϩ Ϫ

http://classic.jimmunol.org CD4 CD25 cells. A, The frequencies of cytokine-producing cells were analyzed by gating on the CD4ϩ subpopulation. Representatives of three independent experiments using paired PB- and LP-sorted CD4ϩCD25Ϫ/ CD4ϩCD25bright cells. B, Culture supernatants were collected from the 4 4 ϩ FIGURE 5. The LP CD4ϩCD25bright but not the CD4ϩCD25Ϫ T cells proliferation cultures (culture supernatants from 1 ϫ 10 or 2 ϫ 10 PB CD4 Ϫ 4 ϩ Ϫ 4 ϩ bright suppress the proliferation of the PB CD4ϩCD25Ϫ T cells in vitro. Different CD25 cells, 1 ϫ 10 PB CD4 CD25 plus 1 ϫ 10 LP CD4 CD25 4 ϩ Ϫ 4 ϩ Ϫ doses of CD4ϩCD25bright or control CD4ϩCD25Ϫ T cells (0, 0.5 ϫ 104, cells, and 1 ϫ 10 PB CD4 CD25 plus 1 ϫ 10 LP CD4 CD25 cells 3 Downloaded from 1 ϫ 104) were added into wells containing 1 ϫ 104 responder PB or LP depicted in Fig. 5 before the addition of [ H]thymidine incorporation. Levels CD4ϩCD25Ϫ cells and were stimulated with 5 ␮g/ml PHA in the presence of of IL-2, IL-4, IL-5, IL-10, TNF-␣, and IFN-␥ were determined from culture 5 ϫ 104 MMC-treated APCs. At 63 h of culture, supernatants were taken for supernatants by a cytometric bead array. Results were similar in four indepen- cytokine analysis and the cells were pulsed with 1 ␮Ci [3H]thymidine for dent experiments. another 9 h. Shown is a representative experiment of seven total performed. A, Responders are PB CD4ϩCD25Ϫ; regulatory cells are PB CD4ϩCD25bright or PB CD4ϩCD25Ϫ (as a control). B, Responders are LP CD4ϩCD25Ϫ; reg- ϩ ϩ Ϫ responder (52). Furthermore, since it has been known that both the ulatory cells are LP CD4 CD25bright or LP CD4 CD25 (as a control). C, Responders are PB CD4ϩCD25Ϫ; regulatory cells are LP CD4ϩCD25Ϫ,or natural-occurring TR and the anergic cells require stimulation ϩ ϩ Ϫ -p Ͻ 0.05. through the TCR to activate its TR program, we used soluble anti ,ء .(LP CD4 CD25bright,orPBCD4 CD25 (as a control CD3 mAb in place of PHA in this setting. Although the PB CD4ϩCD25bright T cells were able to signiﬁcantly suppress the cells and anergic cells, it is well known that in vitro suppressive proliferation of the PB CD4ϩCD25Ϫ cells when cocultured even at ϩ ϩ activity of naturally arising PB CD4 CD25 TR cells usually can a low ratio of 0.06 TR:1 responder (Fig. 8A), the LP Ͼ ϩ bright occur even if the TR:responder ratio is 0.1 TR:1 responder (49), CD4 CD25 T cells could suppress only at a relatively higher Ͼ Ͼ whereas that of anergic cells can occur only if the ratio is 1TR:1 ratio of 0.25 TR:1 responder (Fig. 8A). We next conducted an ϩ bright 3126 LP CD4 CD25 TR CELLS

FIGURE 7. Expression of GITR/ Foxp3 on/in LP T cells. A, Expression of CD25 and GITR on CD45ROϩ CD45ROϪ or CD71ϩCD71Ϫ cells. Freshly isolated LPMCs from normal individuals were stained with CyChrome- anti-CD4, FITC-anti-CD25 or FITC-anti- GITR, and PE-anti-CD45RO or PE-anti- CD71 mAbs and the expression of CD25/GITR on the indicated cells was analyzed. Results were similar in four independent experiments. B, Expression of Foxp3 in CD45ROϩCD45ROϪ or CD71ϩCD71Ϫ cells. After staining with various mAbs, the indicated cells were sorted by a FACSVantage, thereafter RT-PCR analyses of Foxp3 were conducted. Results were similar in four independent experiments. C, Quantiﬁca- tion of relative Foxp3 mRNA levels in indicated CD4ϩ T cell subsets (n ϭ 5). cDNA samples were subjected to real- time semiquantitative PCR analyses, and the relative quantity of Foxp3 in each sample was normalized to the relative ,p Ͻ 0.05. D ,ء .quantity of G3PDH Expression of CD25 and GITR on freshly isolated and anti-CD3/anti- CD28-stimulated LP CD4ϩCD25Ϫ and CD4ϩCD25bright cells. Freshly isolated and anti-CD3/anti-CD28-stimulated LP CD4ϩCD25Ϫ and CD4ϩCD25bright cells were stained with FITC-anti-CD25 or

