Tolerogenic properties of CD206+ macrophages appeared in the sublingual mucosa after repeated antigen-painting.

The sublingual mucosa (SLM) in the oral cavity is utilized as the site for sublingual immunotherapy to induce tolerance against allergens. We previously reported that CD206+ round-type macrophage-like cells were induced in the SLM after repeated antigen (e.g. cedar pollen or fluorescein isothiocyanate (FITC))-painting. In this study, we examined the phenotypic and functional properties of CD206+ cells induced by repeated FITC-painting on the SLM. CD206+ cells after the repeated FITC-painting possessed a macrophage-like CD11b+Ly6C+ F4/80+CD64+ phenotype and expressed TIM-4, which was expressed in tolerogenic tissue-resident macrophages, at a high level. SLM CD206+ cells preferentially expressed molecules related to endocytosis and homeostatic processes, including the novel B7 family of immune checkpoint molecules, as assessed by microarray analyses. SLM CD206+ cells showed preferential expression of M2-related genes such as Fizz1, Aldh1a1 and Aldh1a2 but not Ym-1 and Arginase-1. A CD206+ cell-rich status inhibited OVA-specific CD4+ T-cell responses but reciprocally enhanced the proportion of both IL-10+CD4+ cells and Foxp3+ regulatory T-cells in regional lymph nodes. Co-culture of CD206+ cells with dendritic cells (DCs) showed that IL-12 production was suppressed in DCs concurrent with the decline of the MHC class IIhiCD86+ population, which was restored by neutralization of IL-10. These results demonstrate SLM CD206+ cells show the feature of tolerogenic macrophages and down-regulate the antigen-presenting cell function of mature DCs resulting in the inhibition of CD4+ T-cell responses.

Establishment and first characterization of a sublingual epithelial and immune cell co-culture model.

We describe here the establishment and first characterization of a co-culture model of human epithelial sublingual cells (HO-1-u-1 cell line) and human dendritic cells derived from human peripheral blood monocytes (PBMC). Cell culture conditions for HO-1-u-1 cells were optimized. First characterization of phenotypic features by electron microscopy and fluorescence imaging revealed resemblance to sublingual tissue specimen from healthy donors. Successful co-culturing of epithelial and dendritic cells (DCs) was confirmed by confocal laser scanning microscopy. Stimulation of HO-1-u1 cells alone and the epithelial/DC co-culture by incubation with liposomes, virosomes and influenza virus lead reproducibly to the release of inflammatory cytokine GM-CSF. This co-culture model may be suitable for elucidation of mechanisms involved in the immune response at the sublingual epithelium as well as for the evaluation of novel topical vaccines, potentially replacing cumbersome ex vivo and in vivo methods currently in place.

Dendritic cells of the oral mucosa.

The oral cavity contains distinct mucosal surfaces, each with its own unique distribution of dendritic cell (DC) subsets. In addition to tissue-specific properties, such organization might confer differential immune outcomes guided by tissue-resident DCs, which translate in the lymph node into an overall immune response. This process is further complicated by continual exposure and colonization of the oral cavity with enormous numbers of diverse microbes, some of which might induce destructive immunity. As a central cell type constantly monitoring changes in oral microbiota and orchestrating T-cell function, oral DCs are of major importance in deciding whether to induce immunity or tolerance. In this review, an overview of the phenotype and distribution of DCs in the oral mucosa is provided. In addition, the role of the various oral DC subsets in inducing immunity vs. tolerance, as well as their involvement in several oral pathologies is discussed.

[Incidence of odontogenic phlegmon associated with polymorphic variant 896A/G of gene TLR4, but not with 2258G/A of gene TLR2].

The problem actuality is caused by significant enhancement of the incidence rate for inflammatory diseases of the head and neck tissues, first of all of the oral cavity floor abscesses and phlegmons, which causes severe forms of mediastinitis while inadequate treatment. The authors have had established, that Toll-like receptors (TLR) initiate a cascade of anti-inflammatory reactions of the inborn immunity, followed by synthesis of a certain cytokines, and their genetic polymorphism changes the immune reactivity of the organism. Trustworthy correlation of the gene TLR4 (rs4986790) polymorphism 896A/G was proved with high risk of the odontogenic phlegmon of the oral cavity floor occurrence, what would permit to prognosticate the disease course in early terms, to optimize the schemes of its prophylaxis and treatment.

Distinct expression of perforin and granzyme B in lip and oral cavity squamous cell carcinoma.

