Volume 131, Issue 2, Pages 428-438 (August 2006) A Unique Dendritic Cell Subset Accumulates in the Celiac Lesion and Efficiently Activates Gluten-Reactive T Cells  Melinda Ráki, Stig Tollefsen, Øyvind Molberg, Knut E.A. Lundin, Ludvig M. Sollid, Frode L. Jahnsen  Gastroenterology  Volume 131, Issue 2, Pages 428-438 (August 2006) DOI: 10.1053/j.gastro.2006.06.002 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 In situ phenotypic characterization of APCs in normal duodenal mucosa. Paired immunofluorescence staining for markers as indicated on cryosections of mucosal tissue. (A) Most HLA-DQ2+ cells co-express CD68+, but (B) a minor population express CD11c+ (arrowheads). (C) Virtually no cells co-express CD68 and CD11c. Note dendritic morphology of CD11c+ cells (arrowhead). (D) A subset of CD11c+ cells co-express CD1c (arrowheads). (E) Most macrophages express DC-SIGN. Surface epithelium is visualized in A–C (autofluorescence at excitation wave length of 650 nm; blue color). Gastroenterology 2006 131, 428-438DOI: (10.1053/j.gastro.2006.06.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 In situ phenotypic characterization of APCs in the celiac lesion. Paired immunofluorescence staining for markers as indicated on cryosections of duodenal mucosa from active celiac lesion. (A) Note an apparent increase in the density of CD11c+ cells compared with that in Figure 1B–D. (B) Some CD14+ monocytes do not express CD68 (arrowhead), indicating DC differentiation. (C) Some DC-SIGN+ cells express CD16 (arrowhead). (D) CD86 is expressed preferentially on CD11c+ DCs in the celiac lesion (arrowhead). Crypt epithelium is indicated with an asterisk (D). The basement membrane of surface epithelium is indicated by a broken line (A and D). Gastroenterology 2006 131, 428-438DOI: (10.1053/j.gastro.2006.06.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Density and phenotypic frequencies of HLA-DQ+ cells in sections from duodenal mucosa of untreated (n = 8) and treated (n = 6) celiac patients, and HLA-DQ2+ (•) and HLA-DQ2− (○) controls (n = 6). HLA-DQ+ cells in the DQ2 negative controls were stained with the clone SPV-L3, which recognizes a common DQ determinant. Medians are indicated by horizontal lines. Gastroenterology 2006 131, 428-438DOI: (10.1053/j.gastro.2006.06.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Density of CD11c+ DCs expressing of CD86+ and DC-LAMP+ in sections from duodenal mucosa of untreated celiac patients and controls. ●, HLA-DQ2+ cells; ○, HLA-DQ2− cells. Medians are indicated by horizontal lines. Gastroenterology 2006 131, 428-438DOI: (10.1053/j.gastro.2006.06.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Antigen-specific T-cell proliferation induced by various subpopulations of biopsy specimen cells. (A) Scheme of the experiment. Duodenal biopsy specimens from untreated patients were incubated with or without the immunodominant 33-mer peptide of α-gliadin. Biopsy specimens then were digested enzymatically into single-cell suspensions, and CD11c+ DCs and DC-SIGN+ macrophages were isolated by magnetic separation. Four cell fractions (total cells, CD11c+ cells, DC-SIGN+ cells, and the remaining cells after the magnetic separation procedure [rest]) were irradiated and used as APCs in coculture with the carboxyfluorescein-diacetate succinimidyl ester-labeled gluten-reactive TCC 430.1.142 or TCC 430.1.135 for 4 days. T-cell activation was measured by proliferative response and cytokine production. (B) Flow cytometric analysis of the purity of the 4 fractions used as APCs in A. Representative results of 3 experiments are shown. Gastroenterology 2006 131, 428-438DOI: (10.1053/j.gastro.2006.06.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Proliferation and cytokine production of gluten-specific T cells after coculture with various fractions of biopsy specimen cells (see Figure 5). (A) Example of dot plot in flow cytometric analysis of T-cell proliferation (% of dividing cells indicated) using unseparated cells from medium-incubated biopsy specimens (left plot), DC- SIGN+ macrophages or CD11c+ DCs isolated from 33-mer-incubated biopsy specimens (middle and right plots, respectively) as APCs at a 1:8 APC–T-cell ratio. (B) T-cell proliferation (upper panels) and concentration of interferon-γ (lower panels) after coculturing TCCs with various cell fractions derived from biopsy specimens stimulated with the 33-mer peptide or medium (see Figure 5). Biopsy specimens of 3 untreated patients (CD563, CD578, and CD559) were analyzed, and TCC 430.1.135 (DQ2-αI-gliadin specific) and TCC 430.1.142 (DQ2-αII-gliadin specific) were used. Gastroenterology 2006 131, 428-438DOI: (10.1053/j.gastro.2006.06.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions