Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals.

Secretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM+ plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM+ B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA+ B cells, memory IgM+ B cells were related to some IgA+ clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM+ B cells and could help SIgA to anchor highly diverse commensal communities to mucus.

Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas).

BACKGROUND: House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. OBJECTIVES: To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. METHODS: Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. RESULTS: The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). A positive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CONCLUSIONS: CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.

Chromatin architecture and the generation of antigen receptor diversity.

The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here, we review recently uncovered mechanisms that underpin long-range genomic interactions and the generation of antigen receptor diversity.

Combined immunodeficiency and impaired PI3K signaling in a patient with biallelic LCP2 variants.

BACKGROUND: Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T- and B-cell defects. OBJECTIVE: We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. METHODS: The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. RESULTS: Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. CONCLUSIONS: Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies.

BACKGROUND: TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. OBJECTIVE: We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. METHODS: Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. RESULTS: Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating B cells but overall normal humoral immune responses. CONCLUSION: Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

COVID-19 Adenoviral Vector Vaccination Elicits a Robust Memory B Cell Response with the Capacity to Recognize Omicron BA.2 and BA.5 Variants.

Following the COVID-19 pandemic, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the SARS-CoV-2 Spike receptor-binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response to mRNA BNT162b2 (Pfizer-BioNTech) vaccination. Whole blood was sampled from 31 healthy adults pre-vaccination and 4 weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each time point. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1 and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.

Clinical and Genetic Characterization of Patients with Artemis Deficiency in Japan.

PURPOSE: Artemis is an exonuclease essential for V(D)J recombination and repair of DNA double-stranded breaks. Pathogenic variants in DCLRE1C encoding Artemis cause T-B-NK+ severe combined immunodeficiency (SCID), and patients with Artemis-deficient SCID (ART-SCID) require definitive therapy with allogeneic hematopoietic cell transplantation (HCT). Here we describe the clinical and genetic characteristics of patients with ART-SCID who were diagnosed in Japan from 2003 to 2022. METHODS: Clinical data of ART-SCID patients who were diagnosed between 2003 and 2022 in Japan were collected from their physicians using a questionnaire. RESULTS: ART-SCID diagnosis was made in eight patients from seven families with severe infections within 6 months of life. Two patients had missense variants, five patients had large genomic deletions, and one patient was compound heterozygous for a missense variant and large genomic deletion. All eight underwent allogeneic HCT within 4 months after the diagnosis, 7 receiving a conditioning regimen containing alkylating agents, and one patient without conditioning due to uncontrolled infection. Two patients with poor performance status (PS) died of complications 410 days and 32 days post-HCT, respectively. Of the six surviving patients with a median follow-up time of 8.3 (0.5-17.9) years, three patients had growth retardation. The patients with PS of 0-2 showed a tendency for better overall survival than those with PS 3-4. CONCLUSION: Large deletions were the most common genetic cause of ART-SCID in Japan. To improve HCT outcome, early diagnosis with newborn screening for SCID is urgently needed.

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.

Multiple sclerosis risk variants influence the peripheral B-cell compartment early in life in the general population.

BACKGROUND AND PURPOSE: Multiple sclerosis (MS) is associated with abnormal B-cell function, and MS genetic risk alleles affect multiple genes that are expressed in B cells. However, how these genetic variants impact the B-cell compartment in early childhood is unclear. In the current study, we aim to assess whether polygenic risk scores (PRSs) for MS are associated with changes in the blood B-cell compartment in children from the general population. METHODS: Six-year-old children from the population-based Generation R Study were included. Genotype data were used to calculate MS-PRSs and B-cell subset-enriched MS-PRSs, established by designating risk loci based on expression and function. Analyses of variance were performed to examine the effect of MS-PRSs on total B-cell numbers (n = 1261) as well as naive and memory subsets (n = 675). RESULTS: After correction for multiple testing, no significant associations were observed between MS-PRSs and total B-cell numbers and frequencies of subsets therein. A naive B-cell-MS-PRS (n = 26 variants) was significantly associated with lower relative, but not absolute, naive B-cell numbers (p = 1.03 × 10-4 and p = 0.82, respectively), and higher frequencies and absolute numbers of CD27+ memory B cells (p = 8.83 × 10-4 and p = 4.89 × 10-3 , respectively). These associations remained significant after adjustment for Epstein-Barr virus seropositivity and the HLA-DRB1*15:01 genotype. CONCLUSIONS: The composition of the blood B-cell compartment is associated with specific naive B-cell-associated MS risk variants during childhood, possibly contributing to MS pathophysiology later in life. Cell subset-specific PRSs may offer a more sensitive tool to define the impact of genetic risk on the immune system in diseases such as MS.

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions.