BACKGROUND: Perforin and granzyme B (GB) are the main constituents of cytotoxic T-lymphocyte granules, and they have important roles in preventing the initiation and progression of cancer. METHODS: The aim of this study was to compare the expression of CD8(+) /perforin(+) double-staining and GB(+) cells, by immunohistochemistry, in primary oral cavity squamous cell carcinoma (OCSCC), lip squamous cell carcinoma (LSCC), non-dysplastic leukoplakia (LK), dysplastic LK, actinic cheilitis (AC), oral lichen planus (LP) and normal oral mucosa. RESULTS: Our results showed a higher expression of CD8(+) /perforin(+) and GB(+) cells in LSCC when compared with the samples of OCSCC, non-dysplastic and dysplastic LK, AC, oral LP and normal oral mucosa. In addition, increased CD8(+) /perforin(+) and GB(+) cell numbers were observed in all pre-malignant lesions (non-dysplastic LK, dysplastic LK, AC) when compared with the control. CONCLUSIONS: Perforin and GB proteins may contribute to antitumoural immunity, leading to the direct killing of tumour cells; however, it seems to occur more effectively in LSCC than OCSCC.

Mapping of the lingual immune system reveals the presence of both regulatory and effector CD4+ T cells.

BACKGROUND: Sublingual immunotherapy (SLIT) is safe and reduces both symptoms and medication requirements in patients with type I respiratory allergies. Nonetheless, immune mechanisms underlying SLIT need to be further documented. OBJECTIVE: A detailed characterization of the lingual immune system was undertaken in mice, to investigate the presence of tolerogenic and pro-inflammatory mechanisms. METHODS: Immune cells were characterized in lingual tissues from BALB/c mice using immunohistology and flow cytometry. Resident CD4(+) T cells were sorted and toll-like receptor (TLR) expression profiles as well as functional characterization were assessed by RT-PCR, T cell suppressive assays and cytokine gene expression, respectively. RESULTS: Eosinophils and mast cells were only detected in submucosal tissues. No NK, NK-T, gamma/delta, CD8(+) T cells, nor B-lymphocytes were detected. Potential antigen presenting cells include various subsets of dendritic cells (CD207(+) Langerhans cells, CD11b(+)CD11c(+) myeloid cells and 120G8(+) plasmacytoid DCs) together with F4/80(+) macrophages. Noteworthy, both CD103(-) and CD103(+) CD4(+) T cells expressing TLR2 and TLR4 receptors are present along the lamina propria, in vicinity of myeloid CD11b(+)CD11c(+/-) dendritic cells. Such resident lingual CD4(+) T lymphocytes comprise both suppressive T cells as well as cells with memory/effector functions (i.e. expressing IFN gamma, IL4, IL10 and IL17 genes following stimulation), irrespective of the presence of the mucosal addressing marker CD103. CONCLUSION: The sublingual route is pertinent to induce antigen-specific tolerance, due to (i) limited numbers of pro-inflammatory cells, rather located in submucosal tissues, (ii) co-localization of APCs and resident CD4(+) T cells with regulatory functions. Since the oral immune system can also elicit pro-inflammatory effector responses, the cytokine milieu in which allergens are presented by sublingual APCs needs to be controlled during immunotherapy (e.g. with adjuvants) in order to favour tolerance over inflammation.

Sensitivity and specificity of fluorescent immunoguided neoplasm detection in head and neck cancer xenografts.

OBJECTIVES: To determine whether fluorescently labeled anti-epidermal growth factor (EGFR) antibody could be used to detect residual disease and to guide surgical resections by comparing the sensitivity and specificity of optical fluorescence imaging with the sensitivity and specificity of histopathologic evaluation. DESIGN: A preclinical model of head and neck squamous cell carcinoma. SUBJECTS: Mice xenografted with SCC-1 tumor cells. INTERVENTIONS: The mice underwent systemic injection with anti-EGFR antibody (cetuximab) conjugated to an optically active fluorophore (Cy5.5). Both a subcutaneous flank model (n = 18) and an orthotopic murine model (n = 15) were used to assess for the presence of residual disease by fluorescent stereomicroscopy after subtotal resections of tumors. Histologic analysis was performed to confirm the presence or absence of disease. RESULTS: In the subcutaneous flank model, a diagnostic dose (50 microg) and therapeutic dose (250 microg) of fluorescent-labeled anti-EGFR were administered. When a diagnostic dose was given, the sensitivity was 86%, which was less than the 91% sensitivity when the higher dose was given. Tumor biopsy specimens in which disease was detected by histologic analysis but not by fluorescence (false-negative result) averaged 166 cells (range, 50-350 cells). The specificity of optical fluorescence to predict the presence of tumor in both groups was 100%. In the floor of the mouth model, we demonstrated a sensitivity of 81% and a specificity of 100%. False-negative results were obtained in a tumor fragment measuring less than 0.5 mm in diameter. CONCLUSION: These data support further investigation of fluorescently labeled anti-EGFR antibody to detect disease in the surgical setting.