Modified Vaccinia Ankara-Vectored Vaccine Expressing Nucleoprotein and Matrix Protein 1 (M1) Activates Mucosal M1-Specific T-Cell Immunity and Tissue-Resident Memory T Cells in Human Nasopharynx-Associated Lymphoid Tissue.

BACKGROUND: Increasing evidence supports a critical role of CD8+ T-cell immunity against influenza. Activation of mucosal CD8+ T cells, particularly tissue-resident memory T (TRM) cells recognizing conserved epitopes would mediate rapid and broad protection. Matrix protein 1 (M1) is a well-conserved internal protein. METHODS: We studied the capacity of modified vaccinia Ankara (MVA)-vectored vaccine expressing nucleoprotein (NP) and M1 (MVA-NP+M1) to activate M1-specific CD8+ T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue from children and adults. RESULTS: After MVA-NP+M1 stimulation, M1 was abundantly expressed in adenotonsillar epithelial cells and B cells. MVA-NP+M1 activated a marked interferon γ-secreting T-cell response to M1 peptides. Using tetramer staining, we showed the vaccine activated a marked increase in M158-66 peptide-specific CD8+ T cells in tonsillar mononuclear cells of HLA-matched individuals. We also demonstrated MVA-NP+M1 activated a substantial increase in TRM cells exhibiting effector memory T-cell phenotype. On recall antigen recognition, M1-specific T cells rapidly undergo cytotoxic degranulation, release granzyme B and proinflammatory cytokines, leading to target cell killing. CONCLUSIONS: MVA-NP+M1 elicits a substantial M1-specific T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue, demonstrating its strong capacity to expand memory T-cell pool exhibiting effector memory T-cell phenotype, therefore offering great potential for rapid and broad protection against influenza reinfection.

Know your enemy: Unexpected, pervasive and persistent viral and bacterial contamination of primary cell cultures.

In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination.

Th17 responses to pneumococcus in blood and adenoidal cells in children.

Pneumococcal infections cause a large global health burden, and the search for serotype-independent vaccines continues. Existing conjugate vaccines reduce nasopharyngeal colonization by target serotypes. Such mucosal effects of novel antigens may similarly be important. CD4+ Th17 cell-dependent, antibody-independent reductions in colonization and enhanced clearance have been described in mice. Here we describe the evaluation of T helper type 17 (Th17) cytokine responses to candidate pneumococcal protein vaccine antigens in human cell culture, using adenoidal and peripheral blood mononuclear cells. Optimal detection of interleukin (IL)-17A was at day 7, and of IL-22 at day 11, in these primary cell cultures. Removal of CD45RO+ memory T cells abolished these responses. Age-associated increases in magnitude of responses were evident for IL-17A, but not IL-22, in adenoidal cells. There was a strong correlation between individual IL-17A and IL-22 responses after pneumococcal antigen stimulation (P < 0·015). Intracellular cytokine staining following phorbol myristate acetate (PMA)/ionomycin stimulation demonstrated that  > 30% CD4+ T cells positive for IL-22 express the innate markers γδT cell receptor and/or CD56, with much lower proportions for IL-17A+ cells (P < 0·001). Responses to several vaccine candidate antigens were observed but were consistently absent, particularly in blood, to PhtD (P < 0·0001), an antigen recently shown not to impact colonization in a clinical trial of a PhtD-containing conjugate vaccine in infants. The data presented and approach discussed have the potential to assist in the identification of novel vaccine antigens aimed at reducing pneumococcal carriage and transmission, thus improving the design of empirical clinical trials.

Plasmacytoid DCs are gentle guardians of tonsillar epithelium.

