Antigen-presenting epithelial cells can play a pivotal role in airway allergy
Authors: Julia Arebro, Lotta Tengroth, Ronia Razavi, Susanna Kumlien Georén, Ola Winqvist, Lars-Olaf Cardell
Journal: Journal of Allergy and Clinical Immunology (2015)

Professional antigen-presenting cells (APCs; ie, dendritic cells, macrophages, and B cells) react against exogenous antigens and initiate an adaptive immune response by presenting antigen peptides in the groove of the MHC class II molecules. During inflammation, ectopic expression of MHC class II has been reported on cells from multiple tissues, including the nasal mucosa, suggesting an antigen-presenting capacity of epithelial cells (ECs).1Dotan I. Allez M. Nakazawa A. Brimnes J. Schulder-Katz M. Mayer L. Intestinal epithelial cells from inflammatory bowel disease patients preferentially stimulate CD4+ T cells to proliferate and secrete interferon-gamma.Am J Physiol Gastrointest Liver Physiol. 2007; 292: 8Crossref Scopus (67) Google Scholar, 2Takizawa R. Pawankar R. Yamagishi S. Takenaka H. Yagi T. Increased expression of HLA-DR and CD86 in nasal epithelial cells in allergic rhinitics: antigen presentation to T cells and up-regulation by diesel exhaust particles.Clin Exp Allergy. 2007; 37: 420-433Crossref PubMed Scopus (23) Google Scholar, 3Goldwich A. Burkard M. Olke M. Daniel C. Amann K. Hugo C. et al.Podocytes are nonhematopoietic professional antigen-presenting cells.J Am Soc Nephrol. 2013; 24: 906-916Crossref PubMed Scopus (103) Google Scholar, 4McDonald G.B. Jewell D.P. Class II antigen (HLA-DR) expression by intestinal epithelial cells in inflammatory diseases of colon.J Clin Pathol. 1987; 40: 312-317Crossref PubMed Scopus (72) Google Scholar The present investigation was designed to examine the contribution of nasal epithelial cells (NECs) to the allergic inflammatory process. The abilities of NECs to take up antigen, express MHC class II and costimulatory molecules, and stimulate antigen-specific activation and proliferation of CD4+ T cells were investigated by using a human mucosal specimen (see the Methods section in this article's Online Repository at www.jacionline.org). First, the cell-surface expression of MHC class II and costimulatory molecules on human and mouse nasal epithelial cells (MNECs) was confirmed (see Figs E1 and E2 in this article's Online Repository at www.jacionline.org). Then the ability of MNECs to present the antigen ovalbumin (OVA) to naive T cells was demonstrated. MNECs from sensitized mice displayed an enhanced MHC class II expression on coculture with OT-II T cells compared with naive cells (Fig 1, A). The total number of OT-II CD4+ T cells in the same cocultures was increased. A tendency toward an increase in CD4+ T-cell counts was also seen when sensitized T cells were used as reporter cells (Fig 1, B). Analysis of T-cell activation revealed a pronounced increase in the total number (see Fig E3, A, in this article's Online Repository at www.jacionline.org) and fraction (Fig 1, C) of activated CD69+ OT-II cells, as well as sensitized T cells, when using sensitized MNECs as APCs. Notably, sensitized MNECs exhibited significantly increased activating capacity, even without added OVA, which was supposedly partially due to the remaining OVA in the MNECs from the sensitization process. In line with this, MNECs from sensitized mice augmented the absolute number (see Fig E3, B) and fraction (Fig 1, D) of CD44+ OT-II cells, as well as sensitized T cells, in a dose-dependent manner. A tendency toward an increased IFN-γ release was simultaneously seen when sensitized MNECs were used as APCs (Fig 1, E). Finally, sensitized MNECs were unable to affect the fraction of CD69+ T cells in cocultures with neutralizing anti–MHC class II antibodies (Fig 1, F). Human nasal epithelial cells (HNECs) were able to endocytose dextran, reaching the intracellular class II loading compartment (Fig 2, A and B, and see Video E1 in this article's Online Repository at www.jacionline.org). Autologous CD4+ T-cell counts increased when cocultured with birch pollen–stimulated HNECs from patients with allergic rhinitis (AR; Fig 2, C), and the number of CD69+ T cells increased