Antigen-presenting epithelial cells can play a pivotal role in airway allergy.

To the Editor:

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).1, 2, 3, 4 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).

Fig E1

A and B, Expression of HLA-DR and CD86 (open histogram) in cultured HNECs from a healthy control subject (Fig E1, A) and a patient with AR (Fig E1, B; representative data from 9 different experiments, respectively). The isotype control is a filled histogram. C, HNECs stained for MHC class II (green). Scale bar = 50 μm. D, Expression of HLA-DR and CD86 on freshly isolated HNECs from healthy control subjects in nasal biopsy specimens (n = 4) and nasal brushings (n = 6).

Fig E2

A 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.

Fig 1

A, MHC class II expression on OVA-stimulated MNECs cocultured with T cells (4 hours). B, CD4+ T-cell counts in cocultures with OVA-stimulated MNECs (24 hours). C and D, Fraction of CD69+/CD4+ (Fig 1, C) and CD44+/CD4+ (Fig 1, D) T cells after coculture with OVA-stimulated MNECs. E, INF-γ release in cocultures (24 hours). F, Cocultures with MNECs and T cells (both from sensitized mice) with anti–MHC class II antibodies (anti-MHC II). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.

Fig E3

Total number of CD69+/CD4+(A) and CD44+/CD4+(B) T cells after coculture with OVA-stimulated MNECs. *P < .05, **P < .01, and ***P < .001.

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).

Fig 2

A, NECs from a patient with AR containing dextran (green) and MHC class II (red). Scale bar = 10 μm. B, Three-dimensional composite image. C, Level of CD4+ T cells after coculture with HNECs. D, Fraction of CD69+/CD4+ T cells after coculture with HNECs. E, Level IL-13 followed by 4 hours of coculture. F and G, Different cell types in nasal mucosa (Fig 2, F) and intensity of HLA-DR (Fig 2, G; n = 7). ∗P < .05.

Fig E4

A, 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.

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. Several viruses, such as human coronavirus causing severe acute respiratory syndrome, have evolved mechanisms to inhibit MHC class II pathways in ECs. 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.

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. 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. 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. 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.