The outcome of the interaction between plasmacytoid dendritic cells (pDCs) and bacteria has been very controversial: pDCs have been reported not to be activated by extracellular bacteria, to be activated but to only produce TNF-α and IL-6, or to be activated and produce IFN-α, the hallmark of pDC activation, but only if the bacteria have first been opsonized. In this issue of the European Journal of Immunology, Soumelis and colleagues [Eur. J. Immunol. 2013. 43: 1264-1273] unequivocally show that both blood and tonsillar pDCs are fully activated by bacteria and can produce IFN-α. They also show that pDCs are found in the stratified mucosal epithelium in human tonsils, and are "educated" by tonsillar epithelial cells not to release inflammatory cytokines, despite still being capable of activating T cells, albeit with no impact on T-cell polarization. Hence, pDCs can respond to bacteria but can be educated by epithelial cells to remain anergic to potential inflammatory signals. These findings support a mechanism by which intraepithelial pDCs, which are exposed to the microbiota colonizing the upper respiratory tract, remain capable of initiating immunity without overreacting to microbial stimulation.

Epithelial control of the human pDC response to extracellular bacteria.

Plasmacytoid pre-dendritic cells (pDCs) are specialized in responding to nucleic acids, and link innate with adaptive immunity. Although the response of pDCs to viruses is well established, whether pDCs can respond to extracellular bacteria remains controversial. Here, we demonstrate that extracellular bacteria such as Neisseria meningitidis, Haemophilus influenzae, and Staphylococcus aureus activate pDCs to produce IFN-α, TNF-α, IL-6, and to upregulate CD86 expression. We observed that pDCs were present within tonsillar crypts and oro-nasopharyngeal epithelium, where they may contact extracellular bacteria, in situ. Tonsil epithelium-conditioned supernatants inhibited IFN-α, TNF-α, and IL-6 triggered by the direct contact of N. meningitidis or S. aureus with pDCs. However, pDC priming of naive T cells was not affected, suggesting that tonsil epithelium micro-environment limits local inflammation while preserving adaptive immunity in response to extracellular bacteria. Our results reveal an important and novel function of pDCs in the initiation of the mucosal innate and adaptive immunity to extracellular bacteria.

Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH).

Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

Isolation of mesenchymal stromal cells (MSCs) from human adenoid tissue.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs) and their characteristics. METHODS: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs) and bone marrow-derived MSCs (BM-MSCs) with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN)-γ stimulation. RESULTS: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1) in A-MSCs were not different from those in T-MSCs and BM-MSCs. CONCLUSIONS: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

Identification of resident memory CD8+ T cells with functional specificity for SARS-CoV-2 in unexposed oropharyngeal lymphoid tissue.

Cross-reactive CD4+ T cells that recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more commonly detected in the peripheral blood of unexposed individuals compared with SARS-CoV-2­reactive CD8+ T cells. However, large numbers of memory CD8+ T cells reside in tissues, feasibly harboring localized SARS-CoV-2­specific immune responses. To test this idea, we performed a comprehensive functional and phenotypic analysis of virus-specific T cells in tonsils, a major lymphoid tissue site in the upper respiratory tract, and matched peripheral blood samples obtained from children and adults before the emergence of COVID-19 (coronavirus disease 2019). We found that SARS-CoV-2­specific memory CD4+ T cells could be found at similar frequencies in the tonsils and peripheral blood in unexposed individuals, whereas functional SARS-CoV-2­specific memory CD8+ T cells were almost only detectable in the tonsils. Tonsillar SARS-CoV-2­specific memory CD8+ T cells displayed a follicular homing and tissue-resident memory phenotype, similar to tonsillar Epstein-Barr virus­specific memory CD8+ T cells, but were functionally less potent than other virus-specific memory CD8+ T cell responses. The presence of preexisting tissue-resident memory CD8+ T cells in unexposed individuals could potentially enable rapid sentinel immune responses against SARS-CoV-2.

Maternal microchimerism in juvenile tonsils and adenoids.