in a dose-dependent manner when stimulating cocultures with Bet v 1, minimizing endotoxin contamination (Fig 2, D, and see Fig E4, A, in this article's Online Repository at www.jacionline.org). IL-13 release was augmented in the same cocultures (Fig 2, E, and see Fig E4, B). Control experiments ruled out that the T-cell activation was mediated by activated MHC class II–expressing T cells (see Fig E4, C). Finally, a high number of HLA-DR–expressing HNECs were found, strengthening the importance of NECs in upper airway antigen presentation (Fig 2, F and G). The OVA peptide amino acid 323-339 encompasses an allergic antigenic epitope of the OVA protein in mice. Thus activation of T-cell receptor (TCR) transgenic OT-II T cells by OVA requires antigen processing. The marked activation and proliferation of OT-II CD4+ T cells when cocultured with OVA-stimulated MNECs from sensitized animals demonstrates the antigen-processing and antigen-presenting capacity of MNECs. An experiment in which splenic cells from sensitized animals containing OVA-specific CD4+ T cells were cocultured in the same manner was used to mimic a more natural process. This resulted in a T-cell response with IFN-γ release, demonstrating the T cell–activating capacity of NECs. The use of a TCR transgenic system and murine ECs made it possible to use naive T cells for the responses, thus making the point that a T-cell response against allergen could be initiated by ECs. This could not be addressed in a human setting. HNECs from sensitized patients with AR demonstrated an allergen-specific increased capacity to activate CD4+ autologous T cells, further corroborating the mouse data (Fig 2). This recall response, which was dependent on the presence of memory T cells, was likely to result in a TH2 response reflected in the detection of IL-13 (Fig 2, E). Professional APCs are believed to mainly present and activate T cells in the lymph nodes. NECs are local, and their numbers in the mucosa far outnumber those of local dendritic cells, making their ability to present allergen and activate T cells an important asset in the first line of defense (Fig 2, F and G). Increased expression of MHC class II in murine type II alveolar ECs has been demonstrated after Mycobacterium tuberculosis infection, further supporting the importance of NECs in the microbial response.5Debbabi H. Ghosh S. Kamath A.B. Alt J. Demello D.E. Dunsmore S. et al.Primary type II alveolar epithelial cells present microbial antigens to antigen-specific CD4+ T cells.Am J Physiol Lung Cell Mol Physiol. 2005; 289: 15Crossref Scopus (84) Google Scholar Several viruses, such as human coronavirus causing severe acute respiratory syndrome, have evolved mechanisms to inhibit MHC class II pathways in ECs.6Josset L. Menachery V.D. Gralinski L.E. Agnihothram S. Sova P. Carter V.S. et al.Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus.MBio. 2013; 4 (e00165-13)Crossref PubMed Scopus (215) Google Scholar Bet v 1 pollen allergen has further been demonstrated to bind and enter ECs within minutes after exposure in patients with AR, even during the nonsymptomatic winter season.7Renkonen J. Mattila P. Lehti S. Makinen J. Sormunen R. Tervo T. et al.Birch pollen allergen Bet v 1 binds to and is transported through conjunctival epithelium in allergic patients.Allergy. 2009; 64: 868-875Crossref PubMed Scopus (26) Google Scholar In summary, for the first time, we have demonstrated the role of antigen-presenting NECs in patients with allergic airway disease. Previous information in the field is scarce, even though it has been shown that intestinal ECs can stimulate CD4+ T cells in vitro to proliferate and secrete cytokines.1Dotan I. Allez M. Nakazawa A. Brimnes J. Schulder-Katz M. Mayer L. Intestinal epithelial cells from inflammatory bowel disease patients preferentially stimulate CD4+ T cells to proliferate and secrete interferon-gamma.Am J Physiol Gastrointest Liver Physiol. 2007; 292: 8Crossref Scopus (67) Google Scholar Furthermore, the importance of a local rapid response that enables antigen presentation and CD4+ T-cell activation was recently demonstrated in an mouse inflammatory bowel disease model in which MHC class II–deficient mice were unable to induce and reinstate tolerance when fed with Helicobacter hepaticus and IL-10 receptor–blocking antibodies.8Thelemann C. Eren R.O. Coutaz M. Brasseit J. Bouzourene H. Rosa M. et al.Interferon-gamma induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.PLoS One. 2014; 9: e86844Crossref PubMed Scopus (93) Google Scholar We demonstrated that MHC class II in NECs can be loaded with exogenous antigens (Fig 2, A and B, and see Video E1), that NECs stimulate allergen-specific CD4+ T-cell responses in mice and human subjects (Fig 1, Fig 2), and that HNECs from patients with AR can amplify the allergic response contributing to the ongoing allergic CD4+ T cell–mediated inflammation, with the latter likely contributing to the high number of activated CD4+ T cells found in nasal mucosa of patients with AR.9Robinson D.S. Hamid Q. Ying S. Tsicopoulos A. Barkans J. Bentley A.M. et al.Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma.N Engl J Med. 1992; 326: 298-304Crossref PubMed Scopus (2606) Google Scholar Hence this is the first time that local immune activity within the nasal mucosa has been directly linked to a rapid and efficient allergen-induced T-cell activation. We thank Olivia Larsson, Cecilia Landberg, and Eric Hjalmarsson for laboratory work; Evelina Lindmark and Jiu Hu for practical assistance with the OT-II mice; Katalin Benedek for assistance with confocal microscopy; and Maria Axelsson and Carina Israelsson for assisting the handling of patients. Nine nonsmoking patients with AR to birch pollen (mean age, 32 years; 3 male and 6 female patients) were included in the study. They were given diagnoses based on clinical history and RAST results for allergen-specific IgE. In addition, 20 healthy nonsmoking control subjects were included (mean age, 29 years; 5 male and 15 female subjects). RAST results for allergen-specific IgE were negative for all healthy control subjects included in the study. The study was approved by the local ethics committee, and all patients provided written informed consent. Primary HNECs were obtained by means of nasal brushing, as previously described.E1Rydberg C. Mansson A. Uddman R. Riesbeck K. Cardell L.O. Toll-like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas.Immunology. 2009; 128: e600-e611Crossref PubMed Scopus (61) Google Scholar None of the patients had symptoms of allergy at the time of nasal brushing because samples were taken before the season, and all but 1 patient had been off antihistamine treatment for more than 1 week. All had been off topical steroids for at least 1 year. Cells were maintained in collagen-coated tissue-culture flasks in complete medium with Keratinocyte Serum-Free Medium (Gibco, Paisley, United Kingdom), 0.005 μg/mL epidermal growth factor, 0.05 mg/mL bovine pituitary extract, 20 U/mL penicillin, and 20 μg/mL streptomycin (Gibco, Grand Island, NY). In addition, 0.0125 μg/mL Fungizone Antimycotic (Gibco) was added to the cells stimulated with Bet v 1. All cells were cultured at 37°C in a humidified 5% CO2 air atmosphere. Cells from passages 3 to 5 were used in the experiment, and they were all positive for epithelial cell adhesion molecule (EpCAM; >90%) and negative for CD11b (<1%) and CD11c (2% to 4%). Peripheral blood samples from all patients with AR and control subjects were obtained approximately 3 weeks after nasal brushing. Blood samples were taken in season, and all patients had nasal allergy symptoms. All patients had been off antihistamine treatment for more than 24 hours. T cells were isolated by using magnetic antibody cell-sorting separation (MACSxpress Pan T Cell Isolation Kit, human; Miltenyi Biotec, Cologne, Germany), according to the manufacturer's instructions. In brief, peripheral blood was added to the Pan T Isolation cocktail, put in the MACSmix Tube Rotator for 5 minutes, and then put in the magnetic field of the MACSxpress Separator for 15 minutes. The supernatant was then collected. Furthermore, erythrocyte depletion was performed on the T cells used for experiments with HNECs stimulated with Bet v 1, according to the manufacturer's instructions. In brief, the MACSxpress Erythrocyte