Tonsil explants as a human in vitro model to study vaccine responses.

Introduction: Vaccination is one of the most effective infection prevention strategies. Viruses with high mutation rates -such as influenza- escape vaccine-induced immunity and represent significant challenges to vaccine design. Influenza vaccine strain selection is based on circulating strains and immunogenicity testing in animal models with limited predictive outcomes for vaccine effectiveness in humans. Methods: We developed a human in vitro vaccination model using human tonsil tissue explants cultured in 3D perfusion bioreactors to be utilized as a platform to test and improve vaccines. Results: Tonsils cultured in bioreactors showed higher viability, metabolic activity, and more robust immune responses than those in static cultures. The in vitro vaccination system responded to various premanufactured vaccines, protein antigens, and antigen combinations. In particular, a multivalent in vitro immunization with three phylogenetically distant H3N2 influenza strains showed evidence for broader B cell activation and induced higher antibody cross-reactivity than combinations with more related strains. Moreover, we demonstrate the capacity of our in vitro model to generate de novo humoral immune responses to a model antigen. Discussion: Perfusion-cultured tonsil tissue may be a valuable human in vitro model for immunology research with potential application in vaccine candidate selection.

Increased autoreactivity and maturity of EBI2+ antibody-secreting cells from nasal polyps.

Elevated numbers of antibody-secreting cells (ASCs) and anti-double-stranded DNA (anti-dsDNA) antibodies are found in nasal polyp (NP) tissue. The presence of anti-dsDNA IgG in tissue prospectively predicts recurrent NP but the characteristics of the source ASCs are unknown. Here, we investigated whether NP B cells expressing the extrafollicular marker EBI2 have increased propensity for autoantibody production and evaluated the molecular characteristics of NP ASCs. NPs showed increased frequencies of anti-dsDNA IgG and total IgG ASCs compared with tonsils, with more pronounced differences among EBI2+ cells. In NPs, EBI2+ cells were frequently double negative (IgD-CD27-) and ASCs. Single-cell RNA-Seq analysis of tonsils and NPs revealed substantial differences in B lineage composition, including differences in percentages of ASCs, germinal centers, proliferative cells, and non-ASCs. NPs exhibited higher expression of specific isotypes (IGHE, IGHA1, IGHA2, and IGHG4) and mature plasma genes, including SDC1 and XBP1, than tonsils. Gene Ontology biological processes indicated upregulated NF-κB and downregulated apoptosis pathways in NP ASCs. Together, these data indicate that NP EBI2+ ASCs secret increased total and anti-dsDNA IgG compared with those from tonsils and had molecular features of mature plasma cell differentiation.

The aberrant tonsillar microbiota modulates autoimmune responses in rheumatoid arthritis.

Palatine tonsils are the only air-contacted lymphoid organs that constantly engage in crosstalk with commensal microorganisms and serve as the first handling sites against microbial antigens. While tonsil inflammations have been implicated in various autoimmune diseases, including rheumatoid arthritis (RA), the precise role of tonsillar microbiota in autoimmune pathogenesis remains inadequately characterized. In this study, we profiled the tonsillar microbiota and identified a notable dysbiosis in patients with RA, particularly within the Streptococcus genus. Specifically, patients with RA exhibited an enrichment of pathogenic Streptococcus species, including S. pyogenes, S. dysgalactiae, and S. agalactiae. Colonization with these bacteria significantly exacerbated arthritis severity and increased autoimmune responses in collagen-induced arthritis (CIA). Furthermore, immunization with peptides derived from these pathogenic Streptococcus species directly induced experimental arthritis. Conversely, patients with RA demonstrated a marked deficiency in commensal Streptococcus members, notably S. salivarius. Treatment of CIA mice with S. salivarius attenuated the progression of arthritis and downregulated autoimmune responses. These findings highlight a pathogenic link of tonsillar microbiota with RA, shedding light on their contribution to autoimmunity.

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.

Using Ex Vivo Tonsil Organoids to Study Memory B Cells.

Tools to study memory B cell (MBC) development and function are needed to understand their role in supporting sustained protection against recurrent infections. While human MBCs are traditionally measured using blood, there is a growing interest in elucidating their behavior within lymphoid tissues, which are the main sites where adaptive immune responses are orchestrated. In this chapter, we introduce a high-throughput organoid system that is derived from primary human lymphoid tissues. The approach can recapitulate many hallmarks of successful adaptive immune responses and capture inter-individual variation in response to a variety of stimuli. Lymphoid tissue organoids enable characterization of pre-existing antigen-specific MBCs within an entirely human system and can provide valuable insights into MBC dynamics.

