Volume 114, Issue 4, Pages 649-656 (April 1998) Major histocompatibility complex class II–dependent antigen presentation by human intestinal endothelial cells  Guttorm Haraldsen, Ludvig M. Sollid, Oddmund Bakke, Inger N. Farstad, Dag Kvale, Øyvind Molberg, Jarle Norstein, Espen Stang, Per Brandtzaeg  Gastroenterology  Volume 114, Issue 4, Pages 649-656 (April 1998) DOI: 10.1016/S0016-5085(98)70578-5 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 HLA class II and Ii expression in HIMECs (○) and HUVECs (●). (A) Time course of protein expression after incubation with IFN-γ (100 U/mL). (B) Dose response measured after 3 days (DR and Ii) or 8 days (DQ and DP). Protein expression measured by cellular ELISA on periodate-lysine-paraformaldehyde–fixed confluent monolayers. Antibodies: MAb B8.11, SPV-L3, B7/21, and BU45, respectively. Representative results from one of at least three similar experiments (mean ± SD of quadruplicate wells). Gastroenterology 1998 114, 649-656DOI: (10.1016/S0016-5085(98)70578-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Fluorescence-activated cell sorter analysis of HLA class II expression induced on (A) HIMECs and (B) HUVECs. Log R-phycoerythrin fluorescence histograms of live cells cultured in medium alone or exposed to IFN-γ (100 U/mL) for 3 days (DR) or 11 days (DP and DQ). Open histograms represent irrelevant control MAb, and shaded histograms represent specific MAb staining. Antibodies: MAb L243, FN-81-1, and B7/21, respectively. Gastroenterology 1998 114, 649-656DOI: (10.1016/S0016-5085(98)70578-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Influence of TNF-α on IFN-γ–stimulated HLA class II expression in HIMECs. (A) Dose response to IFN-γ in the presence or absence of TNF-α (30 U/mL). Total cellular protein expression measured by cellular ELISA after incubation for 8 days with increasing doses of IFN-γ alone (●) or in combination with TNF-α (○) . Representative results from one of three similar experiments (mean ± SD of quadruplicate wells). (B) Flow-cytometric analysis of HIMECs after treatment with IFN-γ alone (filled histograms, DR and DP, 10 U/mL; DQ, 100 U/mL); or TNF-α alone (shaded histograms, 30 U/mL) or both cytokines combined (open histograms) for 8 days. Representative results from one of three similar experiments. Antibodies: MAb B8.11, SPV-L3, and B7/21, respectively. Gastroenterology 1998 114, 649-656DOI: (10.1016/S0016-5085(98)70578-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Ultrastructural localization of HLA-DR and Ii in HUVECs. Before fixation, IFN-γ–treated HUVECs were incubated with 5-nm BSA-gold particles for 3 hours followed by overnight chase in gold-free medium before final incubation with 10-nm BSA-gold particles for 30 minutes. Cells were sectioned and immunolabeled as described in Materials and Methods. (A) Overview of part of cell double labeled for Ii (MAb BU45, large, 20-nm gold particles) and DR (rabbit anti-human HLA-DR, 15-nm gold particles, some indicated by arrowheads). Labeling for Ii is restricted to the Golgi apparatus (G) (see B), to small vesicles located close to the Golgi (large arrows), and to endosomes containing endocytosed 10-nm BSA-gold (asterisks). Labeling of DR is seen in vesicles close to the Golgi, in endosomes containing 10-nm BSA-gold, and in larger vesicular compartments (LV) containing endocytosed 5-nm BSA-gold (chase overnight, open arrows), as well as at the plasma membrane. N, Nucleus. (B) Detailed view of the ultrastructure and labeling of the multivesicular endosomal compartments containing 10-nm BSA-gold (and small amounts of 5-nm BSA-gold, open arrows), documenting colocalization of Ii (large, 20 nm-gold particles) and DR (15-nm gold particles, arrowheads). Parts of two large DR- positive vesicular compartments and Ii labeling in the Golgi (G) are also shown. Bars = 500 nm. Gastroenterology 1998 114, 649-656DOI: (10.1016/S0016-5085(98)70578-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Antigen-induced IFN-γ production in T-cell clones mediated by HIMECs. Effect of chloroquine and inhibitory MAbs (10 μg/mL) to DR (B8.11) or DQ (SPV-L3). Confluent HIMECs (1.2 × 104) stimulated with IFN-γ (100 U/mL, 3 days) were incubated with T-cell clones (TCC) (5 × 104 cells) and PPD (10 μg/mL), or with T-cell clones (5 × 104 cells) and peptide 3-13 (10 μg/mL). Representative results from one of two similar experiments with T-cell clone RN 4.26 (□) and T-cell clone RN 4.97 (▩) are shown. Data given as median and range of quadruplicate wells. Gastroenterology 1998 114, 649-656DOI: (10.1016/S0016-5085(98)70578-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Antigen-presenting capacity of HIMECs compared with professional APCs. Confluent HIMECs (1.2 × 104) stimulated with IFN-γ (100 U/mL, 3 days) were compared with increasing numbers of an Epstein–Barr virus B lymphoblastoid cell line (EB) or mononuclear cells from peripheral blood depleted of T cells (nonT). PPD (10 or 100 μg/mL) was added 24 hours before addition of T-cell clone (clone 4.97, 5 × 104 cells). Representative results from one of three similar experiments (median and range of triplicate wells). Gastroenterology 1998 114, 649-656DOI: (10.1016/S0016-5085(98)70578-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions