Cells In Tonsils Research Articles

Stress Can Induce Bovine Alpha-Herpesvirus 1 (BoHV-1) Reactivation from Latency

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are importantsites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral heterochromatin, remodel chromatin, and activate gene expression. Thus, wepredict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency.

CD27 is not an ideal marker for human memory B cells and can be modulated by IL-21 upon stimulated by Anti-CD40

B cells play a key role in humoral immune responses by producing antibodies. Although there are numerous research on memory B cells definition markers and cytokines on B cell development, different studies have yielded contradictory conclusions due to species studied, the different cells and stimulating agents used. In the current study, we conducted a detailed characterization of B cells in human CBMCs, PBMCs and tonsil, including expression of Igs, activation and memory markers. Furthermore, we found that considerable amounts of IgA and IgG were expressed by CD27− B cells. These “Atypical” memory B cells corresponded to approximately 50% of IgG+ and IgA+B cells in blood, this proportion even reached 90% in tonsil. In addition, we investigated the effect of IL-21 and TGF-β1 on the membrane-bound form and secreted form of Igs using PBMCs and purified blood B cells. There were actual differences between the effect of cytokines on Igs secretion and surface expression. Our study will be helpful to advance the knowledge and understanding of humoral memory.

Inhibition of human cytomegalovirus entry into mucosal epithelial cells

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

Oligoclonal CD4+CXCR5+ T cells with a cytotoxic phenotype appear in tonsils and blood

In clinical situations, peripheral blood accessible CD3+CD4+CXCR5+ T-follicular helper (TFH) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of TFH cells in peripheral blood versus tonsils, CD3+CD4+CD45RA–CXCR5+ cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3+CD4+CXCR5+ T-cell subpopulations also appear in tonsils, there with some supplementary TFH characteristics, while peripheral blood-derived TFH cells display markers of proliferation and migration. Three further subsets of TFH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4+CXCR5+ subpopulation presents pronounced cytotoxic properties. Those ‘killer TFH (TFK) cells’ can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other TFH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4+CXCR5+ T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts.

Development of FluoAHRL: A Novel Synthetic Fluorescent Compound That Activates AHR and Potentiates Anti-Inflammatory T Regulatory Cells.

Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.

Age-dependent distribution of IgA and IgG antibody-secreting cells in the pharyngeal tonsil of the Bactrian camel

The pharyngeal tonsil, located in the nasopharynx, can effectively defend against pathogens invading the body from the upper respiratory tract and play a crucial role in mucosal immunity of the respiratory tract. Immunoglobulin A (IgA) and Immunoglobulin G (IgG) serve as key effector molecules in mucosal immunity, exhibiting multiple immune functions. This study aimed to investigate the distribution patterns and age-related alterations of IgA and IgG antibody-secreting cells (ASCs) in the pharyngeal tonsils of Bactrian camels. Twelve Alashan Bactrian camels were categorized into four age groups: young (1–2 years, n=3), pubertal (3–5 years, n=3), middle-aged (6–16 years, n=3) and old (17–20 years, n=3). The distribution patterns of IgA and IgG ASCs in the pharyngeal tonsils of Bactrian camels of different ages were meticulously observed, analyzed and compared using immunohistochemical and statistical methods. The results revealed that IgA ASCs in the pharyngeal tonsils of all age groups were primarily clustered or diffusely distributed in the reticular epithelium and its subepithelial regions (region A) and around the glands (region C), scattered in the subepithelial regions of non-reticular epithelium (region B), and sporadically distributed in the interfollicular regions (region D). Interestingly, the distribution pattern of IgG ASCs in the pharyngeal tonsils closely mirrored that of IgA ASCs. The distribution densities of IgA and IgG ASCs in these four regions were significantly decreased in turn (P<0.05). However, IgA ASCs exhibited significantly higher densities than IgG ASCs in the same region (P<0.05). Age-related alterations indicated that the distribution densities of IgA and IgG ASCs in each region of the pharyngeal tonsils exhibited a trend of initially increasing and subsequently decreasing from young to old camels, reaching a peak in the pubertal group. As camels age, there was a significant decrease in the densities of IgA and IgG ASCs in all regions of the pharyngeal tonsils (P<0.05). The results demonstrate that the reticular epithelium and its subepithelial regions in the pharyngeal tonsils of Bactrian camels are the primary regions where IgA and IgG ASCs colonize and exert their immune functions. These regions play a pivotal role in inducing immune responses and defending against pathogen invasions in the pharyngeal tonsils. IgA ASCs may be the principal effector cells of the mucosal immune response in the pharyngeal tonsils of Bactrian camels. Aging significantly reduces the densities of IgA and IgG ASCs, while leaving their distribution patterns unaffected. These findings will provide valuable insights for further investigations into the immunomorphology, immunosenescence, and response mechanisms of the pharyngeal tonsils in Bactrian camels.

IMPACT OF A GEL COMPOSITION CONTAINING VARIOUS SUBSTANCES ON INDICES OF IMMUNITY AND TISSUE INFLAMMATION IN PALATINE TONSILS OF PATIENTS WITH CATARRHAL GINGIVITIS DURING CHRONIC TONSILLITIS

The study of pharmacotherapeutic (immunomodulating) action in conditions of in vitro cell cultures is currently considered appropriate and justified for determining the action of drugs at the preclinical level. It is also important to address the pathogenetic nature in using such agents, especially fro local application. This study investigated the efficacy of a multicomponent gel containing decamethoxine and hyaluronic acid in treating chronic catarrhal gingivitis among patients with chronic tonsillitis. The purpose of the work is to assess the efficacy of a gel containing decamethoxine and hyaluronic acid on the tissue of the palatine tonsils in the therapy of chronic catarrhal gingivitis in patients with chronic tonsillitis.&#x0D; Materials and methods. To investigate the effects of the main active components in the gel, we followed the recommendations of I.P. Kaidashev et al. for testing pharmacological preparations in in vitro cultures. Tissue samples from the palatine tonsils were obtained from 20 patients with chronic tonsillitis and chronic catarrhal gingivitis undergoing tonsillectomy. All procedures adhered to the guidelines set by the Laboratory of Pathophysiology and Immunology at Prof. O.S. Kolomiichenko Institute of Otolaryngology, National Academy of Sciences of Ukraine. Palatine tonsil tissue was incubated in 199 medium (Serva, Germany) containing gentamicin sulfate (Darnytsa, Ukraine) at a concentration of 120 μg/ml for 20 minutes at 4-8°C. After that, the hemorrhagic parts were removed from the whole tonsil and placed into Eagle's medium (Sigma, USA) and rinsed three to four times. Subsequently, the prepared tissue was placed in sterile vials containing enriched Eagle's medium supplemented with L-glutamine, sodium bicarbonate, 100x vitamin concentrate, 5% fetal calf serum, and 60 μg/mL gentamicin. Tonsil tissue was dissected using scissors and then washed repeatedly (5-7 times) with fresh medium to remove loosely attached cells, then, the resulting cell suspension was filtered. All procedures were conducted within a sterile laminar flow cabinet. Cell viability was assessed using light microscopy (Olympus-23, Japan) and a hemocytometer. A final cell suspension containing 2.5 million lymphocytes per milliliter of medium was prepared. Suspensions with a viability of less than 90% (blue, "dead" cells) were excluded from further experiments.&#x0D; Results. Treatment of in vitro palatine tonsil cells from patients with chronic catarrhal gingivitis and recurrent tonsillitis with the main components of the developed gel (decamethoxine and hyaluronic acid) did not induce significant changes in the concentration of α and γ-interferons. Our findings suggest that the gel composition reduces the levels of pro-inflammatory factors: interleukin-1β and immune complexes. Additionally, it appears to stimulate the production of the antimicrobial factor antistreptolysin-O by tonsil cells, potentially leading to increased antibody production against hemolytic streptococcus antigens.&#x0D; Conclusion. Based on the in vitro findings, the investigated gel composition containing decamethoxine and hyaluronic acid demonstrates potential anti-inflammatory and antimicrobial properties that stimulate the production of the antimicrobial factor antistreptolysin-O by tonsil cells, which might lead to increased antibody production against hemolytic streptococcus antigens.

An atlas of cells in the human tonsil

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource byannotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

IL-17 and IFN-γ-producing Respiratory Tissue-Resident Memory CD4 T Cells Persist for Decades in Adults Immunized as Children With Whole-Cell Pertussis Vaccines.

The objective was to determine if antigen-specific tissue-resident memory T (TRM) cells persist in respiratory tissues of adults immunized as children with whole-cell pertussis (wP) or acellular pertussis (aP) vaccines. Mononuclear cells from tonsil or nasal tissue cells were cultured with Bordetella pertussis antigens and TRM cells quantified by flow cytometry. Adults immunized with wP vaccines as children had significantly more interleukin 17A (IL-17A) and interferon-γ (IFN-γ)-producing TRM cells that respond to B. pertussis antigens in respiratory tissues when compared with aP-primed donors. Our findings demonstrate that wP vaccines induce CD4 TRM cells that can persist in respiratory tissues for decades.

Susceptibility of primary ovine dorsal soft palate and palatine tonsil cells to FMDV infection.

Foot and mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals. This disease is one of the most important in animal health due to its significant socio-economic impact, especially in case of an outbreak. One important challenge associated with this disease is the ability of the FMD virus (FMDV) to persist in its hosts through still unresolved underlying mechanisms. The absence of relevant in vitro models is one factor preventing advancement in our understanding of FMDV persistence. While a primary bovine cell model has been established using cells from FMDV primary and persistence site in cattle, it appeared interesting to develop a similar model based on ovine anatomical sites of interest to compare host-pathogen interactions. Thus, epithelial cells derived from the palatine tonsils and the dorsal soft palate were isolated and cultured. Their epithelial nature was confirmed using immunofluorescence. Following monolayer infection with FMDV O/FRA/1/2001 Clone 2.2, the FMDV-sensitivity of these cells was evaluated. Dorsal soft palate (DSP) cells were also expanded in multilayers at the air-liquid interface to mimic a stratified epithelium sensitive to FMDV infection. Our investigation revealed the presence of infectious virus, as well as viral antigens and viral RNA, up to 35 days after infection of the cell multilayers. Further experiment with DSP cells from different individuals needs to be reproduced to confirm the robustness of the new model of persistence in multilayer DSP. The establishment of such primary cells creates new opportunities for FMDV research and analysis in sheep cells.

Novel AHR ligand AGT-5 ameliorates type 1 diabetes in mice through regulatory cell activation in the early phase of the disease.

Type 1 diabetes (T1D) is an autoimmune disease with a strong chronic inflammatory component. One possible strategy for the treatment of T1D is to stimulate the regulatory arm of the immune response, i.e. to promote the function of tolerogenic dendritic cells (tolDC) and regulatory T cells (Treg). Since both cell types have been shown to be responsive to the aryl hydrocarbon receptor (AHR) activation, we used a recently characterized member of a new class of fluorescent AHR ligands, AGT-5, to modulate streptozotocin-induced T1D in C57BL/6 mice. Prophylactic oral administration of AGT-5 reduced hyperglycemia and insulitis in these mice. Phenotypic and functional analysis of cells in the pancreatic infiltrates of AGT-5-treated mice (at the early phase of T1D) revealed a predominantly anti-inflammatory environment, as evidenced by the upregulation of tolDC and Treg frequency, while CD8+ cell, Th1 and Th17 cells were significantly reduced. Similarly, AGT-5 enhanced the proportion of Treg and tolDC in small intestine lamina propria and suppressed the activation status of antigen-presenting cells through down-regulation of co-stimulatory molecules CD40, CD80 and CD86. The expression levels of Cyp1a1, controlled by the AHR, were increased in CD4+, CD8+ and Treg, confirming the AHR-mediated effect of AGT-5 in these cells. Finally, AGT-5 stimulated the function of regulatory cells in the pancreatic islets and lamina propria by upregulating indoleamine 2,3-dioxigenase 1 (IDO1) in tolDC. These findings were supported by the abrogation of AGT-5-mediated in vitro effects on DC in the presence of IDO1 inhibitor. AGT-5 also increased the expression of CD39 or CD73 ATP-degrading ectoenzymes by Treg. The increase in Treg is further supported by the upregulated frequency of IL-2-producing type 3 innate lymphoid cells (ILC3) in the lamina propria. Anti-inflammatory effects of AGT-5 were also validated on human tonsil cells, where in vitro exposure to AGT-5 increased the proportion of immunosuppressive dendritic cells and ILC3. These results suggest that AGT-5, by stimulating AHR, may promote a general immunosuppressive environment in the pancreas and small intestine lamina propria at the early phase of disease, and thereby inhibit the severity of T1D in mice.

Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet’s ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Aberrant follicular regulatory T cells associate with immunoglobulin hyperproduction in nasal polyps with ectopic lymphoid tissues

Ectopic lymphoid tissues (eLTs) and associated follicular helper T (TFH) cells contribute to local immunoglobulin hyperproduction in nasal polyps (NPs). Follicular regulatory T (TFR) cells in secondary lymphoid organs counteract TFH cells and suppress immunoglobulin production; however, the presence and function of TFR cells in eLTs in peripheral diseased tissues remain poorly understood. We sought to investigate the presence, phenotype, and function of TFR cells in NPs. The presence, abundance, and phenotype of TFR cells in NPs were examined using single-cell RNA sequencing, immunofluorescence staining, and flow cytometry. Sorted polyp and circulating T-cell subsets were cocultured with autologous circulating naïve B cells, and cytokine and immunoglobulin production were measured by ELISA. TFR cells were primarily localized within eLTs in NPs. TFR cell frequency and TFR cell/TFH cell ratio were decreased in NPs with eLTs compared with NPs without eLTs and control inferior turbinate tissues. TFR cells displayed an overlapping phenotype with TFH cells and FOXP3+ regulatory T cells in NPs. Polyp TFR cells had reduced CTLA-4 expression and decreased capacity to inhibit TFH cell-induced immunoglobulin production compared with their counterpart in blood and tonsils. Blocking CTLA-4 abolished the suppressive effect of TFR cells. Lower vitamin D receptor expression was observed on polyp TFR cells compared with TFR cells in blood and tonsils. Vitamin D treatment upregulated CTLA-4 expression on polyp TFR cells and restored their suppressive function invitro. Polyp TFR cells in eLTs have decreased CLTA-4 and vitamin D receptor expression and impaired capacity to suppress TFH cell-induced immunoglobulin production, which can be reversed by vitamin D treatment invitro.

Innate adaptive immune cell dynamics in tonsillar tissues during chronic SIV infection.

HIV-infected patients are at higher risk of developing oral mucosal infection and Epstein-Barr virus (EBV)-associated B cell malignancies. However, the potential role of oral immunity in the pathogenesis of oral lesions is unknown. Tonsils are oral-pharyngeal mucosal-associated lymphoid tissues that play an important role in oral mucosal immunity. In this study, we investigated the changes of innate and adaptive immune cells in macaque tonsils during chronic SIV infection. We found significantly higher frequencies of classical monocytes, CD3+CD56+ (NKT-like) cells, CD3+CD4+CD8+ (DP), and CD161+ CD4 T cells in tonsils from chronic infected compared to naïve animals. On the contrary, intermediate monocytes and CD3+CD4-CD8- (DN) cells were lower in chronic SIV-infected macaques. We further confirmed a recently described small B-cell subset, NKB cells, were higher during chronic infection. Furthermore, both adaptive and innate cells showed significantly higher TNF-α and cytotoxic marker CD107a, while IL-22 production was significantly reduced in innate and adaptive immune cells in chronic SIV-infected animals. A dramatic reduction of IFN-γ production by innate immune cells might indicate enhanced susceptibility to EBV infection and potential transformation of B cells in the tonsils. In summary, our observation shows that the SIV-associated immune responses are distinct in the tonsils compared to other mucosal tissues. Our data extends our understanding of the oral innate immune system during SIV infection and could aid future studies in evaluating the role of tonsillar immune cells during HIV-associated oral mucosal infections.