Depletion Reagent was added to the supernatant containing T cells, put in the MACSmix Tube Rotator for 5 minutes, and then put in the magnetic field of the MACSxpress Separator for 10 minutes. The supernatant was then collected, washed, and centrifuged. The isolated cells were routinely screened for expression of CD3 by using flow cytometry, revealing highly purified T-cell preparations (>98%). A coculture of HNECs and T cells was set up to investigate T-cell activation. The cultured HNECs (250,000 cells/mL) were stimulated with 0, 100, 1,000, or 10,000 SQ-E/mL birch pollen extract (Aquagen SQ Birch; ALK-Abelló, Hørsholm, Denmark) or 0, 10, 50, or 100 μg/mL Bet v 1 (Biomay, Vienna, Austria) for 24 hours at 37°C in a humidified 5% CO2 atmosphere. T cells for control experiments (Fig E4, C) were stimulated with 10,000 SQ-E/mL birch pollen extract. HNECs were then washed thoroughly with PBS 4 times. Purified primary T cells were added to each well (0.5 × 106 to HNECs stimulated with birch pollen extract and 0.3 × 106 to HNECs stimulated with Bet v 1) and resuspended in RPMI medium (Gibco) containing 2 mmol/L l-glutamine (Gibco), 2% FBS (Gibco), 20 U/mL penicillin, 20 μg/mL streptomycin, and 0.0125 μg/mL Fungizone Antimycotic. On 4, 24, and 48 hours of coculture, T cells, HNECs, and supernatants were separated and collected. Cocultured control cells were kept on ice. Nasal biopsy specimens were taken from inferior turbinates of healthy nonsmoking control subjects after achievement of local anesthesia, as previously described.E2Fransson M. Benson M. Erjefalt J.S. Jansson L. Uddman R. Bjornsson S. et al.Expression of Toll-like receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis.Respir Res. 2007; 8: 17Crossref PubMed Scopus (51) Google Scholar The control subjects had no history of nasal or sinus disease, and RAST results for allergen-specific IgE were negative. Tissue was put through a 100-μm cell strainer (BD Falcon, Franklin Lakes, NJ) into Dulbecco modified Eagle medium and Nutrient Mixture Ham F12 medium (DMEM/F-12, Gibco) containing 10% FBS and incubated in room temperature for 5 minutes. Cells were washed and centrifuged once, and the supernatant was subsequently aspirated and discarded. Cells were highly positive for EpCAM (approximately 50%). Eighty-seven C57BL/6 mice (female) aged 8 to 10 weeks were purchased from Charles River (Sulzfeld, Germany). The animals were allowed pelleted food and water ad libitum and housed in a climate-controlled environment with a 12-hour daylight cycle. The spleens of 4 OT-II TCR transgenic mice recognizing OVA peptide amino acid 323-339 in the context of H2-IAb were used with the permission of Dr F. Carbone.E3Barnden M.J. Allison J. Heath W.R. Carbone F.R. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.Immunol Cell Biol. 1998; 76: 34-40Crossref PubMed Scopus (1220) Google Scholar The study was approved by the Swedish Animal Experimental Review Board (N153-11 regarding C57BL/6 mice and N558/12 regarding OT-II mice). Fifty-nine C57BL/6 mice were sensitized by means of intraperitoneal injection of 10 μg of grade V OVA and 4 mg/mL Al(OH)3 (Sigma-Aldrich, St Louis, Mo) suspended in 0.2 mL of PBS administered in the morning on days 0 and 7. All mice were killed by means of cervical dislocation. Septal mucosae were dissected from 79 mice 14 days after sensitization, as previously described.E4Antunes M.B. Woodworth B.A. Bhargave G. Xiong G. Aguilar J.L. Ratner A.J. et al.Murine nasal septa for respiratory epithelial air-liquid interface cultures.Biotechniques. 2007; 43: 195-196Crossref PubMed Scopus (95) Google Scholar In brief, the skull was sectioned in the coronal plane, and the brain and posterior part of the skull were removed. The septum was separated from the nasal lateral walls. Nasal septums from 12 to 14 mice were pooled and put in DMEM/F-12 (Gibco) and then into dissociation medium for 1 hour containing minimal essential medium (Gibco), 100 IU/mL penicillin/100 μg/mL streptomycin, 1.4 mg/mL Pronase, and 0.1 μg/mL DNase (Sigma-Aldrich). DMEM/F12 containing 10% FBS was added to stop the reaction. Tissue was put through a cell strainer twice and thereafter in complete