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. FITC-anti-GITR on the indicated cells. Results were similar in four independent experiments. E, Expression of Foxp3 in freshly isolated and anti-CD3/anti- CD28-stimulated LP CD4ϩCD25Ϫ and CD4ϩCD25bright cells. Results were similar in three independent experiments. F, Quantiﬁcation of relative Foxp3 mRNA levels in indicated CD4ϩ T cell subsets (n ϭ 4). cDNA samples http://classic.jimmunol.org were subjected to real-time semiquantitative PCR analyses, and the relative quantity of Foxp3 in each sample was normalized to the relative quantity of .p Ͻ 0.05 ,ء .G3PDH Downloaded from

IL-2 consumption assay because it has been previously shown that cells and the PB CD4ϩCD25bright cells regarding regulatory ac-

low amounts of IL-2 abrogate the inhibition of the responder cells tivity at a 1:1 responder:TR ratio and at any concentrations of ex- by anergic cells (52), but not by natural-occurring CD4ϩCD25ϩ ogenously added IL-2. ϩ bright TR cells (56). As shown in Fig. 8B, the LP CD4 CD25 cells ϩ bright ϩ bright LP CD4 CD25 T cells from patients with active IBD also as well as the PB CD4 CD25 cells could inhibit the prolif- ϩ Ϫ erative responses of the PB CD4ϩCD25Ϫ cells in the presence of suppress the PB CD4 CD25 T cell proliferation low concentrations of IL-2 (2, 5, and 10 U/ml), but not a high Finally, we assessed the role of CD4ϩCD25ϩ and CD4ϩ concentration of IL-2 (100 U/ml), in combination with soluble CD25bright T cells in the LP from patients with active IBD to anti-CD3 mAb (HIT3a, 1 ␮g/ml) and MMC-treated APCs. Impor- determine whether the LP CD4ϩCD25ϩ/CD4ϩCD25bright T cells ϩ bright tantly, there were no differences between the LP CD4 CD25 in IBD patients are activated pathogenic T cells or TR cells, which The Journal of Immunology 3127

expression in IBD patients (data not shown). Similarly to the normal LP CD4ϩ T cells, the LP CD4ϩCD25Ϫ T cells as well as the LP CD4ϩCD25ϩ and CD4ϩCD25bright T cells from IBD patients did express intracellular CTLA-4 and surface GITR, although their expression was apt to be less than in/on the LP CD4ϩCD25bright/CD4ϩCD25ϩ T cells (Fig. 9D). Furthermore, the LP CD4ϩCD25bright, but not CD4ϩCD25Ϫ T cells from active IBD patients, did suppress the proliferation of the paired PB CD4ϩ CD25Ϫ T cells to an extent similar to that of the normal LP CD4ϩ CD25bright T cells (Fig. 9E), indicating that the LP CD4ϩCD25bright ϩ bright T cells from IBD patients contain protective CD4 CD25 TR cells.

Discussion In the present study, we demonstrated that the LP CD4ϩCD25bright T cells isolated from normal human individuals possessed regulatory activity. Of particular importance, although many mucosal immunologists believed so far that the activated LP CD25ϩ T cells that were accumulated in the inflamed mucosa of IBD patients should be pathogenic, we also demonstrated that not only these cells and particularly their subpopulation of CD4ϩCD25bright T cells from IBD patients were significantly increased as compared with those from normal individuals, but also the LP CD4ϩCD25bright T cells from IBD patients retained regulatory activity to an extent similar to that of normal individuals. These ϩ bright results indicate that the LP CD4 CD25 TR cells should be involved at least in part in the local T cell homeostasis both in normal and inflammatory conditions. Although accumulating evidence shows that human PB and thy- mic CD4ϩCD8ϪCD25ϩ T cells possess regulatory activity (46Ð 49) with the same characteristics in mice, Baecher-Allan et al. (44) recently demonstrated that only CD4ϩCD25bright, but not the whole CD4ϩCD25ϩ T cells, have regulatory activity. This indi- by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. cates that the whole CD4ϩCD25ϩ T cells contain a relatively high FIGURE 8. T activity of LP CD4ϩCD25bright cells needs a higher T : proportion of previously activated T cells rather than naturally oc- R R ϩ bright responder ratio to suppress responder cells, but cannot be reversed by ad- curring CD4 CD25 TR cells, thereby the freshly isolated ϩ ϩ dition of lower concentrations of IL-2. A, The regulatory activities of the whole CD4 CD25 T cells cannot suppress the proliferation of ϩ Ϫ PB CD4ϩCD25bright cells and the LP CD4ϩCD25bright cells were exam- autologous CD4 CD25 T cells in coculture. Consistent with ϩ Ϫ ϫ 4 ined. The PB CD4 CD25 responder cells (1 10 cells/well) and var- their report, we also detected TR activity in the fraction of the PB ϩ ϩ ious numbers of the PB CD4 CD25bright cells or the LP CD4 CD25bright and LP CD4ϩCD25bright, but not in the whole CD4ϩCD25ϩ T 4 cells (0Ð1 ϫ 10 cells/well) were stimulated with soluble anti-CD3 mAb cells. Furthermore, the evidence that the LP CD4ϩCD25bright T (HIT-3a, 1 ␮g/ml) and MMC-treated autologous APCs (5 ϫ 104 cells/ ϩ ϩ http://classic.jimmunol.org cell fraction rather than whole CD4 CD25 T cells in patients well). The mean percent inhibition of the proliferative response by ϩ bright with IBD was significantly increased as compared with normal CD4 CD25 cells was calculated. B, The effects of culturing the PB ϩ bright CD4ϩCD25Ϫ responder cells (1 ϫ 104 cells/well) with MMC-treated individuals may indicate that the LP CD4 CD25 T cells func- APCs (5 ϫ 104 cells/well) and increasing concentrations of exogenous tion to control or down-modulate the excessive immune responses IL-2 in the presence of the same numbers of the PB CD4ϩCD25bright cells in inflamed mucosa of IBD patients. In support of this, it has re- ϩ bright ϫ 4 ϩ ϩ or the LP CD4 CD25 cells (1 10 cells/well) is shown. Results cently been shown that CD4 CD25 TR cells can reverse the es- p Ͻ 0.05. ϩ high ,ء .were similar in four independent experiments Downloaded from tablished intestinal inflammation in CD4 CD45RB T cell- transferred colitic mice (58). However, the question arises as to why the patients with active should be cells opposite of each other in terms of their pathogen- IBD in this study were obligated to receive the surgical operation esis. As shown in Fig. 9, A and B, both the LP CD4ϩCD25ϩ and because of the limitation by the conventional drug therapy to CD4ϩCD25bright T cells were significantly increased in patients control the severe inflammation, although the increased LP ϩ bright with active CD or active UC as compared with normal individuals. CD4 CD25 TR cells resided in the inflamed mucosa. We Of note, the percentages of the LP CD4ϩCD25bright T cells per believe that the answer lies in the fact that the high levels of total LP CD4ϩCD25ϩ T cells were also significantly increased in various cytokines (e.g., IL-2, IL-6, and IL-15) that are produced in IBD patients compared with those of normal individuals (Fig. 9C). severely inflamed mucosa from active IBD patients (40) and the Although Barrat et al (57) recently reported a combination of up-regulated expression of costimulatory molecules, such as CD80

immunosuppressive drugs, vitamin D3 and dexamethasone, in- and CD86, and class II MHC molecules (40), thereby these would ϩ ϩ bright duced human and mouse naive CD4 T cells to differentiate in abolish the regulatory activity of the LP CD4 CD25 TR cells vitro into IL-10-producing regulatory T cells (named Tr1), there and enhance the LP CD4ϩ T cell activation in situ. In line of this were no correlations between the medical treatments and the CD25 hypothesis, ﬁrst, two reports (59, 60) revealed that not only a high ϩ bright 3128 LP CD4 CD25 TR CELLS

dose of IL-2 along with TCR stimulation triggers their proliferation and neutralizes the suppressive activity during their proliferation, but also upon removal of IL-2, the suppression-broken CD4ϩCD25ϩ T cells revert to their original suppressive state. Second, IL-6 produced by activated dendritic cells in response to TLR ligand during chronic inﬂammation is critical for T cell

activation and efficient blockade of TR activity (61). Thus, the possibility cannot be excluded that the increased LP ϩ bright CD4 CD25 TR cells do not function well at the site of severe inflammation, thereby our patients could not overcome their intestinal inflammation by the conventional drug therapy, such as corticosteroids and immunosuppressants. We are now investigat- ing whether additional IL-2 or IL-6 affects the suppressive activity of the LP CD4ϩ T cells in an in vitro assay and the correlation ϩ bright between the number of the LP CD4 CD25 TR cells and clinical course, especially the onset of IBD. Furthermore, it was very important to determine whether or not ϩ bright the LP CD4 CD25 TR cells were identical to the PB ϩ bright CD4 CD25 TR cells originally described by others (46Ð49) ϩ bright in terms of their origin. Unlike the PB CD4 CD25 TR cells that could suppress responder cells even at a TR:responder ratio of 0.06 TR:1 responder in the presence of soluble anti-CD3 mAb, the ϩ bright LP CD4 CD25 TR cells needed a relatively higher responder:TR ratio of 0.25 TR:1 responder to suppress the responders in the ϩ bright same setting. Like the PB CD4 CD25 TR cells, however, ϩ bright the suppression by the LP CD4 CD25 TR cells could not be reversed by addition of lower amounts of IL-2 (2Ð10 U/ml). Although it might well be that in this special situation, mucosal

TR activity is relatively mild as compared with the PB ϩ bright ϩ bright CD4 CD25 TR cells, it is likely that the LP CD4 CD25 TR cells are derived mainly from natural-occurring TR cells be- Ͻ cause the suppression by the LP TR cells was observed at 1TR:1 responder (0.5 TR:1 responder and 0.25 TR:1 responder) and was not caused by IL-2 consumption in our analysis. ϩ bright by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. Besides the natural-occurring CD4 CD25 TR cells and the anergic cells, the existence of other distinct groups of TR cells, such as Tr1 cells and Th3 cells and their role in controlling intestinal inﬂammation along with suppressor cytokines IL-10 and TGF-␤, should also be considered (4). As a possibility, two groups have recently reported data suggesting that contact with ϩ ϩ Ϫ CD4 CD25 TR cells causes CD25 responder T cells to become suppressive themselves, the mechanism of which is referred to as “infectious tolerance,” although its cytokine dependency is still http://classic.jimmunol.org controversial (37, 62). This mechanism of infectious tolerance could explain not only how such a small LP CD4ϩCD25bright population (1Ð2%) of cells can regulate a much larger population of responder cells in vivo, but also how the LP CD4ϩCD25Ϫ and CD4ϩCD25int T cells could express CTLA-4, GITR, and Foxp3. The functions of mucosal T cells are largely uncertain, but cells

Downloaded from with a “memory” phenotype predominate in both the epithelium and the LP, indicating that they have been exposed to Ags (40). Thus, LP CD4ϩ T cells may be of particular importance to local

T cells in total CD4ϩ cells isolated from normal, UC, and CD patients were /p Ͻ 0.05. C, The frequency of LP CD4ϩCD25ϩ ,ء .determined ,ء .CD4ϩCD25bright T cells in obtained from normal, UC, and CD patients p Ͻ 0.05. D, Representative data showing the expression of CTLA-4 (upper) and GITR (lower) on the LP CD4ϩCD25Ϫ, CD4ϩCD25ϩ, and FIGURE 9. LP CD4ϩCD25bright T cells from IBD patients also possess CD4ϩCD25bright T cells from normal, UC, and CD patients. E, The LP regulatory activity. A, The percentages of the LP CD4ϩCD25ϩ T cells in CD4ϩCD25bright cells (lower), but not CD4ϩCD25Ϫ T cells (upper) from total CD4ϩ cells isolated from normal individuals (NL, n ϭ 15), patients IBD patients, could also suppress the proliferation of the PB CD4ϩCD25Ϫ with UC (n ϭ 13), and patients with CD (n ϭ 17) were determined by a responder cells to an extent similar to that of normal individuals. Results .p Ͻ 0.05. B, The percentages of the LP CD4ϩCD25bright were similar in four independent experiments ,ء .FACSCalibur The Journal of Immunology 3129