Effect of chicken infectious bronchitis vaccine on morphogenesis and differentiation of cells in caecal tonsils

The study of the chickens’ immune system morphofunctional state allows assessing critical periods of their development and the body as a whole, as well as the effectiveness of vaccine prevention methods. The purpose of this study was to identify morphological and immunohistochemical changes in the caecal tonsils of chickens aged 8, 20, 40, 90, 110 days for vaccine prevention of infectious bronchitis. During the study, the following research methods were used: cytological, histological, immunohistochemical, morphometric, light-optical, statistical. Histological preparations of caecal tonsils of poultry aged 8, 20, 40, 90, 110 days of vaccinated and unvaccinated groups were analysed and studied. Up to 20 days of age, no lymphoid nodules were detected in the caecal tonsils of chickens, both vaccinated and non-vaccinated groups. Histological and morphometric studies were conducted, which showed that vaccination of poultry accelerates the development of lymphoid formations in the early stages of the postnatal period of ontogenesis, especially in chickens aged 20 days, which is manifested by an increase in the number and size of lymphoid nodules of caecal tonsils relative to the control group. Immunohistochemical studies have established that the placement and accumulation of T-lymphocytes with markers CD4+ , CD8+ , as well as B-lymphocytes (CD20+ ) and CD45RA+ cells in caecal tonsils, namely in lymphoid nodules and diffuse accumulation of lymphoid cells, depend on the age of chickens and multiplicity of vaccinations. It was proved that in chickens aged 8 days, the number of T-lymphocyte subpopulations with surface markers CD4+ , CD8+ prevailed over CD45RA+ and CD20+ . With the increase in the number of immunizations (after three-fold vaccination against infectious bronchitis of chickens), immunocytometric studies indicated that in vaccinated chickens aged 40 and 90 days, there was a clear increase in the number of mature B-lymphocytes by 1.58 and 1.37 times, respectively. Considering the fact that the number of CD8+ lymphocytes in vaccinated chickens aged 40 days was 1.49 times greater than the number of CD4+ cells, this led to a sharp decrease in the immunoregulatory index of the caecal tonsils, which must be factored in when carrying out preventive vaccinations

PI16+ reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

Fibroblastic reticular cells (FRCs) direct the interaction and activation of immune cells in discrete microenvironments of lymphoid organs. Despite their important role in steering innate and adaptive immunity, the age- and inflammation-associated changes in the molecular identity and functional properties of human FRCs have remained largely unknown. Here, we show that human tonsillar FRCs undergo dynamic reprogramming during life and respond vigorously to inflammatory perturbation in comparison to other stromal cell types. The peptidase inhibitor 16 (PI16)-expressing reticular cell (PI16+ RC) subset of adult tonsils exhibited the strongest inflammation-associated structural remodeling. Interactome analysis combined with ex vivo and in vitro validation revealed that T cell activity within subepithelial niches is controlled by distinct molecular pathways during PI16+ RC–lymphocyte interaction. In sum, the topological and molecular definition of the human tonsillar stromal cell landscape reveals PI16+ RCs as a specialized FRC niche at the core of mucosal immune responses in the oropharynx.

Phenotypic and transcriptional heterogeneity of memory CXCR5+ CD8+ T cells in human blood and lymphoid tissue

Abstract CXCR5+ CD8+ T cells are involved in antiviral and -tumor immunosurveillance. Animal models have demonstrated that CXCR5+ CD8+ T cells have multiple roles, including cytotoxic, stem-like, and regulatory functions. Many of these functions might be disease-dependent, where human studies are lacking to understand if CXCR5+ CD8+ T cells show signs of significant heterogeneity. To this end, we performed simultaneous single-cell (sc) RNA−, TCR−, and CITE-seq on CXCR5+ and CXCR5− memory CD8+ T cells from matched tonsils and blood to unravel if CXCR5+ CD8+ T cells show potential differences between human lymphoid tissue and blood. Our analysis indicates that a high proportion of CXCR5+ CD8+ T cells in tonsils show tissue residency-like (TRM) features, including CD69 and CD103 expression. Instead, blood CXCR5+ CD8+ T cells showed increased signs of cytotoxic functions in blood compared to their counterparts in tonsils. GSEA analysis identified groups of genes differentially expressed, such as the p53 pathway enriched in tonsils compared to ribosome-related pathways in blood CXCR5+ memory CD8+ T cells. Additionally, with CITE-seq markers, we identified the protein landscape of sub-populations and found increased PD-1 and CD45RO expression in tonsils compared to blood CXCR5+ memory CD8+ T cells. Overall, these findings indicate that CXCR5+ CD8+ T cells are heterogeneous with potentially distinct functional subsets that patrol blood and lymphoid tissues.

