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Peripheral and Intestinal Regulatory CD4+CD25high T Cells in Inflammatory Bowel Disease

2005; Elsevier BV; Volume: 128; Issue: 7 Linguagem: Inglês

10.1053/j.gastro.2005.03.043

1528-0012

Jochen Maul, Christoph Loddenkemper, Pamela Mundt, Erika Gebel Berg, Thomas Giese, Andreas Stallmach, Martin Zeitz, Rainer Duchmann,

Inflammatory Bowel Disease

Background & Aims: Regulatory CD25+ T cells (Treg) are effective in the prevention and down-regulation of inflammatory bowel disease (IBD) in animal models. Functional Treg cells are characterized by the expression of the transcription factor FOXP3 and show a CD4+CD25high phenotype in humans. The aim of this study was to determine whether disease activity in IBD correlates with changes in frequency of Treg cells and their distribution in the intestinal mucosa. Methods: Treg cells were analyzed from peripheral blood and from biopsy specimens of IBD patients, inflammatory controls, and healthy volunteers by flow cytometry (CD4+CD25high), immunochemistry (FOXP3), and real-time PCR (FOXP3). Regulatory properties of purified peripheral CD4+CD25high Treg cells were determined by their suppressive effect on the proliferation of CD4+CD25− T cells. Results: In peripheral blood, CD4+CD25high T cells from IBD patients retain their suppressive activity. CD4+CD25high and FOXP3+ Treg cells are increased during remission but decreased during active disease. This contrasts with their strong increase in peripheral blood of patients with acute diverticulitis. Different than peripheral blood, inflamed IBD mucosa contains an increased number of CD4+CD25high T cells, FOXP3+ T cells, and transcripts for FOXP3 compared with noninflamed mucosa. However, the increase of FOXP3+ T cells in IBD lesions is significantly lower compared with inflammatory controls. Conclusions: The frequency of CD4+CD25+ Treg cells varies with IBD activity. Active IBD is not associated with a functional defect but with a contraction of the peripheral blood Treg pool and an only moderate expansion in intestinal lesions. Thus, compensatory mechanisms, numerically, are not successfully achieved in these diseases. Background & Aims: Regulatory CD25+ T cells (Treg) are effective in the prevention and down-regulation of inflammatory bowel disease (IBD) in animal models. Functional Treg cells are characterized by the expression of the transcription factor FOXP3 and show a CD4+CD25high phenotype in humans. The aim of this study was to determine whether disease activity in IBD correlates with changes in frequency of Treg cells and their distribution in the intestinal mucosa. Methods: Treg cells were analyzed from peripheral blood and from biopsy specimens of IBD patients, inflammatory controls, and healthy volunteers by flow cytometry (CD4+CD25high), immunochemistry (FOXP3), and real-time PCR (FOXP3). Regulatory properties of purified peripheral CD4+CD25high Treg cells were determined by their suppressive effect on the proliferation of CD4+CD25− T cells. Results: In peripheral blood, CD4+CD25high T cells from IBD patients retain their suppressive activity. CD4+CD25high and FOXP3+ Treg cells are increased during remission but decreased during active disease. This contrasts with their strong increase in peripheral blood of patients with acute diverticulitis. Different than peripheral blood, inflamed IBD mucosa contains an increased number of CD4+CD25high T cells, FOXP3+ T cells, and transcripts for FOXP3 compared with noninflamed mucosa. However, the increase of FOXP3+ T cells in IBD lesions is significantly lower compared with inflammatory controls. Conclusions: The frequency of CD4+CD25+ Treg cells varies with IBD activity. Active IBD is not associated with a functional defect but with a contraction of the peripheral blood Treg pool and an only moderate expansion in intestinal lesions. Thus, compensatory mechanisms, numerically, are not successfully achieved in these diseases. Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory condition of the gastrointestinal tract that manifests as 2 distinct but sometimes overlapping clinical entities, Crohn’s disease (CD) and ulcerative colitis (UC). Both diseases are frequently associated with extraintestinal manifestations and are thus systemic in nature.1Blumberg R. Strober W. Prospects for research in inflammatory bowel disease.JAMA. 2001; 285: 643-647Crossref PubMed Scopus (133) Google Scholar Although messenger RNA (mRNA) profiles of important immune mediators in IBD tissue are quite similar for CD and UC,2Autschbach F. Giese T. Gassler N. Sido B. Heuschen G. Heuschen U. Zuna I. Schulz P. Weckauf H. Berger I. Otto H.F. Meuer S.C. Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn’s disease.Virchows Arch. 2002; 441: 500-513Crossref PubMed Scopus (94) Google Scholar there is a large body of data from animal models and functional studies in humans indicating that the inflammatory process is in fact channelled through different abnormalities in immune function with an excessive T helper (Th)1 response in CD and a Th2-like response in UC.1Blumberg R. Strober W. Prospects for research in inflammatory bowel disease.JAMA. 2001; 285: 643-647Crossref PubMed Scopus (133) Google Scholar, 3Strober W. Fuss I.J. Blumberg R.S. The immunology of mucosal models of inflammation.Annu Rev Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1142) Google Scholar In addition, there is increasing awareness that IBD may be more completely described as a dysbalance of the immune system, caused by an excess of inflammatory stimuli and mediators and an inadequately low function or number of cellular components that down-regulate mucosal immune responsiveness. CD4+CD25+ regulatory T cells (Treg) control immune responses to self- and foreign antigens and are likely candidates to search for inadequate counter regulation in IBD. In more detail, they prevent organ-specific autoimmune diseases such as autoimmune thyroiditis, autoimmune gastritis, insulitis, and arthritis4Sakaguchi S. Sakaguchi N. Asano M. Itoh M. Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J Immunol. 1995; 155: 1151-1164PubMed Google Scholar, 5Suri-Payer E. Amar A.Z. Thornton A.M. Shevach E.M. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells.J Immunol. 1998; 160: 1212-1218PubMed Google Scholar and inhibit the rejection of transplanted organs.6Graca L. Thompson S. Lin C.Y. Adams E. Cobbold S.P. Waldmann H. Both CD4(+)CD25(+) and CD4(+)CD25(-) regulatory cells mediate dominant transplantation tolerance.J Immunol. 2002; 168: 5558-5565Crossref PubMed Scopus (336) Google Scholar, 7Hara M. Kingsley C.I. Niimi M. Read S. Turvey S.E. Bushell A.R. Morris P.J. Powrie F. Wood K.J. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo.J Immunol. 2001; 166: 3789-3796Crossref PubMed Scopus (691) Google Scholar, 8Kingsley C.I. Karim M. Bushell A.R. Wood K.J. CD25+CD4+ regulatory T cells prevent graft rejection CTLA-4- and IL-10- dependent immunoregulation of alloresponses.J Immunol. 2002; 168: 1080-1086Crossref PubMed Scopus (650) Google Scholar They are also involved in oral tolerance,9Zhang X. Izikson L. Liu L. Weiner H.L. Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration.J Immunol. 2001; 167: 4245-4253PubMed Google Scholar inhibit the activation of Th1 cells against enteric bacterial antigens,10Gad M. Brimnes J. Claesson M.H. CD4+ T regulatory cells from the colonic lamina propria of normal mice inhibit proliferation of enterobacteria-reactive, disease-inducing Th1- cells from SCID mice with colitis.Clin Exp Immunol. 2003; 131: 34-40Crossref PubMed Scopus (25) Google Scholar and prevent11Read S. Malmstrom V. Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.J Exp Med. 2000; 192: 295-302Crossref PubMed Scopus (1849) Google Scholar and treat established12Mottet C. Uhlig H.H. Powrie F. Cutting edge cure of colitis by CD4(+)CD25(+) regulatory T cells.J Immunol. 2003; 170: 3939-3943PubMed Google Scholar colitis in animal models of IBD. Because murine CD25+ T cells form a uniform population on flow cytometry, it is impossible to use this marker to separate activated from regulatory T cells. Further phenotypic markers such as CD45RBlow11Read S. Malmstrom V. Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.J Exp Med. 2000; 192: 295-302Crossref PubMed Scopus (1849) Google Scholar or the integrin13Lehmann J. Huehn J. de la Rosa M. Maszyna F. Kretschmer U. Krenn V. Brunner M. Scheffold A. Hamann A. Expression of the integrin α Eβ 7 identifies unique subsets of CD25+ as well as CD25- regulatory T cells.Proc Natl Acad Sci U S A. 2002; 99: 13031-13036Crossref PubMed Scopus (408) Google Scholar αEβ7 can help to identify CD25+ Treg cells in mice. Regulatory T cells are also present in human peripheral blood,14Baecher-Allan C. Brown J.A. Freeman G.J. Hafler D.A. CD4+CD25 high regulatory cells in human peripheral blood.J Immunol. 2001; 167: 1245-1253PubMed Google Scholar, 15Dieckmann D. Plottner H. Berchtold S. Berger T. Schuler G. Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood.J Exp Med. 2001; 193: 1303-1310Crossref PubMed Scopus (971) Google Scholar, 16Jonuleit H. Schmitt E. Stassen M. Tuettenberg A. Knop J. Enk A.H. Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood.J Exp Med. 2001; 193: 1285-1294Crossref PubMed Scopus (1065) Google Scholar, 17Levings M.K. Sangregorio R. Roncarolo M.G. Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function.J Exp Med. 2001; 193: 1295-1302Crossref PubMed Scopus (858) Google Scholar, 18Wing K. Ekmark A. Karlsson H. Rudin A. Suri-Payer E. Characterization of human CD25+ CD4+ T cells in thymus, cord and adult blood.Immunology. 2002; 106: 190-199Crossref PubMed Scopus (196) Google Scholar, 19Stephens L.A. Mottet C. Mason D. Powrie F. Human CD4(+)CD25(+) thymocytes and peripheral T cells have immune suppressive activity in vitro.Eur J Immunol. 2001; 31: 1247-1254Crossref PubMed Scopus (434) Google Scholar intestinal lamina propria,20Makita S. Kanai T. Oshima S. Uraushihara K. Totsuka T. Sawada T. Nakamura T. Koganei K. Fukushima T. Watanabe M. CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells.J Immunol. 2004; 173: 3119-3130PubMed Google Scholar tonsils,21Taams L.S. Smith J. Rustin M.H. Salmon M. Poulter L.W. Akbar A.N. Human anergic/suppressive CD4(+)CD25(+) T cells a highly differentiated and apoptosis-prone population.Eur J Immunol. 2001; 31: 1122-1131Crossref PubMed Scopus (397) Google Scholar and thymus.18Wing K. Ekmark A. Karlsson H. Rudin A. Suri-Payer E. Characterization of human CD25+ CD4+ T cells in thymus, cord and adult blood.Immunology. 2002; 106: 190-199Crossref PubMed Scopus (196) Google Scholar, 19Stephens L.A. Mottet C. Mason D. Powrie F. Human CD4(+)CD25(+) thymocytes and peripheral T cells have immune suppressive activity in vitro.Eur J Immunol. 2001; 31: 1247-1254Crossref PubMed Scopus (434) Google Scholar As in their rodent counterparts, the suppressive effect of human Treg depends on cell contact. However, it is not mediated via CTLA-4 and cannot be blocked by antibodies (Abs) to interleukin (IL)-4, IL-10, or transforming growth factor (TGF)-β.14Baecher-Allan C. Brown J.A. Freeman G.J. Hafler D.A. CD4+CD25 high regulatory cells in human peripheral blood.J Immunol. 2001; 167: 1245-1253PubMed Google Scholar, 21Taams L.S. Smith J. Rustin M.H. Salmon M. Poulter L.W. Akbar A.N. Human anergic/suppressive CD4(+)CD25(+) T cells a highly differentiated and apoptosis-prone population.Eur J Immunol. 2001; 31: 1122-1131Crossref PubMed Scopus (397) Google Scholar In contrast to the situation in mice described above, human Treg cells form a subpopulation to the right on CD25+ flow cytometry and can thus be identified by their high density of the IL-2-receptor α-chain.14Baecher-Allan C. Brown J.A. Freeman G.J. Hafler D.A. CD4+CD25 high regulatory cells in human peripheral blood.J Immunol. 2001; 167: 1245-1253PubMed Google Scholar, 22Cao D. Malmstrom V. Baecher-Allan C. Hafler D. Klareskog L. Trollmo C. Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis.Eur J Immunol. 2003; 33: 215-223Crossref PubMed Scopus (378) Google Scholar Recent reports, however, show that the transcription factor FOXP3 might be a specific marker for CD4+CD25+ regulatory cells.23Hori S. Nomura T. Sakaguchi S. Control of regulatory T-cell development by the transcription factor Foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (49) Google Scholar In contrast to most phenotypic markers, which are quite likely surrogate markers related to the differentiation of regulatory T cells, the expression of FOXP3 seems to be crucial for their functional characteristics. This was shown by introducing the FOXP3 gene into naive CD4+CD25− T cells, which subsequently displayed suppressive function.23Hori S. Nomura T. Sakaguchi S. Control of regulatory T-cell development by the transcription factor Foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (49) Google Scholar, 24Yagi H. Nomura T. Nakamura K. Yamazaki S. Kitawaki T. Hori S. Maeda M. Onodera M. Uchiyama T. Fujii S. Sakaguchi S. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells.Int Immunol. 2004; 16: 1643-1656Crossref PubMed Scopus (683) Google Scholar Considering the potent regulatory capacity of CD4+CD25high peripheral blood T cells in healthy human individuals, we determined whether abnormalities in suppressor function or number of CD25+ Treg cells in the peripheral blood and in the intestinal mucosa correlate with IBD activity. Peripheral blood from 46 patients with CD (24 patients with active and 22 patients with inactive disease; 22 women, 24 men; mean age, 42 years; range, 18–74 years), 28 patients with UC (14 patients with active and 14 patients with inactive disease; 13 women, 15 men; mean age, 41 years; range, 25–89 years), 11 patients with acute diverticulitis (7 women, 4 men; mean age, 59 years; range, 45–73 years), and 16 healthy volunteers (9 women, 7 men; mean age, 31 years; range, 23–57 years) were investigated. Active CD was defined as a Crohn’s disease activity index (CDAI) >150, inactive disease as a CDAI <150.25Best W.R. Becktel J.M. Singleton J.W. Rederived values of the eight coefficients of the Crohn’s disease activity index (CDAI).Gastroenterology. 1979; 77: 843-846PubMed Scopus (519) Google Scholar Active UC was defined according to Truelove and Witts.26Truelove S.C. Witts L.J. Cortisone in ulcerative colitis final report on a therapeutic trial.Br Med J. 1955; : 1041-1048Crossref PubMed Scopus (2179) Google Scholar Blood samples from patients with acute diverticulitis were taken in the first 48 hours of hospital admission and before the second day of antibiotic therapy. Matched biopsy specimens, if available, from macroscopically inflamed and noninflamed areas from patients with UC (n = 14), patients with CD (n = 20), patients with diverticulitis or infectious enteritis (n = 14), and healthy patients undergoing screening colonoscopy for colorectal cancer (n = 17) were taken during ileocolonoscopy. In patients with IBD, mucosal biopsy specimens with severe inflammatory features were compared with matched biopsy specimens with little or no inflammation. The study was performed according to established ethical guidelines (Ethical Comittee of Medicine, Free University of Berlin, Germany), and the patients were informed and gave written consent before they were included in the study. Peripheral blood mononuclear cells (PBMC) were isolated from freshly drawn blood by Ficoll density gradient centrifugation. For isolation of lamina propria mononuclear cells (LPMC), freshly taken biopsy specimens were washed in PBS and incubated with EDTA for 30 minutes at 4°C. After an additional washing in PBS, tissue samples were digested in 0.1 mg/mL Collagenase, 200 μg/mL Hyaluronidase (both from Sigma-Aldrich Chemie, Steinheim, Germany), and 100 μg/mL DNAse (Roche Diagnostics, Mannheim, Germany) in HBSS. Tissue was disintegrated using 35 μm Medicons with a Medimachine (DakoCytomation, Hamburg, Germany) and filtered through a 70-μm cell strainer (BD Biosciences, Heidelberg, Germany). Finally, isolated cells were purified by Ficoll density centrifugation. CD4+, CD4+CD25−, and CD4+CD25+ cells were isolated from PBMC by positive selection using a multisort kit (Miltenyi Biotech, Bergisch Gladbach, Germany). For isolation of CD4+CD25+ subsets, CD4+ PBMC were positively selected using anti-CD4 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) after staining with fluorescein isocyanate (FITC)-labelled anti-CD4 Ab (clone M-T466, Miltenyi Biotech). CD4+ PBMC were then