During pregnancy small amounts of cells pass between the mother and the fetus, and this transfer may give rise to a chimeric state that persist for years in both individuals. Both fetal and maternal microchimerism (MMc) have been associated with different autoimmune disorders. Information about MMc in tissues of healthy individuals is sparse but is important when looking for maternal cells within affected tissues of certain diseases. The aim of this study was to investigate the occurrence of maternal cells in tonsils and adenoids of 20 healthy children between the ages of 2 and 15 years. All the children underwent surgery because of recurrent tonsillitis or respiratory obstruction. MMc was detected using an RT-PCR assay based on differences in gene polymorphisms between mother and child. We found maternal cells in the tonsils and/or adenoids in four of 20 children. This frequency is less than the frequency of maternal cells found in the peripheral blood of healthy adults but in agreement with the previously reported frequency of maternal chimerism in control tissues

Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

Unresponsiveness to inhaled antigen is governed by conventional dendritic cells and overridden during infection by monocytes.

The nasal-associated lymphoid tissues (NALTs) are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage. NALTs represent a known site for the deposition of inhaled antigens, but little is known of the mechanisms involved in the induction of immunity within this lymphoid tissue. We find that during the steady state, conventional dendritic cells (cDCs) within the NALTs suppress T cell responses. These cDCs, which are also prevalent within human NALTs (tonsils/adenoids), express a unique transcriptional profile and inhibit T cell proliferation via contact-independent mechanisms that can be diminished by blocking the actions of reactive oxygen species and prostaglandin E2 Although the prevention of unrestrained immune activation to inhaled antigens appears to be the default function of NALT cDCs, inflammation after localized virus infection recruited monocyte-derived DCs (moDCs) to this region, which diluted out the suppressive DC pool, and permitted local T cell priming. Accommodating for inflammation-induced temporal changes in NALT DC composition and function, we developed an intranasal vaccine delivery system that coupled the recruitment of moDCs with the sustained release of antigen into the NALTs, and we were able to substantially improve T cell responses after intranasal immunization. Thus, homeostasis and immunity to inhaled antigens is tuned by inflammatory signals that regulate the balance between conventional and moDC populations within the NALTs.

Complex human adenoid tissue-based ex vivo culture systems reveal anti-inflammatory drug effects on germinal center T and B cells.

BACKGROUND: Human immunology research is often limited to peripheral blood. However, there are important differences between blood immune cells and their counterparts residing in secondary lymphoid organs, such as in the case of germinal center (GC) T follicular helper (Tfh) cells and GC B cells. METHODS: We developed a versatile ex vivo lymphoid organ culture platform that is based on human pharyngeal tonsils (adenoids) and allows for drug testing. We systematically phenotyped Tfh and GC B cell subsets in explant- and suspension cultures using multicolor flow cytometry and cytokine multiplex analysis. FINDINGS: Phenotypic changes of certain ex vivo cultured immune cell subsets could be modulated by cytokine addition. Furthermore, we optimized an activation-induced marker assay to evaluate the response to T cell stimulation. We provide proof-of-concept that Tfh and GC B cells could be modulated in these cultures by different anti-inflammatory drugs in unstimulated states and upon activation with vaccine-derived antigens. For example, GC B cells were lost upon CD40L blockade, and clinically approved JAK inhibitors impacted Tfh and GC B cells, including down-regulation of their key transcription factor BCL6. BCL6 regulation was affected by IL-6 signaling in T cells and IL-4 in B cells, respectively. Furthermore, we demonstrated that JAK signaling and TNF signaling contributed to the stimulation-induced activation of tonsil-derived T cells. INTERPRETATION: Our optimized methods, assays, and mechanistic findings can contribute to a better understanding of human GC responses. These insights may be relevant for improving autoimmune disease therapy and vaccination efficacy. FUNDING: This work was supported by a project grant under the joint research cooperation agreement of LMU Munich, LMU University Hospital, and Sanofi-Aventis Deutschland GmbH, as well as by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Emmy Noether Programme BA 5132/1-1 and BA 5132/1-2 (252623821), SFB 1054 Project B12 (210592381), and SFB 914 Project B03 (165054336).

