Volume 128, Issue 7, Pages 1868-1878 (June 2005) Peripheral and Intestinal Regulatory CD4+CD25high T Cells in Inflammatory Bowel Disease  Jochen Maul, Christoph Loddenkemper, Pamela Mundt, Erika Berg, Thomas Giese, Andreas Stallmach, Martin Zeitz, Rainer Duchmann  Gastroenterology  Volume 128, Issue 7, Pages 1868-1878 (June 2005) DOI: 10.1053/j.gastro.2005.03.043 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Representative FACS-analysis of CD4+CD25+ T-cell subgroups. Freshly isolated peripheral blood mononuclear cells were stained with anti-CD4-PE and anti-CD25-FITC. Lymphocytes were gated via forward and side scatter characteristics. (A) Highly suppressive CD4+CD25high Treg cells appear as a distinct subpopulation to the right from the major population containing CD4+CD25low and CD4+CD25− T cells. (B) In Crohn’s disease (CD) and ulcerative colits (UC), CD4+CD25high Treg cells are less frequent in active disease compared with inactive disease (top and middle). This contrasts with an increased frequency of CD4+ CD25high Treg cells in patients with acute diverticulitis (bottom). Gastroenterology 2005 128, 1868-1878DOI: (10.1053/j.gastro.2005.03.043) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Regulatory T cells in peripheral blood from IBD patients and controls. (A) The ratio of CD4+CD25high/CD4+CD25low cells was analyzed in peripheral blood mononuclear cells from patients with active (n = 20) and inactive (n = 22) Crohn’s disease (CD), active (n = 13) and inactive (n = 12) ulcerative colitis (UC), acute diverticulitis (DC; n = 11), and healthy controls (HC; n = 14) by flow cytometry. (B) FOXP3+/100 CD3+ T cells were analyzed on cytospins of peripheral blood mononuclear cells from patients with active (n = 8) and inactive (n = 7) Crohn’s disease (CD), active (n = 5) and inactive (n = 5) ulcerative colitis (UC), acute diverticulitis (DC; n = 6), and healthy controls (HC; n = 4) by immunochemistry; horizontal line: mean of that group. Gastroenterology 2005 128, 1868-1878DOI: (10.1053/j.gastro.2005.03.043) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 CD4+CD25high T cells from peripheral blood of IBD patients retain their capacity to suppress the proliferation of CD4+CD25− responder cells. Peripheral blood mononuclear cells were presorted with anti-CD4-MACS beads. Cells were then stained with anti-CD25-APC and further purified by FACS. CD4+CD25− responder T cells (5 × 104 cells/well) were stimulated with a submaximal dose of soluble anti-CD3 (OKT3, 10 μg/mL) and heterologous T-cell-depleted accessory cells (50 Gy, 5 × 104 cells/well) alone (1:0) and in the presence of suppressive CD4+CD25high Treg cells at different responder/Treg ratios. Proliferation (triplicate cultures) was measured by 3[H]-thymidin incorporation. Results (mean cpm) represent data from 6 patients with IBD (n = 4 for CD, n = 2 for UC) and 4 healthy controls (HC). Suppressive capacity of CD4+CD25high Treg cells was similar in patients with IBD compared with HC. Gastroenterology 2005 128, 1868-1878DOI: (10.1053/j.gastro.2005.03.043) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 CD4+ CD25high T cells are increased in IBD lesions. Biopsy specimens from macroscopically inflamed and noninflamed areas from inflammatory controls (IC; diverticulitis, n = 12, enteritis, n = 2), patients with Crohn’s disease (CD, n = 20) or ulcerative colitis (UC, n = 14) and from healthy controls (HC, n = 17) were taken during ileocolonoscopy. Matched biopsy specimens are connected by a line (black for diverticulitis and CD, grey for UC). Isolated LPMC cells were stained for CD3 and FOXP3 on cytospins (upper left) and with antibodies against CD4 and CD25 by flow cytometry (upper right). Sections from intestinal biopsies were stained for CD3 and FOXP3 (middle left). mRNA isolated from intestinal biopsies was subjected to real-time PCR and analyzed for FOXP3, CD25, and IL-8 transcripts (lower left, middle right, and lower right). In all matched samples, the percentage of CD4+CD25high or FOXP3+ T cells and the transcripts for FOXP3, CD25, and IL-8 were increased in inflamed compared with noninflamed mucosa. FOXP3+ T cells in both cytospins and sections from intestinal IBD lesions were significantly decreased compared with inflammatory controls; ND, not done. Gastroenterology 2005 128, 1868-1878DOI: (10.1053/j.gastro.2005.03.043) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Immunohistochemistry for CD3+FOXP3+ cells. Representative H&E staining of matched mucosal biopsy specimens from a patient with CD shows little chronic inflammation in the colon (A) and an intense inflammatory infiltrate in the ileal lamina propria and the submucosa (C). Biopsy specimen from the colon of a patient with UC showing numerous crypt abscesses (E) and large bowel specimen with a diverticulum from a patient with acute diverticulitis (G). Corresponding double-immunoenzymatic labeling of CD3+ T cells (red, membranous) with a subpopulation of T cells exhibiting additional expression of FOXP3 (brown, nuclear) shows no CD3+FOXP3+ cells in the mucosal biopsy specimen from the patient with CD in an area of little inflammation (B). Biopsy specimens from CD (D) and UC (F) patients in areas of severe inflammation show only a moderate amount of CD3+FOXP3+ cells in the lamina propria. The number of T cells with coexpression of CD3/FOXP3 is highest in diverticulitis (H). Representative cells costaining for CD3 (membranous) and FOXP3 (nuclear) are indicated by arrows. All FOXP3+ cells stained positive for CD25 (insert in F). Gastroenterology 2005 128, 1868-1878DOI: (10.1053/j.gastro.2005.03.043) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Immunocytochemistry for FOXP3/CD3 and FOXP3/CD25. Cytospin showing costaining for FOXP3/CD3 (A) and FOXP3/CD25 (B). Gastroenterology 2005 128, 1868-1878DOI: (10.1053/j.gastro.2005.03.043) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Gastroenterology 2005 128, 1868-1878DOI: (10. 1053/j. gastro. 2005. 03 Copyright © 2005 American Gastroenterological Association Terms and Conditions