High-dimensional single-cell analysis of human natural killer cell heterogeneity.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Distinct Localization, Transcriptional Profiles, and Functionality in Early Life Tonsil Regulatory T Cells.

CD4+ regulatory T cells (Tregs) are key orchestrators of the immune system, fostering the establishment of protective immunity while preventing deleterious responses. Infancy and childhood are crucial periods of rapid immunologic development, but how Tregs mediate immune responses at these earliest timepoints of human life is poorly understood. In this study, we compare blood and tissue (tonsil) Tregs across pediatric and adult subjects to investigate age-related differences in Treg biology. We observed increased FOXP3 expression and proportions of Tregs in tonsil compared with paired blood samples in children. Within tonsil, early life Tregs accumulated in extrafollicular regions with cellular interactions biased toward CD8+ T cells. Tonsil Tregs in both children and adults expressed transcriptional profiles enriched for lineage defining signatures and canonical functionality compared with blood, suggesting tissue as the primary site of Treg activity. Early life tonsil Tregs transcriptional profiles were further defined by pathways associated with activation, proliferation, and polyfunctionality. Observed differences in pediatric tonsil Treg transcriptional signatures were associated with phenotypic differences, high proliferative capacity, and robust production of IL-10 compared with adult Tregs. These results identify tissue as a major driver of Treg identity, provide new insights into developmental differences in Treg biology across the human lifespan, and demonstrate unique functional properties of early life Tregs.

Tissue-specific sex differences in pediatric and adult immune cell composition and function.

Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in peripheral blood, but little is known about sex differences within human lymphoid tissues. Here, we characterized the composition and phenotypes of adaptive immune cells from male and female ex vivo tonsils and evaluated their responses to influenza antigens using an immune organoid approach. In a pediatric cohort, female tonsils had more memory B cells compared to male tonsils direct ex vivo and after stimulation with live-attenuated but not inactivated vaccine, produced higher influenza-specific antibody responses. Sex biases were also observed in adult tonsils but were different from those measured in children. Analysis of peripheral blood immune cells from in vivo vaccinated adults also showed higher frequencies of tissue homing CD4 T cells in female participants. Together, our data demonstrate that distinct memory B and T cell profiles are present in male vs. female lymphoid tissues and peripheral blood respectively and suggest that these differences may in part explain sex biases in response to vaccines and viruses.

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.

Accelerated involution of germinal center in palatine tonsils in IgA nephropathy.

BACKGROUND: Altered immunological responses in the palatine tonsils may be involved in the pathogenesis of IgA nephropathy (IgAN). The germinal center serves as the site for antigen-specific humoral immune responses in the palatine tonsils. Germinal center involution is frequently observed in the palatine tonsils of IgAN (IgAN tonsils). However, the pathogenic significance of these characteristic changes remains unclear. This study aimed to investigate the morphological changes in secondary lymphoid follicles in IgAN tonsils and to evaluate the correlation between the morphometric results and the clinicopathological severity of IgAN. METHODS: The tonsils of age-matched patients with recurrent tonsillitis (RT tonsils) were used as controls. The correlation between the degree of lymphoid follicular involution and histopathological severities in clinical or kidney biopsy was evaluated. RESULTS: In total, 87 patients with IgAN were included (48% male, median age 35 years, median estimated glomerular filtration rate: 74 mL/min/1.73 m2). Compared to RT tonsils, IgAN tonsils showed smaller median sizes of lymphoid follicles and germinal centers (P < 0.001). The relative areas of lymphoid follicles (%LFA) and germinal centers (%GCA) in the total tonsillar tissue were smaller in the IgAN tonsils than in the RT tonsils (P < 0.001). In contrast, the median proportion of mantle zones in the total tonsillar tissue was comparable between the groups. A lower %LFA was associated with a longer period from the onset of urinary abnormalities to biopsy diagnosis and higher urinary protein excretion (P = 0.01). %LFA showed significant negative correlations with frequencies of glomeruli with both global and segmental sclerosis. CONCLUSIONS: The present study confirmed accelerated germinal center involution in the tonsils of patients with IgAN. This characteristic change in the IgAN tonsil correlates with heavy proteinuria and advanced chronic histopathological changes in the kidneys, thereby suggesting the involvement of repeated tonsillar immunoreactions during IgAN progression.