stained with allophycocyanin (APC)-labelled CD25 Ab (clone 2A3, Becton Dickinson, Heidelberg, Germany). CD4+CD25high Treg cells were sorted according to the gates indicated in Figure 1A using a FACSDiva (Becton Dickinson, Heidelberg, Germany). T-cell depleted accessory cells were obtained by negative selection of PBMC using anti-CD2 beads (Miltenyi Biotech) and irradiation at 50 Gy. For immunostaining phycoerythrin (PE)-conjugated or phycoerythrin-cyanin (PC7)-conjugated Abs against CD4 (clones 13B8.2 and SFCI12T4D11, Beckman Coulter, Krefeld, Germany), FITC- and APC-conjugated Abs against CD25 (clones 2A3 and M-A251, Becton Dickinson), and respective mouse isotype controls were employed. Cells were washed and stained for 10 minutes on ice with optimal dilution of each Ab. Cells were then washed again and analyzed by flow cytometry (FACSCalibur and CellQuest software; Becton Dickinson) using a live gate set around viable lymphocytes based on their forward scatter/side scatter (FCS/SSC) characteristics. For analysis of CD25high T cells, large activated cells as determined by forward and side scatter properties were excluded. CD25high T cells were brighter than the CD25 low expressing CD4-CD25+ (most likely B cells) and generally showed a lower CD4 expression than the CD4+CD25low or CD4+CD25− population. For assessment of regulatory properties of CD4+CD25+ subpopulations, 5 × 104 autologous CD4+CD25− T cells were cultured in RPMI 1640 (PAA Laboratories, Linz, Austria) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 mmol/L glutamine. Cells were stimulated with irradiated heterologous T cell-depleted accessory cells and soluble anti-CD3 (10 μg/mL, OKT3, gift of Dr. J. Hoffmann, Berlin, Germany) in triplicate in 96-well U-bottomed plates (NUNC, Wiesbaden, Germany). MACS-sorted CD4+CD25+, FACS sorted CD4+CD25−, or CD4+CD25high cells were added. CD4+CD25high cells were seeded at different responder:Treg ratios. After 72 hours of culture, 0.5 μCi /well of 3[H]-thymidin (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) was added for additional 18 hours. Cells were harvested on glass fiber membranes, and incorporated 3[H]-thymidin was detected by liquid scintillation counting (LKB Wallace, Turku, Finland). Isolated PBMC were lysed in 400 μL lysis buffer from the MagnaPure mRNA Isolation Kit I (Roche Diagnostics) according to the manufacturer’s instruction. Mucosal biopsy specimens were collected in RNA-later (AMBION, Huntingdon, United Kingdom). The samples were then transferred into RiboLyser tubes “Green” (ThermoHYBAID, Heidelberg, Germany) containing 400 μL lysis buffer from the MagnaPure mRNA Isolation Kit II (Roche Diagnostics). Tissue was disrupted by 1 run with the RiboLyser (ThermoHYBAID). After each run, the RiboLyser tubes were centrifuged at 4°C for 1 minute at 13,000 rpm. Lysates from both PBMC and mucosal biopsy specimens were collected and stored at −80°C until mRNA isolation. The samples were thawed at room temperature. To each sample, 600 μL capture buffer containing oligo-dT was added. After centrifugation, this mix was transferred into a MagnaPure samples cartridge, and the mRNA was isolated with the MagnaPure-LC device using the mRNA-II standard protocol. RNA was reverse transcribed using AMV-RT and oligo-(dT) as primer (First Strand cDNA synthesis kit, Roche Diagnostics) according to the manufacturer’s protocol in a thermocycler. After termination of the cDNA synthesis, the reaction mix was diluted in an appropriate volume and stored at −20°C until PCR analysis. Parameter-specific primer sets optimized for the LightCycler (RAS, Mannheim, Germany) were purchased from SEARCH-LC GmbH, Heidelberg, Germany. The PCR was performed with the LightCycler FastStart DNA Sybr GreenI kit (RAS) according to the protocol provided. To control for specificity of the amplification products, a melting curve analysis was performed. No amplification of unspecific products was observed. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of 4 different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. RNA input was normalized by the average expression of the 2 housekeeping genes β-Actin and Cyclophilin B. The data of 2 independent analyses for each sample and parameter were averaged and presented as adjusted transcripts per μL cDNA. For immunostaining, 4μm-thick sections from mucosal biopsy specimens were cut, deparaffinized, and subjected to a heat-induced epitope retrieval step before incubation with antibodies. Furthermore, cytospins from PBMC and LPMC were air-dried, fixed for 10 minutes in acetone, and stored at −80°C. For immunostaining, the cytospins were fixed in buffered formalin. Sections and cytospins were immersed in sodium citrate buffer solutions at pH 6.0 and heated in a high-pressure cooker. The slides were rinsed in cool running water, washed in Tris-buffered saline (pH 7.4), blocked using a commercial peroxidase-blocking reagent (DakoCytomation, Glostrup, Denmark), and incubated for 30 minutes with the goat polyclonal antibody against the C terminus of the FOXP3 protein (ab2481, dilution 1:50) obtained from Abcam (Abcam Limited, Cambridge, United Kingdom), followed by rabbit anti-goat antibody and the EnVision peroxidase kit (DakoCytomation). The slides were then incubated for an additional 30 minutes with the second antibody against CD3 (clone UCHT1, 1:25, DakoCytomation), and, for detection, the alkaline phosphatase anti-alkaline phosphatase (APAAP) method was used. For double labeling of FOXP3/CD25, the anti-CD25 antibody (clone 4C9, 1:100, Novocastra) was incubated for 30 minutes as second primary antibody and detected using biotinylated donkey anti-mouse antibody (1:200, Dianova) and streptavidin-AP (DakoCytomation). Tonsillar tissue with follicular hyperplasia served as positive controls, displaying scattered T cells in the interfollicular area with nuclear expression of FOXP3. Negative controls were performed by omitting the primary antibodies. For all studies, data are expressed as mean ± SD; differences were analyzed by using the nonparametric Mann-Whitney rank test (SPSS, v11.5; SPSS Inc, Chicago, IL). P values <.05 were considered significant. Similar to published data,14Baecher-Allan C. Brown J.A. Freeman G.J. Hafler D.A. CD4+CD25 high regulatory cells in human peripheral blood.J Immunol. 2001; 167: 1245-1253PubMed Google Scholar, 18Wing K. Ekmark A. Karlsson H. Rudin A. Suri-Payer E. Characterization of human CD25+ CD4+ T cells in thymus, cord and adult blood.Immunology. 2002; 106: 190-199Crossref PubMed Scopus (196) Google Scholar, 22Cao D. Malmstrom V. Baecher-Allan C. Hafler D. Klareskog L. Trollmo C. Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis.Eur J Immunol. 2003; 33: 215-223Crossref PubMed Scopus (378) Google Scholar we found that CD4+CD25high Treg cells are present in peripheral blood lymphocytes of healthy individuals at a mean of 1.64% (range, 0.6%–3.17%). On flow cytometry, these human Treg cells appear as a subpopulation to the right from the major population of CD4+CD25− and CD4+CD25low cells (Figure 1A). To determine whether changes in frequency of CD25high Treg cells are present in IBD and correlate with disease activity, we analyzed CD25high surface expression in 36 patients with active and 34 patients with inactive CD or UC (Figure 1B and Table 1). We found significant differences in CD4+CD25high Treg cell frequencies, resulting from lower levels in active and higher levels in inactive IBD (0.73% ± 0.39% vs 1.83% ± 0.98%, respectively; P < .001). In inactive CD, the percentage of CD4+CD25high Treg cells increased 3-fold compared with active disease (0.68% ± 0.40% vs 2.04% ± 1.06%, respectively; P < .001). UC patients showed similar results (0.80% ± 0.39% vs 1.44% ± 0.69%, respectively; P < .05), but changes were less prominent. In contrast to CD, in which the frequency of CD4+CD25high Treg cells in active (0.68% ± 0.40%) and inactive (2.04% ± 1.06%) disease oscillated around their normal expression in healthy controls (1.64% ± 0.72%), CD4+CD25high Treg cells were not more frequent in patients with UC in remission (1.44% ± 0.69%) than in healthy controls. Interestingly, subsequent studies in inflammatory controls demonstrated that the decreased frequency of CD4+CD25high Treg cells was a