RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect.

BACKGROUND: Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG4 . IgG4 competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy. METHODS: Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort. RESULTS: Four months of SLIT increased RGP-specific IgE and IgG4 in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG4 ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1+ Bmem expressing surface IgG4 , CD23, and CD29 after SLIT. CONCLUSIONS: A clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation.

The proliferation and differentiation of antigen-specific B cells, including the generation of germinal centers (GC), are prerequisites for long-lasting, antibody-mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell-derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL-21-mediated promotion of plasma cell differentiation. Collectively, our data establish that IL-21 acts from the outset of a T cell-dependent immune response to increase cell cycle progression and fuel cyclic re-entry of B cells, thereby regulating the initial GC size and early plasma cell output.

The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21.

Humoral immune responses require germinal centres (GC) for antibody affinity maturation. Within GC, B cell proliferation and mutation are segregated from affinity-based positive selection in the dark zone (DZ) and light zone (LZ) substructures, respectively. While IL-21 is known to be important in affinity maturation and GC maintenance, here we show it is required for both establishing normal zone representation and preventing the accumulation of cells in the G1 cell cycle stage in the GC LZ. Cell cycle progression of DZ B cells is unaffected by IL-21 availability, as is the zone phenotype of the most highly proliferative GC B cells. Collectively, this study characterises the development of GC zones as a function of time and B cell proliferation and identifies IL-21 as an important regulator of these processes. These data help explain the requirement for IL-21 in normal antibody affinity maturation.

Advances in allergen-specific immune cell measurements for improved detection of allergic sensitization and immunotherapy responses.

Over the past two decades, precision medicine has advanced diagnostics and treatment of allergic diseases. Component-resolved analysis of allergen sensitization facilitates stratification of patients. Furthermore, new formulations of allergen immunotherapy (AIT) products can more effectively deliver the relevant components. Molecular insights from the identification of allergen component sensitization and clinical outcomes of treatment with new AIT formulations can now be utilized for a deeper understanding of the nature of the pathogenic immune response in allergy and how this can be corrected by AIT. Fundamental in these processes are the allergen-specific B and T cells. Within the large B- and T-cell compartments, only those that specifically recognize the allergen with their immunoglobulin (Ig) or T-cell receptor (TCR), respectively, are of clinical relevance. With peripheral blood allergen-specific B- and T-cell frequencies below 1%, bulk cell analysis is typically insufficiently sensitive. We here review the latest technologies to detect allergen-specific B and T cells, as well as new developments in utilizing these tools for diagnostics and therapy monitoring to advance precision medicine for allergic diseases.

pH and Proton Sensor GPR65 Determine Susceptibility to Atopic Dermatitis.

pH sensing by GPR65 regulates various inflammatory conditions, but its role in skin remains unknown. In this study, we performed a phenome-wide association study and report that the T allele of GPR65-intronic single-nucleotide polymorphism rs8005161, which reduces GPR65 signaling, showed a significant association with atopic dermatitis, in addition to inflammatory bowel diseases and asthma, as previously reported. Consistent with this genetic association in humans, we show that deficiency of GPR65 in mice resulted in markedly exacerbated disease in the MC903 experimental model of atopic dermatitis. Deficiency of GPR65 also increased neutrophil migration in vitro. Moreover, GPR65 deficiency in mice resulted in higher expression of the inflammatory cytokine TNF-α by T cells. In humans, CD4+ T cells from rs8005161 heterozygous individuals expressed higher levels of TNF-α after PMA/ionomycin stimulation, particularly under pH 6 conditions. pH sensing by GPR65 appears to be important for regulating the pathogenesis of atopic dermatitis.

CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers.

BACKGROUND: Diagnostic tests for allergy rely on detecting allergen-specific IgE. Component-resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care. OBJECTIVE: To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry. METHODS: Recombinant forms of Lol p 1 and Lol p 5 proteins from ryegrass pollen (RGP) and Api m 1 from honeybee venom (BV) were produced, biotinylated, and tetramerized with streptavidin-fluorochrome conjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic, and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation. RESULTS: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single-tube assay enabled rapid detection of sensitization to both Lol p 1 and Lol p 5 in RGP-allergic patients and discriminated between controls, BV-allergic, and RGP-allergic patients. CONCLUSION: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease, and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.

An acetate-yielding diet imprints an immune and anti-microbial programme against enteric infection.