Age-related changes in the morphology and the distribution of IgA and IgG in the pharyngeal tonsils of yaks (Bos grunniens).

To evaluate age-related changes in the morphology as well as the expression and localization of IgA and IgG in yak pharyngeal tonsils, 20 healthy yaks were divided into four age groups [newborn (1-7 days old), juvenile (5-7 months old), adult (3-6 years old) and old (7-10 years old)]. Morphologic characteristics were observed by histological techniques. The expression and localization of IgA and IgG in pharyngeal tonsils were detected by enzyme linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The results showed that the epithelium of the pharyngeal tonsils included nonreticular epithelium with an intact basement membrane and reticular epithelium with a discontinuous basement membrane and nonepithelial cell infiltration. In newborn yaks, only primary lymphoid follicles were observed in pharyngeal tonsils. In other age groups, both primary and secondary lymphoid follicles were observed, but some of the lymphoid follicles in the old yaks were degenerated. The number of lymphoid follicles increased from the newborn to the adult group and peaked in the adult group, but the number decreased in the old group. In addition, the age-related trends of IgA and IgG protein expression were similar to those of the number of lymphoid follicles. The concentration of IgG was significantly higher than that of IgA in all age groups. Both IgA and IgG antibody secreting cells (ASCs) were distributed in the subepithelial region of the nonreticular epithelium, the reticular epithelium, the lymphoid follicles, the interfollicular areas and in between the salivary glands. The densities of IgA and IgG ASCs in pharyngeal tonsils were similar to the expression trend of both proteins in each age group. The results indicate that the morphology and amount of lymphoid follicles in yak pharyngeal tonsils vary with age. Pharyngeal tonsils produce more IgG than IgA, indicating that IgG could be significant component of mucosal immune responses in yaks.

Expression of tight junction proteins in epithelium including Ck20-positive M-like cells of human adenoids in vivo and in vitro.

The human adenoid epithelium forms a continuous barrier against a wide variety of exogenous antigens. In this study, to elucidate the structures of the epithelial barrier in the human adenoid, including M-cells, we identified M-cells using an anti-cytokeratin 20 (Ck20) antibody and investigated expression of tight junction proteins in human adenoid epithelium in vivo and in vitro. In human adenoid epithelium and primary cultures, mRNAs of occludin, junctional adhesion molecule-A, ZO-1, and claudin-1, -4, -7, and -8 were detected by reverse transcription-polymerase chain reaction, whereas claudin-2 and -9 were expressed in vitro. In the epithelium in vivo, some Ck20-positive cells were randomly observed and indicated pocket-like structures, whereas Ck7 was positive in almost cells. Transmission electron microscopy revealed that Ck20-associated gold particles could be identified in M-like cells which had short microvilli and harboured the lymphocyte in the pocket-like structure. In primary cultures in vitro, Ck20-positive cells were also detected and had a function to take up fluorescent microparticles. In Ck20-positive cells in vivo and in vitro, expression of occludin, ZO-1, claudin-1 and -7 were observed at cell borders. These results indicate that the epithelial barrier of the human adenoid is stably maintained by expression of tight junction proteins in the epithelium including Ck20-positive M-like cells.

Differential cellular composition of human palatine and pharyngeal tonsils.