immune regulation. They are generally anergic to TCR-mediated 4. Bouma, G., and Strober, W. 2003. The immunological and genetic basis of in- proliferative signals in normal mucosal conditions (28, 40, 63). testinal bowel disease. Nat. Rev. Immunol. 3:521. 5. Abbas, A. K., K. M. Murphy, and A. Sher. 1996. Functional diversity of helper The role of CTLA-4 that is expressed in activated/memory, but not T lymphocytes. Nature 383:787. naive T cells, in maintaining peripheral tolerance is well estab- 6. O’Garra, A. 1998. Cytokines induce the development of functionally heteroge- lished (64, 65). Nevertheless, to our best knowledge, our observa- neous T helper cell subsets. Immunity 8:275. 7. Gershon, R. K. 1975. A disquisition on suppressor T cells. Transplant. Rev. tion that CTLA-4 is constitutively expressed in the LP 26:170. CD4ϩCD25Ϫ as well as CD4ϩCD25bright T cells, but not in the PB 8. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, ϩ Ϫ Y. Kuniyasu, T. Nomura, M. Toda, and T. Takahashi. 2001. Immunologic tol- CD4 CD25 T cells is the first report and may be in line with the erance maintained by CD25ϩCD4ϩ regulatory T cells: their common role in general view on the acquisition and maintenance of mucosal tol- controlling autoimmunity, tumor immunity, and transplantation tolerance. Immu- nol. Rev. 182:18. erance. Further study will be needed to determine whether or not ϩ blockade of CTLA-4 abolishes the anergic condition of the LP 9. Curotto de Lafaille, M. A., and J. J. Lafaille. 2002. CD4 regulatory T cells in ϩ Ϫ autoimmunity and allergy. Curr. Opin. Immunol. 14:771. CD4 CD25 T cells. 10. Powrie, F., and M. W. Leach. 1995. Genetic and spontaneous models of inflam- Based on the evidence that the LP CD4ϩCD25Ϫ T cells not only matory bowel disease in rodents: evidence for abnormalities in mucosal immune regulation. Ther. Immunol. 2:115. expressed CTLA-4, GITR, and Foxp3, but also showed anergic ϩ Ϫ 11. Blumberg, R. S., L. J. Saubermann, and W. Strober. 1999. Animal models of cells as compared with the paired PB CD4 CD25 T cells, we mucosal inflammation and their relation to human inflammatory bowel disease. ϩ Ϫ Curr. Opin. Immunol. 11:648. questioned whether the LP CD4 CD25 T cells are also TR cells ϩ bright 12. Strober, W., I. J. Fuss, R. S. 2002. Blumberg. The immunology of mucosal as well as the LP CD4 CD25 cells. Unexpectedly, although models of inflammation. Annu. Rev. Immunol. 20:495. ϩ Ϫ we could not detect regulatory activity in the LP CD4 CD25 13. Maloy, K., and F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2:816. subpopulation in our in vitro system at a ratio of 1 TR:1 responder, ϩ Ϫ 14. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immuno- it is possible that only a part of the LP CD4 CD25 subpopulation logical self-tolerance maintained by acute T cells expressing IL-2 receptor ␣ expressed CTLA-4, GITR, and Foxp3 and retains TR activity, chains (CD25): breakdown of a single mechanism of self-tolerance causes vari- thereby none of the LP CD4ϩCD25Ϫ cells showed regulatory ous autoimmune diseases. J. Immunol. 155:1151. 15. Shevach, E. M. 2002. CD4ϩCD25ϩ suppressor T cells: more questions than function in vitro by the indicated ratio. However, it should be answers. Nat. Rev. Immunol. 2:389. ϩ Ϫ noted that anti-CD3/CD28-stimulated LP CD4 CD25 T cells 16. Battaglia, M., B. R. Blazar, and M. G. Roncarolo. 2002. The puzzling world of expressed Foxp3 to an extent similar to the stimulated LP murine T regulatory cells. Microbes Infect. 4:559. ϩ bright 17. Sakaguchi, S. 2000. Regulatory T cells: key controllers of immunological self- CD4 CD25 TR cells. Consistent with this, Walker et al. (55) tolerance. Cell 101:455. have very recently demonstrated that anti-CD3/CD28-stimulated 18. Sakaguchi, S., T. Takahashi, S. Yamazaki, Y. Kuniyasu, M. Itoh, N. Sakaguchi, CD4ϩCD25Ϫ peripheral T cells express Foxp3 and that these and J. Shimizu. 2001. Immunologic self tolerance maintained by T-cell-mediated control of self-reactive T cells: implications for autoimmunity and tumor immu- activated cells are equally as suppressive as natural-occurring nity. Microbes Infect. 3:911. ϩ ϩ peripheral CD4 CD25 T cells. In addition, increasing evidence 19. Read, S., V. Malmstrom, and F. Powrie. 2000. Cytotoxic T lymphocyte-associ- R ϩ ϩ suggests that regulatory CD4ϩ T cells exist in the CD25Ϫ ated antigen 4 plays an essential role in the function of CD4 CD25 regulatory cells that control intestinal inflammation. J. Exp. Med. 193:295. compartment in mice as well (26, 30Ð34), although it is widely 20. Takahashi, T. T. Tagami, S. Yamazaki, T. Ueda, J. Shimizu, N. Sakaguchi, accepted that CD4ϩCD25ϩ T cells are the major population that T. W. Mak, and S. Sakaguchi. Immunologic self-tolerance maintained by CD4ϩCD25ϩ regulatory T cells constitutively expressing cytotoxic T lympho- contains TR cells. In particular, three independent groups, cyte-associated antigen 4. J. Exp. Med. 193: 303.