OBJECTIVES: During gastrointestinal infection, dysbiosis can result in decreased production of microbially derived short-chain fatty acids (SCFAs). In response to the presence of intestinal pathogens, we examined whether an engineered acetate- or butyrate-releasing diet can rectify the deficiency of SCFAs and lead to the resolution of enteric infection. METHODS: We tested whether a high acetate- or butyrate-producing diet (HAMSA or HAMSB, respectively) condition Citrobacter rodentium infection in mice and assess its impact on host-microbiota interactions. We analysed the adaptive and innate immune responses, changes in gut microbiome function, epithelial barrier function and the molecular mechanism via metabolite sensing G protein-coupled receptor 43 (GPR43) and IL-22 expression. RESULTS: HAMSA diet rectified the deficiency in acetate production and protected against enteric infection. Increased SCFAs affect the expression of pathogen virulence genes. HAMSA diet promoted compositional and functional changes in the gut microbiota during infection similar to healthy microbiota from non-infected mice. Bacterial changes were evidenced by the production of proteins involved in acetate utilisation, starch and sugar degradation, amino acid biosynthesis, carbohydrate transport and metabolism. HAMSA diet also induced changes in host proteins critical in glycolysis, wound healing such as GPX1 and epithelial architecture such as EZR1 and PFN1. Dietary acetate assisted in rapid epithelial repair, as shown by increased colonic Muc-2, Il-22, and anti-microbial peptides. We found that acetate increased numbers of colonic IL-22 producing TCRαß+CD8αß+ and TCRγδ+CD8αα+ intraepithelial lymphocytes expressing GPR43. CONCLUSION: HAMSA diet may be an effective therapeutic approach for fighting inflammation and enteric infections and offer a safe alternative that may impact on human health.

Induction of IgG2 and IgG4 B-cell memory following sublingual immunotherapy for ryegrass pollen allergy.

BACKGROUND: While treatment for atopic rhinitis is aimed mostly to relieve symptoms, only allergen-specific immunotherapy (AIT) is targeted to modify the natural history of allergic diseases. This results in sustained clinical tolerance, even when treatment has stopped. The immunomodulatory effects of AIT are attributed mainly to increased regulatory T-cell function and increased allergen-specific IgG4 , yet little is known about the effect on the memory B-cell compartment. OBJECTIVE: We aimed to examine the effects of AIT on the IgE- and IgG subclass-expressing memory B cells. METHODS: We recruited 29 patients with atopic seasonal rhinoconjunctivitis and performed a longitudinal analysis of the peripheral immune compartment before, during, and after sublingual immunotherapy (SLIT) for allergy to temperate grass pollen, predominantly to ryegrass pollen (RGP; Lolium perenne). Using flow cytometry on peripheral blood mononuclear cells and serum immunoassays, we analyzed the effects of a 4 months preseasonal treatment regimen comprising two or three courses in consecutive years on circulating IgE+ and IgG+ memory B cells and allergen-specific Ig levels. RESULTS: SLIT increased RGP-specific serum IgG2 and IgG4 , as well as the frequencies of IgG2+ and IgG4+ memory B cells, whereas no effect was observed on the IgE+ memory B-cell compartment. Furthermore, SLIT enhanced proportions of regulatory T cells specific to RGP. These changes were associated with clinical improvement. CONCLUSION: Our data provide evidence for immunological effects of SLIT on B-cell memory. Skewing responses toward IgG2 and IgG4 subclasses might be a mechanism to suppress IgE-mediated allergic responses.

Recent developments and highlights in immune monitoring of allergen immunotherapy.

Allergic diseases are the most common chronic immune-mediated disorders and can manifest with an enormous diversity in clinical severity and symptoms. Underlying mechanisms for the adverse immune response to allergens and its downregulation by treatment are still being revealed. As a result, there have been, and still are, major challenges in diagnosis, prediction of disease progression/evolution and treatment. Currently, the only corrective treatment available is allergen immunotherapy (AIT). AIT modifies the immune response through long-term repeated exposure to defined doses of allergen. However, as the treatment usually needs to be continued for several years to be effective, and can be accompanied by adverse reactions, many patients face difficulties completing their schedule. Long-term therapy also potentially incurs high costs. Therefore, there is a great need for objective markers to predict or to monitor individual patient's beneficial changes in immune response during therapy so that efficacy can be identified as early as possible. In this review, we specifically address recent technical developments that have generated new insights into allergic disease pathogenesis, and how these could potentially be translated into routine laboratory assays for disease monitoring during AIT that are relatively inexpensive, robust and scalable.

Impaired memory B-cell development and antibody maturation with a skewing toward IgE in patients with STAT3 hyper-IgE syndrome.

BACKGROUND: Signal transducer and activator of transcription 3 hyper-IgE syndrome (STAT3-HIES) is caused by heterozygous mutations in the STAT3 gene and is associated with eczema, elevated serum IgE, and recurrent infections resembling severe atopic dermatitis, while clinically relevant specific IgE is almost absent. METHODS: To investigate the impact of STAT3 signaling on B-cell responses, we assessed lymph node and bone marrow, blood B and plasma cell subsets, somatic hypermutations in Ig genes, and in vitro proliferation and antibody production in STAT3-HIES patients and healthy controls. RESULTS: Lymph nodes of STAT3-HIES patients showed normal germinal center architecture and CD138+ plasma cells residing in the paracortex, which expressed IgE, IgG, and IgM but not IgA. IgE+ plasma cells were abundantly present in STAT3-HIES bone marrow. Proliferation of naive B cells upon stimulation with CD40L and IL-4 was similar in patients and controls, while patient cells showed reduced responses to IL-21. IgE, IgG1, IgG3 and IgA1 transcripts showed reduced somatic hypermutations. Peripheral blood IgE+ memory B-cell frequencies were increased in STAT3-HIES, while other memory B-cell frequencies except for IgG4+ cells were decreased. CONCLUSIONS: Despite impaired STAT3 signaling, STAT3-HIES patients can mount in vivo T-cell-dependent B-cell responses, while circulating memory B cells, except for those expressing IgG4 and IgE, were reduced. Reduced molecular maturation demonstrated the critical need of STAT3 signaling for optimal affinity maturation and B-cell differentiation, supporting the need for immunoglobulin substitution therapy and explaining the high IgE serum level in the majority with absent allergic symptoms.

Avenues to autoimmune arthritis triggered by diverse remote inflammatory challenges.

Environmental factors contribute to development of autoimmune diseases. For instance, human autoimmune arthritis can associate with intestinal inflammation, cigarette smoking, periodontal disease, and various infections. The cellular and, molecular pathways whereby such remote challenges might precipitate arthritis or flares remain unclear. Here, we used a transfer model of self-reactive arthritis-inducing CD4(+) cells from KRNtg mice that, upon transfer, induce a very mild form of autoinflammatory arthritis in recipient animals. This model enabled us to identify external factors that greatly aggravated disease. We show that several distinct challenges precipitated full-blown arthritis, including intestinal inflammation through DSS-induced colitis, and bronchial stress through Influenza infection. Both triggers induced strong IL-17 expression primarily in self-reactive CD4(+) cells in lymph nodes draining the site of inflammation. Moreover, treatment of mice with IL-1ß greatly exacerbated arthritis, while transfer of KRNtg CD4(+) cells lacking IL-1R significantly reduced disease and IL-17 expression. Thus, IL-1ß enhances the autoaggressive potential of self-reactive CD4(+) cells, through increased Th17 differentiation, and this influences inflammatory events in the joints. We propose that diverse challenges that cause remote inflammation (lung infection or colitis, etc.) result in IL-1ß-driven Th17 differentiation, and this precipitates arthritis in genetically susceptible individuals. Thus the etiology of autoimmune inflammatory arthritis likely relates to diverse triggers that converge to a common pathway involving IL-1ß production and Th17 cell distribution.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk.

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.

GPR43 - A Prototypic Metabolite Sensor Linking Metabolic and Inflammatory Diseases.

Short-chain fatty acids (SCFAs) are released upon fermentation of dietary fiber by gut bacteria. G protein-coupled receptor 43 (GPR43), a key receptor for SCFAs, is expressed on cell types important for immunity and metabolism. GPR43 modulates both inflammatory and metabolic processes, and is crucial for understanding the pathogenesis of 'Western lifestyle' diseases.

Evidence that asthma is a developmental origin disease influenced by maternal diet and bacterial metabolites.

Asthma is prevalent in Western countries, and recent explanations have evoked the actions of the gut microbiota. Here we show that feeding mice a high-fibre diet yields a distinctive gut microbiota, which increases the levels of the short-chain fatty acid, acetate. High-fibre or acetate-feeding led to marked suppression of allergic airways disease (AAD, a model for human asthma), by enhancing T-regulatory cell numbers and function. Acetate increases acetylation at the Foxp3 promoter, likely through HDAC9 inhibition. Epigenetic effects of fibre/acetate in adult mice led us to examine the influence of maternal intake of fibre/acetate. High-fibre/acetate feeding of pregnant mice imparts on their adult offspring an inability to develop robust AAD. High fibre/acetate suppresses expression of certain genes in the mouse fetal lung linked to both human asthma and mouse AAD. Thus, diet acting on the gut microbiota profoundly influences airway responses, and may represent an approach to prevent asthma, including during pregnancy.