OBJECTIVE: The goal of this study was to gain a better understanding of the potential functional specialization of palatine and pharyngeal tonsils, by comparing their cellular composition in paired specimens from a large cohort of adenotonsillectomy patients. DESIGN: Resident B cell, T cell, dendritic cell, and stromal cell subsets were characterized using multicolor flow cytometry in palatine and pharyngeal tonsil specimens from 27 patients, age 2-34 years. RESULTS: Paired comparisons showed highly significant intra-individual differences in resident cell subsets of palatine and pharyngeal tonsils. Palatine tonsils harbored higher fractions of germinal center B cells/plasmablasts and IgD- CD27- double-negative B cells, and conversely lower fractions of IgD + CD38- resting naïve B cells compared to pharyngeal tonsils. Palatine tonsils also showed lower fractions of plasmacytoid dendritic cells, and higher percentages of two subsets of stromal cells - fibroblastic reticular cells and lymphatic endothelial cells - compared to pharyngeal tonsils from the same individual. CONCLUSIONS: Despite their physical proximity and histological similarities, palatine and pharyngeal tonsils display marked intra-individual differences in their cellular composition with regard to functionally important immune and stromal subsets. These differences are likely to have immunologic, pathologic, and physiologic significance.

Pancreatic changes in cases of death due to hypothermia.

Several morphological alterations of the pancreatic tissue have been described as common findings in hypothermia (e.g. bleedings, pancreatitis, vacuoles). The frequency of these findings varies a lot. It was the aim of this study to clarify the kind and frequency of pancreatic changes in cases of death due to hypothermia. The autopsy reports of 143 cases of fatal hypothermia were, retrospectively, evaluated with regard to describe macroscopic findings in the pancreas. Additionally, microscopic investigations of tissue samples of the pancreas were carried out in 62 cases. As a control group, pancreatic samples of 25 autopsy cases without hypothermia and without alcoholism were collected. Additionally, pancreatic samples of 25 further autopsy cases with an alcoholic disease in the case history were investigated. In only 5 out of 143 cases of the study group, macroscopic bleedings in the pancreas were described. One case of acute and one of chronic pancreatitis was found in the autopsy reports. In 11 (17.7%) out of 62 cases, microscopic investigations yielded bleedings in the pancreatic tissue and in 24 (38.7%) out of 62 cases, optically empty vacuoles in the adenoid cells were found. In 15 out of 62 cases (24.2%), autolysis was too pronounced to gain utilisable results. In the control group without alcoholism, 12 out of 25 cases (48%) were diagnosed without pathological findings, five cases showed bleedings, one case an acute pancreatitis, one case a chronic pancreatitis and in six cases, the pancreatic tissue was autolytic. Vacuoles in the adenoid cells were not found. In the additional collective with alcoholism in the case history, 13 cases presented signs of an acute or a chronic pancreatitis. In 3 out of these 13 cases, vacuoles in the adenoid cells were found, but no case with vacuoles and without signs of a chronic pancreatitis was observed. The high frequency of pancreatic bleedings in cases of fatal hypothermia as described in the literature cannot be confirmed by our investigations. Only the vacuoles in the adenoid cells of the pancreas seem to be an additional sign of death due to hypothermia or associated with hypothermia.

Histological studies on the ontogeny of bovine palatine and pharyngeal tonsil: germinal center formation, IgG, and IgA mRNA expression.

The development and distribution of lymphocyte subsets in calf palatine and pharyngeal tonsil were examined. During prenatal development, B cells were distributed in the subepithelial area, and T cells and MHC class II(+) cells were found in the deep layer of B-cell area, respectively, in both tonsils. At neonatal stage, lymphoid follicle containing a few CD4(+) cells have been formed in both tonsils. IgG(+) and IgA(+) cells were found in the parafollicular and epithelial area. At 3 months old, many germinal centers were recognized in both tonsils. CD4(+) cells and IgG mRNA expression were detected in light zone of germinal centers. Many IgG, and IgA mRNA expressions also could be detected in the parafollicular and subepithelial area of both tonsils. The data suggest that both tonsils have an important role of local immune defense against invading antigen after birth. The comparison of the histological characteristics of tonsil and Peyer's patch during ontogeny is also discussed.

Distribution of leucocyte subsets in the canine pharyngeal tonsil.