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

Anti-tissue transglutaminase antibodies (TG2A) positivity and the risk of vitamin D deficiency among children - a cross-sectional study in the generation R cohort.

BACKGROUND: Suboptimal vitamin D status is common in people with celiac disease (CeD), a disease that can be characterized by the presence of serum anti-tissue transglutaminase antibodies (TG2A) (i.e., TG2A positivity). To date, it remains unclear whether childhood TG2A positivity is associated with vitamin D status and how this potential association can be explained by other factors than malabsorption only, since vitamin D is mainly derived from exposure to sunlight. The aim of our study was therefore to assess whether childhood TG2A positivity is associated with vitamin D concentrations, and if so, to what extent this association can be explained by sociodemographic and lifestyle factors. METHODS: This cross-sectional study was embedded in the Generation R Study, a population-based prospective cohort. We measured serum anti-tissue transglutaminase antibodies (TG2A) concentrations and serum 25-hydroxyvitamin D (25(OH)D) concentrations of 3994 children (median age of 5.9 years). Children with serum TG2A concentrations ≥ 7 U/mL were considered TG2A positive. To examine associations between TG2A positivity and 25(OH)D concentrations, we performed multivariable linear regression, adjusted for sociodemographic and lifestyle factors. RESULTS: Vitamin D deficiency (serum 25(OH)D < 50 nmol/L) was found in 17 out of 54 TG2A positive children (31.5%), as compared to 1182 out of 3940 TG2A negative children (30.0%). Furthermore, TG2A positivity was not associated with 25(OH)D concentrations (ß -2.20; 95% CI -9.72;5.33 for TG2A positive vs. TG2A negative children), and this did not change after adjustment for confounders (ß -1.73, 95% CI -8.31;4.85). CONCLUSIONS: Our findings suggest there is no association between TG2A positivity and suboptimal vitamin D status in the general pediatric population. However, the overall prevalence of vitamin D deficiency in both populations was high, suggesting that screening for vitamin D deficiency among children, regardless of TG2A positivity, would be beneficial to ensure early dietary intervention if needed.

Altered leukocyte subsets and immune proteome indicate proinflammatory mechanisms in mastocytosis.

BACKGROUND: Indolent systemic mastocytosis (ISM) is characterized by pathologic accumulation of mast cells. The mechanism behind its phenotypic heterogeneity is not well understood. Interaction of mast cells with other immune cells might cause systemic inflammation and thereby associated symptoms. OBJECTIVE: We investigated peripheral leukocyte compartments and serum immune proteome in ISM. METHODS: Peripheral blood leukocyte phenotyping using flow cytometry in a cohort of 18 adults with ISM and 12 healthy controls. Targeted proteomics was performed to measure 169 proteins associated with inflammation on serum of another 20 ISM patients and 20 healthy controls. RESULTS: Proportions of plasmacytoid dendritic cells and monocytes were significantly decreased while TH2 cells were increased in peripheral blood of ISM patients. Furthermore, a shift from naive to memory T cells was observed. Hierarchical clustering of the serum proteome revealed 2 distinct subgroups within ISM patients. In subgroup A (n = 8), 62 proteins were significantly overexpressed, whereas those of subgroup B (n = 12) were comparable to healthy controls. Patients in subgroup A displayed upregulated signaling pathways downstream of Toll-like receptor 4, TNF-α, and IFN-γ. Fatigue was more often present in subgroup A compared to B (75% vs 33% respectively, P = .06). CONCLUSIONS: Altered distribution of leukocyte subsets and a proinflammatory proteome were observed in subsequent 2 cohorts of ISM patients. We hypothesize that neoplastic mast cells recruit and activate plasmacytoid dendritic cells, monocytes, and T cells, leading to a vicious cycle of inflammation.

Recurrent tandem duplication of UNC13D in familial hemophagocytic lymphohistiocytosis type 3.

Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes Munc13-4. One published case was reported to carry a tandem duplication of UNC13D exons 7-12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.

The association of Epstein-Barr virus infection with CXCR3+ B-cell development in multiple sclerosis: impact of immunotherapies.

Epstein-Barr virus (EBV) infection of B cells is associated with increased multiple sclerosis (MS) susceptibility. Recently, we found that CXCR3-expressing B cells preferentially infiltrate the CNS of MS patients. In chronic virus-infected mice, these types of B cells are sustained and show increased antiviral responsiveness. How EBV persistence in B cells influences their development remains unclear. First, we analyzed ex vivo B-cell subsets from MS patients who received autologous bone marrow transplantation (n = 9), which is often accompanied by EBV reactivation. The frequencies of nonclass-switched and class-switched memory B cells were reduced at 3-7 months, while only class-switched B cells returned back to baseline at 24-36 months posttransplantation. At these time points, EBV DNA load positively correlated to the frequency of CXCR3+ , and not CXCR4+ or CXCR5+ , class-switched B cells. Second, for CXCR3+ memory B cells trapped within the blood of MS patients treated with natalizumab (anti-VLA-4 antibody n = 15), latent EBV infection corresponded to enhanced in vitro formation of anti-EBNA1 IgG-secreting plasma cells under GC-like conditions. These findings imply that EBV persistence in B cells potentiates brain-homing and antibody-producing CXCR3+ subsets in MS.

The benefit of boosters: diversity and inclusion in the COVID-19 memory response.

In a new study, a group evaluate immune responses against SARS-CoV-2 in vaccinated individuals and find evidence of durable immune memory for at least 6 months, irrespective of former infection.

Humoral immunity and B-cell memory in response to SARS-CoV-2 infection and vaccination.

Natural infection with SARS-CoV-2 induces a robust circulating memory B cell (Bmem) population, which remains stable in number at least 8 months post-infection despite the contraction of antibody levels after 1 month. Multiple vaccines have been developed to combat the virus. These include two new formulations, mRNA and adenoviral vector vaccines, which have varying efficacy rates, potentially related to their distinct capacities to induce humoral immune responses. The mRNA vaccines BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) elicit significantly higher serum IgG and neutralizing antibody levels than the adenoviral vector ChAdOx1 (AstraZeneca) and Ad26.COV2.S (Janssen) vaccines. However, all vaccines induce Spike- and RBD-specific Bmem, which are vital in providing long-lasting protection in the form of rapid recall responses to subsequent infections. Past and current SARS-CoV-2 variants of concern (VoC) have shown the capacity to escape antibody neutralization to varying degrees. A booster dose with an mRNA vaccine following primary vaccination restores antibody levels and improves the capacity of these antibodies and Bmem to bind viral variants, including the current VoC Omicron. Future experimental research will be essential to evaluate the durability of protection against VoC provided by each vaccine and to identify immune markers of protection to enable prognostication of people who are at risk of severe complications from COVID-19.

New insights into human immune memory from SARS-CoV-2 infection and vaccination.

Since early 2020, the world has been embroiled in an ongoing viral pandemic with SARS-CoV-2 and emerging variants resulting in mass morbidity and an estimated 6 million deaths globally. The scientific community pivoted rapidly, providing unique and innovative means to identify infected individuals, technologies to evaluate immune responses to infection and vaccination, and new therapeutic strategies to treat infected individuals. Never before has immunology been so critically at the forefront of combatting a global pandemic. It has now become evident that not just antibody responses, but formation and durability of immune memory cells following vaccination are associated with protection against severe disease from SARS-CoV-2 infection. Furthermore, the emergence of variants of concern (VoC) highlight the need for immunological markers to quantify the protective capacity of Wuhan-based vaccines. Thus, harnessing and modulating the immune response is key to successful vaccination and treatment of disease. We here review the latest knowledge about immune memory generation and durability following natural infection and vaccination, and provide insights into the attributes of immune memory that may protect from emerging variants.

Coordinated IgG2 and IgE responses as a marker of allergen immunotherapy efficacy.

BACKGROUND: IgG2 responses are associated with repeated antigen exposure and display highly mutated variable domains. A recent study highlighted a role of IgG2+ memory B cells and allergen-specific IgG2 levels after a 3rd consecutive pre-seasonal sublingual allergen immunotherapy (AIT) with grass pollen tablet. Herein, we aim to explore changes in allergen-specific IgG2 in individuals undergoing house dust mite immunotherapy (HDM-AIT) and explore whether the interrelationship with other humoral responses (i.e., IgG4 and IgE) may discriminate between high and low responders. METHODS: Levels of serum Dermatophagoides pteronyssinus and Dermatophagoides farinae-specific IgG2, IgG4, and IgE antibodies were measured by ELISA or ImmunoCap in a sub-group of individuals enrolled in a randomized, double-blind, placebo-controlled, sublingual AIT study evaluating the safety and efficacy of a 300 IR HDM tablet. RESULTS: After 1-year sublingual AIT, HDM-specific serum IgG2 responses increase mostly in high versus low responders and are distinctive according to the clinical benefit. Higher correlation between HDM-specific IgG2, IgE, and/or IgG4 responses is seen in subjects benefiting the most from HDM-AIT as indicated by changes in Average Total Combined Scores. More strikingly, statistically significant correlation between HDM-specific IgG2 and IgE responses is only observed in individuals stratified as high responders. CONCLUSIONS: We provide evidence for coordinated serum immune responses upon AIT in HDM-allergic subjects exhibiting high clinical benefit when compared with low responders. Assessing HDM-specific IgE, IgG2, and IgG4 in serum could be used as follow-up combined markers to support decision as to AIT continuation and/or adaptation.

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.

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.