Mapping Human Immunity and the Education of Waldeyer's Ring.

The development and deployment of single-cell genomic technologies have driven a resolution revolution in our understanding of the immune system, providing unprecedented insight into the diversity of immune cells present throughout the body and their function in health and disease. Waldeyer's ring is the collective name for the lymphoid tissue aggregations of the upper aerodigestive tract, comprising the palatine, pharyngeal (adenoids), lingual, and tubal tonsils. These tonsils are the first immune sentinels encountered by ingested and inhaled antigens and are responsible for mounting the first wave of adaptive immune response. An effective mucosal immune response is critical to neutralizing infection in the upper airway and preventing systemic spread, and dysfunctional immune responses can result in ear, nose, and throat pathologies. This review uses Waldeyer's ring to demonstrate how single-cell technologies are being applied to advance our understanding of the immune system and highlight directions for future research.

Evaluation of the local and systemic pattern of sensitization to allergens in patients with adenotonsillar hypertrophy.

PURPOSE: Adenotonsillar hypertrophy (ATH) is a medical condition characterized by the enlargement or swelling of the tonsils. The role of allergy in ATH has not been persuasively evidenced. Therefore, we investigated the state of humoral immunity and the presence of specific immunoglobulin E (sIgE) in tissues and sera in children suffering from TH. METHODS: According to the skin prick test (SPT) result, 44 ATH children were divided into the atopic and non-atopic groups. The level of sIgE against 30 inhalants and food allergens in the sera and tissue homogenates was measured by a commercial allergy immunoblotting kit. In addition, we evaluated the following variables in both tonsillar tissue homogenates and serum: total IgE, IgA, IgM, IgG, and tissue eosinophil counts. RESULTS: Our results showed that 21 (47.7%) of patients with ATH were sensitized to at least one allergen in the adenotonsillar sample and/or sera. Only two patients were negative for sIgE in the atopic group, but in the non-atopic group, only one had positive sIgE results. In the atopic group, 19 (86.4%) patients had positive sIgE in tonsillar tissues, and 18 (81.8%) had sensitized serum. There were no statistical differences in the case of other antibodies except IgE levels between the two groups. The average eosinophilic count was significantly higher in atopic patients than in the non-atopic group. CONCLUSION: The results of this study support the role of allergy in the pathogenesis of ATH and confirmed local allergic inflammation in tonsillar tissue.

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.

Spatial profiling of chromatin accessibility in mouse and human tissues.

Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2-5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

Clinical and Pathological Implications of Increases in Tonsillar CD19+CD5+ B Cells, CD208+ Dendritic Cells, and IgA1-positive Cells of Immunoglobulin A Nephropathy.

OBJECTIVE: Several studies indicated that tonsillectomy can improve the prognosis of patients with immunoglobulin A nephropathy (IgAN). However, the relationship between tonsillar immunity and IgAN is still unclear. METHODS: A total of 14 IgAN patients were recruited in the current study from May 2015 to April 2016 in Tongji Hospital. B cells, dendritic cells (DCs), and IgA1 positive cells in human tonsils were detected using immunofluorescence and immunohistochemistry. Correlations between these cells and clinicopathologic features were evaluated. RESULTS: CD19+CD5+ B cells were predominantly located in germinal centers and mantle zones of lymphoid follicles, the CD208+ DCs were distributed in the interfollicular and subepithelial area, and IgA1-positive cells were predominantly detected in mantle zones of lymphoid follicles and subepithelial tissues. The numbers of CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues from IgAN patients were significantly higher than those in the normal controls (P<0.01, respectively). CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues were significantly associated with 24-h proteinuria levels and tubular atrophy/interstitial fibrosis of IgAN. CONCLUSION: CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues might be involved in the pathogenesis of IgAN.

Small centers of defense.

Deciphering immune responses to viruses and vaccines using human tonsil organoids.

Role of Palatine Tonsil and Epipharyngeal Lymphoid Tissue in the Development of Glomerular Active Lesions (Glomerular vasculitis) in Immunoglobulin A Nephropathy.

