Distinct microbial and immune niches of the human colon.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species.

Single cell sequencing data identify distinct B cell and fibroblast populations in stricturing Crohn's disease.

Single cell RNA sequencing of human full thickness Crohn's disease (CD) small bowel resection specimens was used to identify potential therapeutic targets for stricturing (S) CD. Using an unbiased approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient-matched SCD and non-stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican-positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C) 12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix and stricture organization associated with C12 and C9, and regulation of WNT pathway genes with C9. Trajectory and differential gene analysis of C12 and C9 identified four sub-clusters. Intra sub-cluster gene analysis detected 13 co-regulated gene modules that aligned along predicted pseudotime trajectories. CXCL14 and ADAMDEC1 were key markers in module 1. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.

Peanut diversity and specific activity are the dominant IgE characteristics for effector cell activation in children.

BACKGROUND: IgE mediates allergic reactions to peanut; however, peanut-specific IgE (sIgE) levels do not always equate to clinical peanut allergy. Qualitative differences between sIgE of peanut-sensitized but tolerant (PS) and peanut-allergic (PA) individuals may be important. OBJECTIVE: We sought to assess the influence of IgE characteristics on effector cell activation in peanut allergy. METHODS: A cohort of 100 children was studied. The levels of IgE to peanut and peanut components were measured. Specific activity (SA) was estimated as the ratio of allergen-sIgE to total IgE. Avidity was measured by ImmunoCAP with sodium thiocyanate. IgE diversity was calculated on the basis of ImmunoCAP-Immuno Solid-phase Allergen Chip assays for 112 allergens or for 6 peanut allergens. Whole-blood basophils and mast cell line Laboratory of Allergic Diseases 2 sensitized with patients' plasma were stimulated with peanut or controls and assessed by flow cytometry. RESULTS: SA to peanut (P < .001), Ara h 1 (P = .004), Ara h 2 (P < .001), Ara h 3 (P = .02), and Ara h 6 (P < .001) and the avidity of peanut-sIgE (P < .001) were higher in PA than in PS individuals. Diversity for peanut allergens was greater in PA individuals (P < .001). All IgE characteristics were correlated with basophil and mast cell activation. Peanut SA (R = 0.447) and peanut diversity (R = 0.440) had the highest standardized ß-coefficients in combined multivariable regression models (0.447 and 0.440, respectively). CONCLUSIONS: IgE specificity, SA, avidity, and peanut diversity were greater in PA than in PS individuals. IgE peanut SA and peanut diversity had the greatest influence on effector cell activation and could be used clinically.

Allergen-specific IgG show distinct patterns in persistent and transient food allergy.

BACKGROUND: Immediate food-allergic reactions are IgE-mediated, but many individuals with detectable allergen-specific IgE do not react to the food. Allergen-specific IgG may interfere with allergen-IgE interaction and/or through intracellular inhibitory signalling to suppress mast cell and basophil response to food allergens. We aimed to understand the role of allergen-specific IgG in food allergy and natural tolerance. METHODS: IgG and IgG isotypes specific to peanut, cow's milk and egg were measured using ImmunoCAP and ELISA respectively in samples of children with suspected food allergies. Expression of IgE and IgG and their receptors and expression of activation markers following allergen stimulation were measured on basophils and mast cells by flow cytometry, with and without blockade of FcγRIIα or FcγRIIß receptors. RESULTS: The levels of peanut-specific IgG, IgG1, IgG2, IgG3 and IgG4 in ELISA were higher in peanut-allergic than in non-peanut-allergic children. No difference in allergen-specific IgG isotypes was observed between allergic and non-allergic children to milk or egg, except for milk-specific IgG4 that was higher in non-cow's milk-allergic than in cow's milk-allergic children. Basophils and LAD2 cells expressed IgG receptors, but IgG and IgA were not detected on the surface of either cell type and blocking FcγRIIα or FcγRIIß did not modify basophil or mast cell activation in response to allergen in allergic or tolerant children. CONCLUSION: Allergen-specific IgG patterns were distinct in persistent (peanut) versus transient (milk and egg) food allergies. We found no evidence that FcγRIIα or FcγRIIß receptors affect allergen-induced activation of mast cells and basophils in food allergy or natural tolerance.

Interplay between Affinity and Valency in Effector Cell Degranulation: A Model System with Polcalcin Allergens and Human Patient-Derived IgE Antibodies.

An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor FcεRI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.

Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition.

Antibodies classically bind antigens via their complementarity-determining regions, but an alternative mode of interaction involving V-domain framework regions has been observed for some B cell "superantigens." We report the crystal structure of an antibody employing both modes of interaction simultaneously and binding two antigen molecules. This human antibody from an allergic individual binds to the grass pollen allergen Phl p 7. Not only are two allergen molecules bound to each antibody fragment (Fab) but also each allergen molecule is bound by two Fabs: One epitope is recognized classically, the other in a superantigen-like manner. A single allergen molecule thus cross-links two identical Fabs, contrary to the one-antibody-one-epitope dogma, which dictates that a dimeric allergen at least is required for this to occur. Allergens trigger immediate hypersensitivity reactions by cross-linking receptor-bound IgE molecules on effector cells. We found that monomeric Phl p 7 induced degranulation of basophils sensitized solely with this monoclonal antibody expressed as an IgE, demonstrating that the dual specificity has functional consequences. The monomeric state of Phl p 7 and two structurally related allergens was confirmed by size-exclusion chromatography and multiangle laser light scattering, and the results were supported by degranulation studies with the related allergens, a second patient-derived allergen-specific antibody lacking the nonclassical binding site, and mutagenesis of the nonclassically recognized allergen epitope. The antibody dual reactivity and cross-linking mechanism not only have implications for understanding allergenicity and allergen potency but, importantly, also have broader relevance to antigen recognition by membrane Ig and cross-linking of the B cell receptor.

Antibodies and superantibodies in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.

The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy.

Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance.

Potential Mechanisms for IgG4 Inhibition of Immediate Hypersensitivity Reactions.

IgG4 is the least abundant IgG subclass in human serum, representing less than 5% of all IgG. Increases in IgG4 occur following chronic exposure to antigen and are generally associated with states of immune tolerance. In line with this, IgG4 is regarded as an anti-inflammatory antibody with a limited ability to elicit effective immune responses. Furthermore, IgG4 attenuates allergic responses by inhibiting the activity of IgE. The mechanism by which IgG4 inhibits IgE-mediated hypersensitivity has been investigated using a variety of model systems leading to two proposed mechanisms. First by sequestering antigen, IgG4 can function as a blocking antibody, preventing cross-linking of receptor bound IgE. Second IgG4 has been proposed to co-stimulate the inhibitory IgG receptor FcγRIIb, which can negatively regulate FcεRI signaling and in turn inhibit effector cell activation. Recent advances in our understanding of the structural features of human IgG4 have shed light on the unique functional and immunologic properties of IgG4. The aim of this review is to evaluate our current understanding of IgG4 biology and reassess the mechanisms by which IgG4 functions to inhibit IgE-mediated allergic responses.

IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.

BACKGROUND: Most children with detectable peanut-specific IgE (P-sIgE) are not allergic to peanut. We addressed 2 non-mutually exclusive hypotheses for the discrepancy between allergy and sensitization: (1) differences in P-sIgE levels between children with peanut allergy (PA) and peanut-sensitized but tolerant (PS) children and (2) the presence of an IgE inhibitor, such as peanut-specific IgG4 (P-sIgG4), in PS patients. METHODS: Two hundred twenty-eight children (108 patients with PA, 77 PS patients, and 43 nonsensitized nonallergic subjects) were studied. Levels of specific IgE and IgG4 to peanut and its components were determined. IgE-stripped basophils or a mast cell line were used in passive sensitization activation and inhibition assays. Plasma of PS subjects and patients submitted to peanut oral immunotherapy (POIT) were depleted of IgG4 and retested in inhibition assays. RESULTS: Basophils and mast cells sensitized with plasma from patients with PA but not PS patients showed dose-dependent activation in response to peanut. Levels of sIgE to peanut and its components could only partially explain differences in clinical reactivity between patients with PA and PS patients. P-sIgG4 levels (P = .023) and P-sIgG4/P-sIgE (P < .001), Ara h 1-sIgG4/Ara h 1-sIgE (P = .050), Ara h 2-sIgG4/Ara h 2-sIgE (P = .004), and Ara h 3-sIgG4/Ara h 3-sIgE (P = .016) ratios were greater in PS children compared with those in children with PA. Peanut-induced activation was inhibited in the presence of plasma from PS children with detectable P-sIgG4 levels and POIT but not from nonsensitized nonallergic children. Depletion of IgG4 from plasma of children with PS (and POIT) sensitized to Ara h 1 to Ara h 3 partially restored peanut-induced mast cell activation (P = .007). CONCLUSIONS: Differences in sIgE levels and allergen specificity could not justify the clinical phenotype in all children with PA and PS children. Blocking IgG4 antibodies provide an additional explanation for the absence of clinical reactivity in PS patients sensitized to major peanut allergens.

Influence of seasonal exposure to grass pollen on local and peripheral blood IgE repertoires in patients with allergic rhinitis.

BACKGROUND: Previous studies of immunoglobulin gene sequences in patients with allergic diseases using low-throughput Sanger sequencing have limited the analytic depth for characterization of IgE repertoires. OBJECTIVES: We used a high-throughput, next-generation sequencing approach to characterize immunoglobulin heavy-chain gene (IGH) repertoires in patients with seasonal allergic rhinitis (AR) with the aim of better understanding the underlying disease mechanisms. METHODS: IGH sequences in matched peripheral blood and nasal biopsy specimens from nonallergic healthy control subjects (n = 3) and patients with grass pollen-related AR taken in season (n = 3) or out of season (n = 4) were amplified and pyrosequenced on the 454 GS FLX+ System. RESULTS: A total of 97,610 IGH (including 8,135 IgE) sequences were analyzed. Use of immunoglobulin heavy-chain variable region gene families 1 (IGHV1) and 5 (IGHV5) was higher in IgE clonotypic repertoires compared with other antibody classes independent of atopic status. IgE repertoires measured inside the grass pollen season were more diverse and more mutated (particularly in the biopsy specimens) and had more evidence of antigen-driven selection compared with those taken outside of the pollen season or from healthy control subjects. Clonal relatedness was observed for IgE between the blood and nasal biopsy specimens. Furthermore in patients with AR, but not healthy control subjects, we found clonal relatedness between IgE and IgG classes. CONCLUSION: This is the first report that exploits next-generation sequencing to determine local and peripheral blood IGH repertoires in patients with respiratory allergic disease. We demonstrate that natural pollen exposure was associated with changes in IgE repertoires that were suggestive of ongoing germinal center reactions. Furthermore, these changes were more often apparent in nasal biopsy specimens compared with peripheral blood and in patients with AR compared with healthy control subjects.

Chronic rhinosinusitis with nasal polyps: eosinophils versus B lymphocytes in disease pathogenesis.

PURPOSE OF REVIEW: To highlight the current evidence that supports the view that eosinophils may not drive disease in chronic rhinosinusitis with nasal polyps (CRSwNP) and the emerging evidence for B cells as an important player in this disease. RECENT FINDINGS: Eosinophil depletion studies in CRSwNP do not fully support a critical role for eosinophils in CRSwNP. Almost complete eosinophil depletion with dexpramipexole had no impact on polyp size reduction or clinical improvement. Anti-interleukin (IL)-5 and IL-5Rα inhibition were more effective though with less clinical impact when compared to anti-immunoglobulin E (IgE) or IL-4Rα inhibition strategies. As IL-5Rα is also expressed on CRSwNP derived IgE+ and IgG4+ plasma cells to the same extent as eosinophils, improvements in CRSwNP with IL-5 inhibition may suggest a role for B cells over eosinophils in CRSwNP. We review both eosinophils and B cells in the context of CRSwNP and highlight the current evidence that supports an emerging role for B cells. SUMMARY: Despite many aspects of immunopathology in CRSwNP explainable by B cell dysfunction, B cells have so far been ignored in CRSwNP. Further work is needed, as targeting B cells may offer an exciting new therapeutic option in the future.

Decoding the language of immunity.

Optimized transfer RNA (tRNA) codon use can speed up antibody generation.

Allergen specificity of IgG(4)-expressing B cells in patients with grass pollen allergy undergoing immunotherapy.

BACKGROUND: Serum IgG(4) responses to allergen immunotherapy are well documented as blocking allergen binding to receptor-bound IgE on antigen-presenting cells and effector cells, but the molecular characteristics of treatment-induced IgG(4), particularly in relation to expressed antibody, are poorly defined. OBJECTIVES: We aimed to clone and express recombinant IgG(4) from patients receiving grass pollen immunotherapy using single B cells to obtain matched heavy- and light-chain pairs. METHODS: IgG(4)(+) B cells were enriched from blood samples taken from 5 patients receiving grass pollen immunotherapy. Matched heavy- and light-chain variable-region sequences were amplified from single IgG(4)(+) B cells. Variable regions were cloned and expressed as recombinant IgG(4). Binding analysis of grass pollen-specific IgG(4) was performed by using surface plasmon resonance. Functional assays were used to determine IgE blocking activity. In a separate experiment grass pollen-specific antibodies were depleted from serum samples to determine the proportion of grass pollen-specific IgG(4) within total IgG(4). RESULTS: Depletion of grass pollen-specific antibodies from serum led to a modest reduction in total IgG(4) levels. Matched heavy- and light-chain sequences were cloned from single IgG(4)(+) B cells and expressed as recombinant IgG(4). We identified an IgG(4) that binds with extremely high affinity to the grass pollen allergen Phl p 7. Furthermore, we found that a single specific mAb can block IgE-mediated facilitated allergen presentation, as well as IgE-mediated basophil activation. CONCLUSION: Although increases in IgG(4) levels cannot be wholly accounted for within the allergen-specific fraction, allergen immunotherapy might result in the production of high-affinity allergen-specific blocking IgG(4).

The impact of sphingosine-1-phosphate receptor modulators on COVID-19 and SARS-CoV-2 vaccination.

BACKGROUND: Sphingosine-one phosphate receptor (S1PR) modulation inhibits S1PR1-mediated lymphocyte migration, lesion formation and positively-impacts on active multiple sclerosis (MS). These S1PR modulatory drugs have different: European Union use restrictions, pharmacokinetics, metabolic profiles and S1PR receptor affinities that may impact MS-management. Importantly, these confer useful properties in dealing with COVID-19, anti-viral drug responses and generating SARS-CoV-2 vaccine responses. OBJECTIVE: To examine the biology and emerging data that potentially underpins immunity to the SARS-CoV-2 virus following natural infection and vaccination and determine how this impinges on the use of current sphingosine-one-phosphate modulators used in the treatment of MS. METHODS: A literature review was performed, and data on infection, vaccination responses; S1PR distribution and functional activity was extracted from regulatory and academic information within the public domain. OBSERVATIONS: Most COVID-19 related information relates to the use of fingolimod. This indicates that continuous S1PR1, S1PR3, S1PR4 and S1PR5 modulation is not associated with a worse prognosis following SARS-CoV-2 infection. Whilst fingolimod use is associated with blunted seroconversion and reduced peripheral T-cell vaccine responses, it appears that people on siponimod, ozanimod and ponesimod exhibit stronger vaccine-responses, which could be related notably to a limited impact on S1PR4 activity. Whilst it is thought that S1PR3 controls B cell function in addition to actions by S1PR1 and S1PR2, this may be species-related effect in rodents that is not yet substantiated in humans, as seen with bradycardia issues. Blunted antibody responses can be related to actions on B and T-cell subsets, germinal centre function and innate-immune biology. Although S1P1R-related functions are seeming central to control of MS and the generation of a fully functional vaccination response; the relative lack of influence on S1PR4-mediated actions on dendritic cells may increase the rate of vaccine-induced seroconversion with the newer generation of S1PR modulators and improve the risk-benefit balance IMPLICATIONS: Although fingolimod is a useful asset in controlling MS, recently-approved S1PR modulators may have beneficial biology related to pharmacokinetics, metabolism and more-restricted targeting that make it easier to generate infection-control and effective anti-viral responses to SARS-COV-2 and other pathogens. Further studies are warranted.

The role of Raptor in lymphocytes differentiation and function.

Raptor, a key component of mTORC1, is required for recruiting substrates to mTORC1 and contributing to its subcellular localization. Raptor has a highly conserved N-terminus domain and seven WD40 repeats, which interact with mTOR and other mTORC1-related proteins. mTORC1 participates in various cellular events and mediates differentiation and metabolism. Directly or indirectly, many factors mediate the differentiation and function of lymphocytes that is essential for immunity. In this review, we summarize the role of Raptor in lymphocytes differentiation and function, whereby Raptor mediates the secretion of cytokines to induce early lymphocyte metabolism, development, proliferation and migration. Additionally, Raptor regulates the function of lymphocytes by regulating their steady-state maintenance and activation.