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. including us, recently demonstrated that several fractions of 21. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, and S. Sakaguchi. 2002. Stim- CD4ϩCD25Ϫ T cells, such as ␣EϩCD4ϩCD25Ϫ (33), ulation of CD25ϩCD4ϩ regulatory T cells through GITR breaks immunological ϩ ϩ Ϫ ϩ ϩ Ϫ self-tolerance. Nat. Immunol. 3:135. LAP CD4 CD25 (66), and GITR CD4 CD25 (27), could 22. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, ϩ high suppress the development of murine CD4 CD45RB T cell- M. Collins, and M. C. Byrne. 2002. CD4ϩCD25ϩ immunoregulatory T cells: transferred colitis. Further studies will be needed to address the gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311. correlation between the anergic memory LP CD4ϩCD25Ϫ T ϩ Ϫ 23. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell de- cells and TR function by sorting CD4 CD25 T cell fractions, velopment by the transcription factor Foxp3. Science 299:1057. ␣ ϩ ϩ Ϫ 24. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the which are enriched TR cells, such as LP E CD4 CD25 , ϩ ϩ ϩ ϩ Ϫ ϩ ϩ Ϫ development and function of CD4 CD25 regulatory T cells. Nat. Immunol. LAP CD4 CD25 , and GITR CD4 CD25 T cells. 4:330. ϩ http://classic.jimmunol.org In conclusion, the LP CD4 CD25bright T cells from both normal 25. Khattri, R., T. Cox, Y. Sue-Ann, and F. Ramsdell. 2003. An essential role for ϩ ϩ individuals and IBD patients were demonstrated to be mucosal T scrurfininCD4 CD25 T regulatory cells. Nat. Immunol. 4:337. R 26. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, cells, indicating that these cells might not only serve as mucosal A. Cumano, and A. Bandeira. 2001. CD25ϩCD4ϩ T cells regulate the expansion immune controllers to maintain T cell homeostasis against many of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008. stimuli, such as intestinal bacteria, but also as ideal cellular im- 27. Uraushihara, K., T. Kanai, K. Ko, T. Totsuka, S. Makita, R. Iiyama, T. Nakamura, and M. Watanabe. 2003. Regulation of murine inflammatory bowel munosuppressive cells as beneficial therapy for IBD patients in disease by CD25ϩ and CD25ϪCD4ϩ glucocorticoid-induced TNF receptor fam- ϩ Downloaded from place of chemical immunosuppressants, which retain many side ily-related gene regulatory T cells. J. Immunol. 171:708. effects. 28. Zeitz, M., T. C. Quinn, and A. S. Graeff. 1988. Mucosal T cells provide helper function but do not proliferative when stimulated by specific antigen in lympho- granuloma venereum proctitis in nonhuman primates. Gastroenterology 94:353. Acknowledgments 29. Choy, M. Y., S. J. Walker, C. B. Williams, and T. T. MacDonald. 1990. Differ- ential expression of CD25 (interleukin-2 receptor) on lamina propria T cells and We express special thanks to Drs. Ryuichi Okamoto, Eriko Okada, Kiichiro macrophages in the intestinal lesions in Crohn’s disease and ulcerative colitis. Tsuchiya, Takahiro Kawamura, Shin Namiki, and Tomoko Matsumoto for Gut 31:1365. the assessment of patients’ records and to Drs. Claudio Fiocchi, Warren 30. Graca, L., S. Thompson, C. Y. Lin, E. Adams, S. P. Cobbold, and H. Waldmann. ϩ ϩ ϩ Ϫ Strober, and Barfor R. Sartor for helpful discussion and encouragement. 2002. Both CD4 CD25 and CD4 CD25 regulatory cells mediate dominant transplantation tolerance. J. Immunol. 168:5558. 31. Van de Keere, F., and S. Tonegawa. 1998. CD4ϩ T cells prevent spontaneous References experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell 1. Holmgren, J., and A. Rudin. 1999. Mucosal immunity and bacteria. In Mucosal receptor transgenic mice. J. Exp. Med. 188:1875. Immunology. P. L. Ogra, M. E. Lamm, J. Bienenstock, J. Mestecky, W. Strober, 32. Apostolou, I., A. Sarukhan, L. Klein, and H. von Boehmer. 2002. Origin of and J. R. McGhee, eds. Academic, San Diego, CA, p. 685. regulatory T cells with known specificity for antigen. Nat. Immunol. 3:756. 2. Asseman, C., S. Fowller, and F. Powrie. 2001. Control of experimental inflam- 33. Lehmann, J., J. Huehn, M. de la Rosa, F. Maszyna, U. Kretschmer, V. Krenn, ␣ ␤ matory bowel disease by regulatory T cells. Immunol. Rev. 182:190. M. Brunner, and A. Hamann. 2002. Expression of the integrin E 7 identifies 3. Annacker, O., and F. Powrie. 2002. Homeostasis of intestinal immune regulation. unique subsets of CD25ϩ as well as CD25 Ϫregulatory T cells. Proc. Natl. Acad. Microbes Infect. 4:567. USA 99:13031. ϩ bright 3130 LP CD4 CD25 TR CELLS

834. Stephens, L. A., and D. Mason. 2000. CD25 is a marker for CD4ϩ thymocytes CD25brightCD4ϩ T cells from the target organ of patients with rheumatoid ar- that prevent autoimmune diseases in rats, but peripheral T cells with this func- thritis. Eur. J. Immunol. 33:215. ϩ Ϫ tion are found in both CD25 and CD25 subpopulations. J. Immunol. 51. Viglietta, V., C. Baecher-Allan, H. L. Weiner, and D. A. Hafler. 2004. Loss of 165:3105. functional suppression by CD4ϩCD25ϩ regulatory T cells in patients with mul- 35. Lombardi, G., I. Sidhu, R. Batchelor, and R. Lechler. 1994. Anergic T cells as tiple sclerosis. J. Exp. Med. 199:971. suppressor cells in vitro. Science 264:1587. 52. Lombardi, G., S. Sidhu, R, Batchelor, and R. Lechler. 1994. Anergic T cells as 36. Jooss, K., B. Gjata, O. Danos, H. Von Boehmer, and A. Sarukhan. 2001. Reg- suppressor cells in vitro. Science 264: 1587. ϩ ulatory function of in vivo anergized CD4 T cells. Proc. Natl. Acad. Sci. USA 53. Schieferdecker, H. L., R. Ullrich, A. N. Weib-Breckwoldt, R. Schwarting, 98:8738. H. Stein, E. O. Riecken, and M. Zeitz. 1990. The HML-1 antigen of intestinal 37. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, and A. H. Enk. 2001. Induction of ϩ lymphocytes is an activation antigen. J. Immunol. 144:2511. interleukin 10-producing, nonproliferating CD4 T cells with regulatory prop- 54. Khoo, U. Y., I. E. Proctor, and A. J. Macpherson. 1997. CD4ϩ T cell down- erties by repetitive stimulation with allogenic immature human dendritic cells. regulation in human intestinal mucosa: evidence for intestinal tolerance to lumi- J. Exp. Med. 192:1213. ϩ Ϫ nal bacterial antigens. J. Immunol. 158:3626. 38. Shimuzu, J., and E. Moriizumi. 2003. CD4 CD25 T cells in aged mice are 55. Walker, M. R., D. J. Kasprowicz, V. H. Gersuk, A. Benard, M. Von Landeghen, hyporesponsive and exhibit suppressive activity. J. Immunol. 170: 1675. J. H. Buckner, and S. F. Ziegler. 2003. Induction of FoxP3 and acquisition of T 39. Qian, L., G. Schurmann, M. Betzler, and S. C. Meuer. 1991. Activation and regulatory activity by stimulating CD4ϩCD25Ϫ T cells. J. Clin. Invest. 112:1437. signaling status of human lamina propria T lymphocytes. Gastroenterology 56. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, 101:1529. J. Shimizu, and S. Sakaguchi. 1998. Immunologic self-tolerance maintained by 40. James, S. P., and H. Kiyono. 1999. Gastrointestinal lamina propria T cells. In CD25ϩCD4ϩ naturally anergic and suppressive T cells: induction of autoimmune Mucosal Immunology. P. L. Ogra, M. E. Lamm, J. Bienenstock, J. Mestecky, disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969. W. Strober, and J. R. McGhee, eds. Academic, San Diego, CA, p. 381. 57. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkout, 41. Sallusto, F., D. Lenig, R. Foster, M. Lipp, and A. Lanzavecchia. 1999. Two R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, and A. O’Garra. 2002. subsets of memory T lymphocytes with distinct homing potentials and effector ϩ In vitro generation of interleukin 10-producing regulatory CD4 T cells is in- functions. Nature 401:708. duced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and 42. Mowat, A. M. 2003. Anatomical basis of tolerance and immunity to intestinal Th2-inducing cytokines. J. Exp. Med. 195:603. antigens. Nat. Rev. Immunol. 3:331. ϩ ϩ 58. Mottet, C., H. H. Uhlig, and F. Powrie. 2003. Cure of colitis by CD4 CD25 43. Kanai, T., M. Watanabe, A. Okazawa, K. Nakamaru, M. Okamoto, regulatory T cells. J. Immunol. 170:3939. M. Naganuma, H. Ishii, M. Ikeda, M. Kurimoto, and T. Hibi. 2000. Interleukin 59. Thornton, A. M., and E. M. Shevach. 1998. CD4ϩCD25ϩ immunoregulatory T 18 is a potent proliferative factor for intestinal mucosal lymphocytes in Crohn’s cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 disease. Gastroenterology 119:1514. production. J. Exp. Med. 188:287. 44. Baecher-Allan, C., J. A. Brown, G. J. Freeman, and D. A. Hafler. 2001. ϩ high 60. Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, and CD4 CD25 regulatory cells in human peripheral blood. J. Immunol. ϩ ϩ 167:1245. S. Sakaguchi. Thymus and autoimmunity: production of CD25 CD4 naturally 45. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, anergic and suppressive T cells as a key function of the thymus in maintaining S. A. Yosayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, and F. Romsdell. 2001. immunologic self-tolerance. J. Immunol. 162: 5317. 61. Pasare, C., and R. Medzhitov. 2003. Toll pathway-dependent blockade of Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal ϩ ϩ lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27:68. CD4 CD25 T cell-mediated suppression by dendritic cells. Science 299:1033. ϩ ϩ 62. Dieckmann, D., C. M. Bruett, H. Ploettner, M. B. Lutz, and G. Schuler. Human 46. Stephens, L. A., C. Mottet, D. Mason, and F. Powrie. 2001. Human CD4 CD25 ϩ ϩ thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur. CD4 CD25 regulatory, contact-dependent T cells induce interleukin 10-pro- J. Immunol. 31:1247. ducing, contact-independent type 1-like regulatory T cells. J. Exp. Med. 196: 247. 47. Dieckmann, D., H. Plottner, S. Berchtold, T. Berger, and G. Schuler. 2001. Ex 63. Targan, S. R., R. L. Deem, M. Liu, S. Wang, and A. Nel. 1995. Definition of a vivo isolation and characterization of CD4ϩCD25ϩ T cells with regulatory prop- lamina propria T cell responsive state: enhanced cytokine responsiveness of T erties from human blood. J. Exp. Med. 193:1303. cells stimulated through the CD2 pathway. J. Immunol. 154:664. 48. Jonuleit, H., E. Schmitt, M. Stassen, A. Tuettenberg, J. Knop, and A. H. Enk. 64. Chambers, C. A., M. S. Kuhns, J. G. Egen, and J. P. Allison. 2001. CTLA-4- 2001. Identification and functional characterization of human CD4ϩCD25ϩ T mediated inhibition in regulation of T cell responses: mechanisms and manipu- cells with regulatory properties isolated from peripheral blood. J. Exp. Med. lation in tumor immunotherapy. Annu. Rev. Immunol. 19:565. 193:1285. 65. Schwartz, R. H. 2003. T cell anergy. Annu. Rev. Immunol. 21:305. ϩ ϩ by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. 49. Baecher-Allan, C., and D. A. Hafler. 2004. Human CD4 CD25 regulatory. 66. Oida, T., X. Zhang, M. Goto, S. Hachimura, M. Totsuka, S. Kaminogawa, and Semin. Immunol. 16:89. H. L. Weiner. 2003. CD4ϩCD25Ϫ T cells that express latency-associated peptide 50. Cao, D., V. Malmstrom, C. Baecher-Allan, D. A. Hafler, L. Klareskog, and on the surface suppress CD4ϩCD45RBhigh-induced colitis by a TGF-␤-depen- C. Trollmo. 2003. Isolation and functional characterization of regulatory dent mechanism. J. Immunol. 170:2516. http://classic.jimmunol.org Downloaded from