culture medium with DMEM/F12 (Gibco) containing 100 IU/mL penicillin–100 μg/mL streptomycin, 10% FBS, and 120 IU/mL ITS Premix (BD Biosciences, San Jose, Calif). The suspension was incubated at 37°C for 2 hours on 100-mm Primaria culture dishes (BD Bioscience). The cell suspension was then collected and cultured in collagen-coated tissue culture flasks containing complete culture medium at 37°C in a humidified 5% CO2 atmosphere seeded at a density of 2.5 × 105 cells/mL. Spleens were taken from 4 OT-II mice, 6 sensitized C57BL/6 mice, and 2 naive C57BL/6 mice. The tissue was put through a 100-μm cell strainer (BD Falcon). Cells were washed and centrifuged once, and the supernatant was subsequently aspirated and discarded. The cell populations contained approximately 15% (OT-II mice), approximately 25% (sensitized C57BL/6 mice), and approximately 12% (naive C57BL/6 mice) CD3+ cells. T cells were defined as responders to stimulation of NECs. Cocultures of MNECs (pooled from 12-14 mice) and mouse splenic cells (from 1-2 mice) were set up to investigate T-cell activation. At epithelial confluence, they were put in DMEM/F-12 (Gibco) containing 2% FBS (Gibco) and 100 IU/mL penicillin–100 μg/mL streptomycin (Sigma-Aldrich). OVA was added at concentrations 1, 10, or 100 mg/mL, and cells were incubated for 24 hours at 37°C in a humidified 5% CO2 atmosphere. They were then washed thoroughly with PBS 4 times. Splenic cells were cocultured with ECs by adding 1 × 106 splenic cells to each well. After 4 or 24 hours of coculture at 37°C in a humidified 5% CO2 air atmosphere, splenic cells, MNECs, and supernatants were collected. Cocultured control cells were put on ice. In the experiments with anti–MHC class II antibodies, 2.5 μg/mL anti-mouse I-A/I-E, or isotype control rat IgG2b, κ (both from BioLegend, San Diego, Calif) was added for 4 hours in addition to the splenic cells (4 hours). The following mAbs were purchased from BD Biosciences: anti-human CD3 (UCHT1, SK7), CD4 (RPA-T4), CD19 (SJ25C1), CD25 (2A3), CD45 (HI30), CD68 (Y1/82A), CD69 (FN50), CD11b (ICRF44), CD11c (B-ly6), EpCAM (EBA-1), HLA-DR (G46-6), CD86 (2331 [FUN-1]), CD80 (BB1), anti-mouse CD4 (RM4-5), CD8a (53-6.7), CD19 (ID3), CD44 (IM7), CD69 (H1.2F3), MHC class II (M5/114.15.2), and CD86 (GL1). Anti-human CD8 (DK25) was purchased from Dako (Glostrup, Denmark). Anti-human HLA-DR (L243) and anti-mouse EpCAM (G8.8), CD80 (16-10A1), and CD86 (GL-1) were purchased from BioLegend. All antibodies were titrated for optimum concentration before use, with dilutions ranging from 1:5 to 1:400. Cells were identified based on forward and side scatter properties. Cells were analyzed on an LRSFortessa analyzer (BD Biosciences), and data were processed with FlowJo software (Tree Star, Ashland, Ore). For optical imaging, HNECs from 1 patient with AR were cultured, as described above. For visualization of MHC class II expression, HNECs were preincubated with 10% normal donkey serum (Abcam, Cambridge, United Kingdom) for 30 minutes to block nonspecific binding. Subsequently, cells were incubated with an anti–MHC class II primary antibody (1:50, EPR11226, Abcam) for 1 hour, followed by an Alexa Fluor 488–labeled anti-rabbit secondary antibody (1:200, Abcam) for 1 hour. Imaging was performed with a Nikon Digital Sight DS-U1 camera coupled to a Nikon Eclipse TE2000-U microscope by using NIS Elements F. 2.20 software. HNECs from another patient with AR were used to study dextran uptake. Fluorescein isothiocyanate–coupled 3000 molecular weight dextran (1 mg/mL D3306; Invitrogen, Carlsbad, Calif) was added to HNECs in complete medium for 2 hours in the dark at 37°C and thereafter washed with PBS to visualize endocytosis.E5Sallusto F. Cella M. Danieli C. Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.J Exp Med. 1995; 182: 389-400Crossref PubMed Scopus (2199) Google Scholar Cells were fixed for 15 minutes in 4% formaldehyde, washed with PBS, and subsequently incubated with 10% normal donkey serum for 30 minutes to block nonspecific binding. MHC class II was visualized with an anti–MHC class II primary antibody (1:50, EPR11226, Abcam) and incubation for 1 hour, followed by an Alexa Fluor 555–labeled anti-rabbit secondary antibody (1:200, Abcam) and incubation for 1 hour. Stained cells were maintained in ProLong Gold antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI; Invitrogen). For laser confocal microscopy, a Nikon Eclipse TE 300 microscope (PerkinElmer, Waltham, Mass) equipped with a solid-state laser (blue, 405 nm; green, 488 nm; and red, 568 nm) and connected to a Hamamatsu ORCA ER camera (Hammamatsu, Hammamatsu City, Japan) was used. Images were analyzed with UltraWIEW software. A 3-dimensional image was created with Image Processing and Analysis in Java (ImageJ; National Institutes of Health, Bethesda, Md) software. Supernatants collected after coculture of stimulated HNECs and autologous T cells (4, 24, and 48 hours) were analyzed with Luminex (Luminex, Austin, Tex) regarding levels of IL-13 and IL-5. A Human Cytokine Standard 17-plex (Bio-Rad Laboratories, Hercules, Calif) was used and run in a Luminex200 system. The analyses were carried out according to the manufacturer's instructions. In brief, specific antibodies labeled with spectrally encoded beads were applied to the samples and standards. After incubation, the beads were washed and mixed with specific biotinylated detector antibodies. Subsequently, excess biotinylated antibodies were removed by using a washing step, followed by addition of streptavidin-R-phycoerythrin to conjugate and label the detector antibodies. By monitoring the spectral properties of the beads and the amount of associated streptavidin-R-phycoerythrin fluorescence with the Bio-Plex System (Bio-Rad Laboratories), cytokine concentrations could be determined. The minimum detectable concentration was 0.12 pg/mL (IL-13) and 0.31 pg/mL (IL-5). IFN-γ levels in supernatants of cocultures of OVA-stimulated MNECs and splenic cells were measured with a mouse IFN-γ–specific ELISA kit (BioLegend), according to the manufacturer's instructions. The minimum detectable concentration of IFN-γ for the kit was 8 pg/mL. Statistical differences were determined by using 1-way repeated-measures ANOVA with the Bonferroni posttest (for more than 2 sets of paired data). For unrepeated measurements, 1-way ANOVA was used together with the Bonferroni posttest. Statistical differences between MNECs and HNECs, respectively, without stimulation were performed by using unpaired t tests. Statistical analyses were performed with GraphPad Prism software (version 6.0; GraphPad Software, La Jolla, Calif). All data are shown as means ± SEMs. For human data, n equals the number of patients. Our initial experiments were carried out to confirm cell-surface expression of MHC class II on isolated NECs from healthy control subjects (Fig E1, A, left panel) and patients with birch pollen–induced AR sampled before the start of the season (Fig E1, B, left panel). The HLA-DR expression on isolated HNECs was verified by means of immunofluorescence staining (Fig E1, C). ECs must express costimulatory molecules, such as CD86 or CD80, to act as APCs. Isolated HNECs from healthy control subjects (Fig E1, A, right panel) and patients with AR (Fig E1, B, right panel) displayed cell-surface expression of the costimulatory molecule CD86. No expression of CD80 was found (data not shown). There were no significant differences in the fraction of HLA-DR or CD86 between healthy control subjects and patients with AR before the season (data not shown). There was no significant difference in the fraction of HLA-DR or CD86 expression between HNECs derived from nasal biopsy specimens or HNECs obtained from nasal brushing, validating the nasal brush method for use in our forthcoming experiments (Fig E1, D). In mice we found a marked H2-IAb expression on MNECs, as well as cell-surface expression of the costimulatory molecules CD86 and CD80 (Fig E2, A). To mimic allergic patients, we sensitized C57BL/6 mice with OVA in conjunction with aluminum hydroxide. NECs from OVA-sensitized mice expressed marked levels of H2-IAb (Fig E2, B, left panel). Expression levels of CD86 were similar to those in naive mice (Fig E2, B, middle panel), whereas we found increased expression levels of the costimulatory molecule CD80 in OVA-sensitized mice (Fig E2, B, right panel). The fraction of MNECs expressing H2-IAb and CD80 was significantly increased on OVA-sensitized epithelium (based on 28 animals) compared with that on naive epithelium (based on 25 animals), whereas no difference was seen in CD86 expression (Fig E2, C). Notably, sensitized MNECs exhibited significantly increased activating capacity demonstrated by the increased number and fraction of CD69 expression on OT-II T cells seen even without OVA stimulation compared with that seen on naive MNECs without OVA stimulation (P < .001 for both number and fraction of CD69; Figs 1, C, and E3, A). Splenic CD4+ T cells from naive mice were added to stimulated naive and sensitized MNECs in all experiments to rule out nonspecific T-cell activation. No signs of T-cell proliferation were seen in these experiments (data not shown), demonstrating the necessity of antigen presentation and engagement of antigen-specific T cells. A neutralizing anti–MCH class II antibody (anti-mouse A-I/E-I) was added to the cocultures to test whether the activation was class II dependent, resulting in no increase of CD69+/CD4+ T-cell counts after 4 hours. When adding an isotype antibody instead, the increase in cell counts persisted. This indicated that activation of T cells was class II dependent. Control experiments were carried out for both the mouse and human coculture experiments in which samples were kept on ice. These experiments revealed no T-cell proliferation or activation (data not shown), supporting the hypothesis that the enhanced T-cell activation is dependent on epithelial antigen processing and presentation. T cells from Bet v 1–sensitized patients harvested in the season demonstrated increased cell-surface expression of CD69, even without the addition of Bet v 1 allergen (Figs 2, D, and E4, A), suggesting that sensitization might activate the ECs and augment their antigen-presenting capacities by, for example, increasing expression of HLA-DR and costimulatory molecules. In the supernatants of the human cocultures, no increase in IL-13 levels was seen on 24 hours of coculture in either of the groups (data not shown). No increase in IL-5 levels was detected in either of the groups on 4, 24, or 48 hours of coculture (data not shown). The frequencies of different cell types in nasal mucosa and the magnitude of HLA-DR were analyzed in the final experiments. These experiments revealed a high number of HLA-DR–expressing NECs (total NECs, 67.313 ± 1.566; Fig 2, F).Fig E2A and B, Expression of H2-IAb, CD86, and CD80 on freshly isolated MNECs from a naive mouse (Fig E2, A) and from a mouse sensitized with OVA (Fig E2, B; representative data from 25-28 animals). C, Expression of H2-IAb, CD86, and CD80 on cultured MNECs from naive and OVA-sensitized mice. *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Total number of CD69+/CD4+ (A) and CD44+/CD4+ (B) T cells after coculture with OVA-stimulated MNECs. *P < .05, **P < .01, and ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4A, Total number of CD69+/CD4+ T cells after coculture with HNECs. B, IL-13 level after 48 hours of coculture. C, Fraction of CD69+/CD4+ T cells in coculture without and with birch pollen–stimulated T cells, ruling out that the T-cell activation was mediated by MHC class II–expressing T cells. *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyNTc0OWMzMjdlNTY5ODA5Yzk1NTRjODU4MWVmNjNkNCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNzE1MjQ1NjE2fQ.da5Z5hH8q0CKQRF8wpWUVyITRFQ7t1m4PossAk4JktvCiRjkzSBHkCr_c0ow4snPfNXnoUFRGez2foM8DylZYuADTkTWSw-I_pjn1ZNdY2Dt0D474BpFC-8_MV0oMB87IF3niluS54gAiEDAKj3PJUej5hStMyj1Vqzw2S6RYlohX8XZJbyxYF9W9bllczvpUKqzSX1YN02r5_cVvw2QekaxHQ5k4srJ7Xo-IUGU5Q8WsS8g7kjPCx8YUl2O0C9chaSvYzx5PYMEd3GTsq0DSeUOzA6SHn3IzVEodJwUqzynHwHM1-L-qFV_R9WYzxcpqOij610shkUyupqyYudmQA Download .mp4 (0.94 MB) Help with .mp4 files Video E1MHC class II molecules (red) were found to be coexpressed in vesicles containing dextran (green) in NECs from patient with AR. The presence of dextran (green) in intracellular vesicles indicates endocytotic uptake of exogenous antigens by NECs. The nucleus was counterstained with 4′-6-diamidino-2-phenylindole (blue).