This report describes the distribution and nature of lymphoid tissue in the nasopharyngeal mucosa of six puppies (mean age+/-SD, 0.3+/-0.25 years) and eight adult dogs (mean age+/-SD, 8.8+/-2.67 years) without respiratory disease. A non-encapsulated area of organized mucosa-associated lymphoid tissue was observed in the caudal part of the posterior wall of the nasopharynx, distal to the openings of the auditory tubes. This structure was consistent with the pharyngeal tonsil and was microscopically more extensive in puppies than in adult dogs. Histochemistry and immunohistochemistry were used to characterize and enumerate the leucocyte subsets in this part of the nasopharynx. Mast cells were found immediately beneath the respiratory epithelium but were also scattered in the glandular and muscular tissue. IgA(+) plasma cells outnumbered IgG(+) and IgM(+) plasma cells, especially in the glandular tissue. All classes of plasma cells were present in significantly greater numbers in adults than in puppies. MHC class II(+) cells were mainly observed in areas containing diffuse and follicular aggregates of lymphoid cells. Both MHC class II(+) cells and CD1c(+) cells with a dendritic morphology were predominantly found immediately beneath or within the epithelium, and cells expressing these markers were more abundant in puppies than in adult dogs. The anti-L1 marker labelled low numbers of cells with a neutrophilic morphology, which were significantly more abundant in puppies than in adult dogs. The majority of lymphoid cells were CD3(+) T lymphocytes and these were particularly abundant in areas containing aggregates of lymphoid cells; CD4(+), CD8(+) and TCR alphabeta(+) cells had the same distribution as the CD3(+) cells. CD4(+) cells were more numerous than CD8(+) cells. The quantitative and qualitative data obtained will enable comparisons to be made with similar studies in dogs suffering from nasopharyngeal diseases, or when the local immune system needs to be investigated.

IgE-positive plasma cells are present in adenoids of atopic children.

CONCLUSIONS: We demonstrated the presence of IgE(+) plasma cells in the adenoids of atopic children. Our data suggest that adenoids are capable of local production of IgE and support the role of adenoids as an inductive site for allergic reactions. OBJECTIVE: We have previously demonstrated increased numbers of IgE(+) cells in the adenoids of atopic children and also found support for an IL-4-induced class switch to IgE production in adenoids. In search of further evidence of adenoids being involved in IgE-mediated sensitization, we investigated the distribution of plasma cells and macrophages positive for IgE in adenoids. MATERIAL AND METHODS: Adenoid tissue from atopic and non-atopic children was examined using immunohistochemical markers for IgE, plasma cells (CD138) and macrophages (CD68). The distribution of positive cells was determined in the extrafollicular area and in the follicles of adenoids. Co-localization of IgE and CD138(+) plasma cells and CD68(+) macrophages was examined using immunohistochemical double-staining methods. RESULTS: Non-atopic adenoids contained no or very few IgE(+) cells. In contrast, IgE(+) cells were found in high numbers in atopic adenoids, mainly in the extrafollicular area. Higher numbers of IgE(+) plasma cells and IgE(+) macrophages were also found in the adenoids of atopic children.

Rapid proliferation of B cells from adenoids in response to Epstein-Barr virus infection.

EBV is a human tumor virus that is associated with different types of tumors. A unique feature of EBV is its capability to infect and immortalize human B cells both in vivo and in vitro. In cell culture, this progress is termed immortalization and infected B cells grow out to permanent, so-called lymphoblastoid cell lines. During our experiments, we observed that B lymphocytes derived from adenoids are infected efficiently by EBV and proliferate much more rapidly than any other known type of B cell. High concentrations of adhesion molecules and of CD21, the EBV receptor, present on these cells may account for this phenomenon. Adenoid B cells may therefore represent a particular subpopulation of preactivated B lymphocytes that can greatly simplify and enhance the production of lymphoblastoid cell lines for, e.g., antigen-presenting cells for gene therapeutic approaches and similar applications.