selective feature of the dysregulated inflammatory response in active IBD. Thus, patients with acute diverticulitis showed the opposite effect, ie, an increased percentage of CD4+CD25high Treg cells (2.03% ± 1.13%). To exclude that changes in frequency of CD4+CD25high Treg cells observed in our study reflect nonspecific effects on total lymphocytes, we also calculated the ratio of CD4+CD25high to CD4+CD25low T cells as an independent measure indicating expansion or contraction of the regulatory T-cell pool (Table 1 and Figure 2, upper panel). Ratios of CD4+CD25high cells (which have regulatory function, see below) to CD4+CD25low cells (which do not have regulatory function) confirmed that CD4+CD25high Treg cells are enriched in inactive vs active IBD (0.33 ± 0.17 vs 0.20 ± 0.08, respectively; P = .026). As expected from the frequency data, both active CD and active UC showed a decreased CD4+CD25high/CD4+CD25low ratio compared with healthy controls (0.22 ± 0.21 vs 0.16 ± 0.09 vs 0.27 ± 0.15, respectively) and patients with inactive CD but not those with inactive UC showed expansion of Treg cells above the level present in healthy controls (0.36 ± 0.17 vs 0.26 ± 0.17 vs 0.27 ± 0.15, respectively). In patients with acute diverticulitis, the CD4+CD25high/CD4+CD25low ratio was strongly increased (0.60 ± 0.29) and not decreased as in IBD. In further experiments, we used FOXP3 as an independent marker for Treg cells. Immuncytochemical analysis of FOXP3/CD3 coexpression on cytospins confirmed that Treg cells are increased in the peripheral blood of IBD patients in remission compared with active disease (P < .001) and decreased in active IBD compared with diverticulitis (P < .001) (Figure 2, lower panel). This same pattern was also seen when FOXP3 transcripts were determined in peripheral blood mononuclear cells by real-time PCR (Table 2).Table 2Adjusted Transcripts per μL cDNA of FOXP3 in Peripheral Blood Mononuclear Cells From Patients With Diverticulitis, Active or Inactive Crohn’s Disease, or Ulcerative Colitis and Healthy ControlsDC (n = 6)Active IBDInactive IBDHC (n = 6)CD (n = 8)UC (n = 5)CD (n = 7)UC (n = 5)FOXP311 ± 56 ± 25 ± 411 ± 49 ± 47 ± 6NOTE. Values are mean ± standard deviation. Open table in a new tab NOTE. Values are mean ± standard deviation. To determine whether the CD4+CD25+ population from IBD patients exhibits regulatory characteristics such as suppression of proliferation and hyporesponsiveness described in healthy human individuals,14Baecher-Allan C. Brown J.A. Freeman G.J. Hafler D.A. CD4+CD25 high regulatory cells in human peripheral blood.J Immunol. 2001; 167: 1245-1253PubMed Google Scholar, 22Cao D. Malmstrom V. Baecher-Allan C. Hafler D. Klareskog L. Trollmo C. Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis.Eur J Immunol. 2003; 33: 215-223Crossref PubMed Scopus (378) Google Scholar in first experiments, MACS-sorted CD4+CD25+ cells from patients with active and inactive Crohn’s disease were cultured alone or together with MACS-sorted autologous CD4+CD25− cells. Indeed, CD4+CD25+ cells did hardly proliferate and effectively suppressed proliferation of CD4+CD25− T cells. No significant difference in suppression between cells from active and inactive disease was observed (data not shown). In subsequent experiments, CD4+CD25high and CD4+CD25− T cells were sorted using gates shown in Figure 1A. Patients with inactive IBD were studied because we knew that CD4+CD25high cells, which can only be purified from peripheral blood with very low yields, are enriched in these patients. To quantitate their regulatory function, autologous responder cells and CD4+CD25high T cells were cocultured at different ratios (responder/suppressor ratios: 1:0, 1:0.125, 1:0.25, 1:0.5, 1:1). CD4+CD25high T cells from patients with IBD and controls potently suppressed proliferation at a 1:1 ratio. Decreasing the ratio of suppressor/responder T cells resulted in less suppression, with no significant difference between patients with IBD and controls (Figure 3). Patients with CD or UC showed similar results (data not shown). As a control, titration of the same dose of CD4+CD25− cells