Preparation of IgE Antibody and Distribution of IgE+ Secretory Cells in the Palatine Tonsil of Bactrian Camel.

Allergic diseases induced by dust have seriously threatened human health, while Bactrian camels can live in a sandy environment for a long time. To prepare rabbit anti-Bactrian camel IgE antibody and explore the distribution characteristics of IgE+ secretory cells in the palatine tonsils, which lays a theoretical foundation for the distribution of local antibodies in the palatal tonsils of Bactrian camel and the study of immune function. In this study, the amino acid sequences of Bactrian camel IgE, IgA, IgM and IgG heavy chain constant regions were compared, and a specific IgE gene fragment were selected (447 bp). The recombinant plasmid pET-28a-IgE was induced in Escherichia coli BL21(DE3) by IPTG and its expression conditions were optimized. The antibody was prepared by immunizing rabbits with purified IgE recombinant protein, its titer and specificity were detected by indirect ELISA and Western blotting. Immunohistochemical and statistical methods investigated the distribution of IgE+ secretory cells in the palatine tonsils. The IgE recombinant protein was expressed in the form of inclusion bodies with a size of 16 kDa. The optimal IPTG induction concentration was 0.7 mmol/L and the induction time was 8 h. The titer of the antibody was 1:16000 by ELISA, and the antibody could specifically bind to the recombinant protein by Western blotting. IgE+ secretory cells were mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil of the Bactrian camel, followed by the subepithelial compartments of stratified squamous epithelium and occasionally in the extrafollicular region. The rabbit anti-Bactrian camel IgE polyclonal antibody was successfully prepared. It is confirmed that IgE exists in the palatine tonsils of Bactrian camels under normal living conditions. In addition, IgE+ secretory cells are mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil, which is consistent with the distribution characteristics of IgG+ and sIgA+ secretory cells in the palatal tonsils of the Bactrian camel.

Follicular regulatory T cells eliminate HIV-1-infected follicular helper T cells in an IL-2 concentration dependent manner.

Follicular helper CD4+ T cells (TFH) are highly permissive to HIV and major foci of virus expression in both untreated and treated infection. Follicular regulatory CD4+ T cells (TFR) limit TFH numbers and function in vitro and in vivo. We evaluated the hypothesis that TFR suppress HIV replication in TFH using a well-established model of ex vivo HIV infection that employs tonsil cells from HIV uninfected individuals spinoculated with CXCR4- and CCR5-tropic HIV-GFP reporter viruses. Both CXCR4 and CCR5-tropic HIV replication were reduced in TFH cultured with TFR as compared to controls. Blocking antibodies to CD39, CTLA-4, IL-10, and TGF-beta failed to reverse suppression of HIV replication by TFR, and there were no sex differences in TFR suppressive activity. TFR reduced viability of TFH and even more so reduced HIV infected TFH as assessed by total and integrated HIV DNA. Exogenous IL-2 enhanced TFH viability and particularly numbers of GFP+ TFH in a concentration dependent manner. TFR reduced productively infected TFH at low and moderate IL-2 concentrations, and this was associated with decreases in extracellular IL-2. Both IL-2 expressing cells and larger numbers of FoxP3+CD4+ cells were detected in follicles and germinal centers of lymph nodes of people living with HIV. TFR may deplete TFH in vivo through restriction of IL-2 and thereby contribute to decay of HIV expressing cells in B cell follicles during HIV infection.