Hematuria is an essential symptom of immunoglobulin A nephropathy (IgAN). Although the etiology of hematuria in IgAN has not been fully elucidated, it is thought that the rupture of the glomerular basement membranes caused by intra-capillary leukocyte influx, so-called glomerular vasculitis, is the pathological condition responsible for severe hematuria. Glomerular vasculitis are active lesions that exist in the glomeruli of acute phase IgAN and it is important because it is suspected to make the transition to segmental glomerular sclerosis (SGS) as a repair scar lesion in the chronic phase, and the progression of SGS would eventually lead to glomerular obsolescence. Worsening of hematuria concomitant with acute pharyngitis is common in patients with IgAN; therefore, elucidating the relationship between the immune system of Waldeyer's ring, including the palatine tonsil and epipharyngeal lymphoid tissue, and the glomerular vasculitis may lead to understanding the nature of IgAN. The epipharynx is an immunologically activated site even under normal conditions, and enhanced activation of innate immunity is likely to occur in response to airborne infection. Hyperactivation of innate immunity via upregulation of Toll-like receptors in the interfollicular area of the palatine tonsil and epipharyngeal lymphoid tissue, followed by enhanced fractalkine/CX3CR1 interactions, appears to play an important role in the development of glomerular vasculitis in IgAN. As latent but significant epipharyngitis is present in most patients with IgAN, it is plausible that acute upper respiratory infection may contribute as a trigger for the innate epipharyngeal immune system, which is already upregulated in a chronically inflamed environment. Given that epipharyngitis and its effects on IgAN are not fully understood, we propose that the so-called "epipharynx-kidney axis" may provide an important focus for future research.

The Tonsil Lymphocyte Landscape in Pediatric Tonsil Hyperplasia and Obstructive Sleep Apnea.

Tonsil hyperplasia is the most common cause of pediatric obstructive sleep apnea (OSA). Despite the growing knowledge in tissue immunology of tonsils, the immunopathology driving tonsil hyperplasia and OSA remains unknown. Here we used multi-parametric flow cytometry to analyze the composition and phenotype of tonsillar innate lymphoid cells (ILCs), T cells, and B cells from pediatric patients with OSA, who had previous polysomnography. Unbiased clustering analysis was used to delineate and compare lymphocyte heterogeneity between two patient groups: children with small tonsils and moderate OSA (n = 6) or large tonsils and very severe OSA (n = 13). We detected disturbed ILC and B cell proportions in patients with large tonsils, characterized by an increase in the frequency of naïve CD27-CD21hi B cells and a relative reduction of ILCs. The enrichment of naïve B cells was not commensurate with elevated Ki67 expression, suggesting defective differentiation and/or migration rather than cellular proliferation to be the causative mechanism. Finally, yet importantly, we provide the flow cytometry data to be used as a resource for additional translational studies aimed at investigating the immunological mechanisms of pediatric tonsil hyperplasia and OSA.

Integrated single-cell transcriptomics and epigenomics reveals strong germinal center-associated etiology of autoimmune risk loci.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type­specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, and BCL6) and transcription factors regulating B cell differentiation (POU2AF1 and HHEX). Together, these analyses provide a powerful new cell type­resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Polarized Secretion of APRIL by the Tonsil Epithelium Upon Toll-Like Receptor Stimulation.

In mucosa such as tonsil, antibody-producing plasmocytes (PCs) lie in sub-epithelium space, which is thought to provide a suitable environment for their survival. A proliferation inducing ligand (APRIL) is one key survival factor for PCs present in this area. According to in situ staining, apical epithelial cells produced APRIL, and the secreted product had to migrate all through the stratified surface epithelium to reach basal cells. A similar process also occurred in the less-organized crypt epithelium. Tonsil epithelial cells captured secreted APRIL, thanks to their surface expression of the APRIL coreceptor, either syndecan-1 or -4 depending on their differentiation stage. In the most basal epithelial cells, secreted APRIL accumulated inside secretory lamp-1+ vesicles in a polarized manner, facing the sub-epithelium. The tonsil epithelium upregulated APRIL production by apical cells and secretion by basal cells upon Toll-like receptor stimulation. Furthermore, LPS-stimulated epithelial cells sustained in vitro PC survival in a secreted APRIL-dependent manner. Taken together, our study shows that the tonsil epithelium responds to pathogen sensing by a polarized secretion of APRIL in the sub-epithelial space, wherein PCs reside.