Type 2-polarized memory B cells hold allergen-specific IgE memory.

Allergen-specific immunoglobulin E (IgE) antibodies mediate pathology in diseases such as allergic rhinitis and food allergy. Memory B cells (MBCs) contribute to circulating IgE by regenerating IgE-producing plasma cells upon allergen encounter. Here, we report a population of type 2-polarized MBCs defined as CD23hi, IL-4Rαhi, and CD32low at both the transcriptional and surface protein levels. These MBC2s are enriched in IgG1- and IgG4-expressing cells while constitutively expressing germline transcripts for IgE. Allergen-specific B cells from patients with allergic rhinitis and food allergy were enriched in MBC2s. Furthermore, MBC2s generated allergen-specific IgE during sublingual immunotherapy, thereby identifying these cells as a major reservoir for IgE. The identification of MBC2s provides insights into the maintenance of IgE memory, which is detrimental in allergic diseases but could be beneficial in protection against venoms and helminths.

Production and use of antigen tetramers to study antigen-specific B cells.

B cells generate antibodies that provide protection from infection, but also cause pathology in autoimmune and allergic conditions. Antigen-specific B cells can be detected by binding their surface antibody receptors with native antigens conjugated to fluorescent probes, a technique that has revealed substantial insight into B cell activation and function. This protocol describes the process of generating fluorescent antigen tetramer probes and delineates a process of enriching large samples based on antigen-specificity for high-resolution analyses of the antigen-specific B cell repertoire. Enrichment of tetramer-binding cells allows for detection of antigen-specific B cells as rare as 1 in 100 million cells, providing sufficient resolution to study naive B cells and IgE-expressing cells by flow cytometry. The generation of antigen tetramers involves antigen biotinylation, assessment of biotin:antigen ratio for optimal tetramer loading and polymerization around a streptavidin-fluorophore backbone. We also describe the construction of a control tetramer to exclude B cells binding to the tetramer backbone. We provide a framework to validate whether tetramer probes are detecting true antigen-specific B cells and discuss considerations for experimental design. This protocol can be performed by researchers trained in basic biomedical/immunological research techniques, using instrumentation commonly found in most laboratories. Constructing the antigen and control tetramers takes 9 h, though their specificity should be assessed before experimentation and may take weeks to months depending on the method of validation. Sample enrichment requires ~2 h but is generally time and cost neutral as fewer cells are run through the flow cytometer.

Heterogeneity, subsets, and plasticity of T follicular helper cells in allergy.

Antibody responses are critical for protection against pathogens. However, diseases such as allergic rhinitis or food allergy result from aberrant production of IgE antibodies against otherwise innocuous environmental antigens. The production of allergen-specific IgE requires interaction between B cells and CD4+ T cells, and a granular understanding of these interactions is required to develop novel therapies for allergic disease. CD4+ T cells are exceptionally heterogeneous in their transcriptional, epigenetic, and proteomic profiles, which poses significant challenges when attempting to define subsets relevant to the study of allergy among a continuum of cells. Defining subsets such as the T follicular helper (TFH) cell cluster provides a shorthand to understand the functions of CD4+ T cells in antibody production and supports mechanistic experimentation for hypothesis-driven discovery. With a focus on allergic disease, this Rostrum article broadly discusses heterogeneity among CD4+ T cells and provides a rationale for subdividing TFH cells into both functional and cytokine-skewed subsets. Further, it highlights the plasticity demonstrated by TFH cells during the primary response and after recall, and it explores the possibility of harnessing this plasticity to reprogram immunity for therapeutic benefit in allergic disease.

Peanut Oral Immunotherapy With or Without H1 and H2 Antihistamine Premedication for Peanut Allergy (PISCES): A Placebo-Controlled Randomized Clinical Trial.

BACKGROUND: Current forms of peanut oral immunotherapy (OIT) are associated with side effects, and there is a lack of evidence addressing how to mitigate them. OBJECTIVE: To determine whether premedication with desloratadine and ranitidine results in fewer side effects during peanut OIT/desensitization. METHODS: A total of 43 patients with peanut allergy (mean age, 7.6 ± 2.1 years, 37% females, 63% males, baseline eliciting dose, 33 ± 26 mg) were randomized to OIT with or without concomitant H1 and H2 antihistamine blockade, or double-placebo. Patients, study staff/investigators, and statisticians were blinded. The primary outcomes were the frequency and severity of OIT-induced adverse events. The secondary outcomes were quality of life and eliciting doses to blinded food challenge. RESULTS: Adverse reactions occurred more in the OIT groups compared with the double-placebo group (OIT with antihistamines vs double-placebo hazard ratio, 3.75 [95% CI, 2.79-4.72]; OIT with placebo antihistamines vs double-placebo, hazard ratio, 4.62 [95% CI, 3.61-5.62]). Patients given antihistamines cotreatment with OIT had a similar risk of adverse events compared with those who did not use antihistamines with OIT (hazard ratio, 1.23 [95% CI, 0.49-1.97]). OIT with and without antihistamines accelerated the incidence rate of adverse events compared with double-placebo (4.8 and 6.4 events per patient vs 3.5 per patient, incidence rate ratio, 2.49 [95% CI, 1.36-4.56] and 2.04 [95% CI, 1.01-4.15], respectively). Antihistamines pretreatment modestly reduced the frequency of moderate to severe adverse reactions among OIT-treated groups (1.9 per patient vs 4.2 per patient, incidence rate ratio, 0.46 [95% CI, 0.24-0.89]), primarily urticaria (0.6 vs 2.1 per patient) followed by abdominal pain (2.6 vs 4.2 per patient), but increased neuropsychiatric adverse events (primarily tiredness and sedation, 2.3 vs 0.7 per patient). Eliciting doses after treatment were similar in all groups. Quality of life improved similarly regardless of treatment with peanut OIT or placebo OIT. CONCLUSIONS: Peanut OIT with antihistamines modestly reduce the skin and gastrointestinal components of the high incidence of adverse reactions during OIT, and there are no clear differences in improvement in quality of life whether treated with OIT, OIT with antihistamines, or placebo OIT despite OIT being effective in inducing desensitization. Safer food allergy treatment approaches that importantly improve quality of life need to be proved in future robust randomized trials.

Type I interferon regulates proteolysis by macrophages to prevent immunopathology following viral infection.

The ability to treat severe viral infections is limited by our understanding of the mechanisms behind virus-induced immunopathology. While the role of type I interferons (IFNs) in early control of viral replication is clear, less is known about how IFNs can regulate the development of immunopathology and affect disease outcomes. Here, we report that absence of type I IFN receptor (IFNAR) is associated with extensive immunopathology following mucosal viral infection. This pathology occurred independent of viral load or type II immunity but required the presence of macrophages and IL-6. The depletion of macrophages and inhibition of IL-6 signaling significantly abrogated immunopathology. Tissue destruction was mediated by macrophage-derived matrix metalloproteinases (MMPs), as MMP inhibition by doxycycline and Ro 28-2653 reduced the severity of tissue pathology. Analysis of post-mortem COVID-19 patient lungs also displayed significant upregulation of the expression of MMPs and accumulation of macrophages. Overall, we demonstrate that IFNs inhibit macrophage-mediated MMP production to prevent virus-induced immunopathology and uncover MMPs as a therapeutic target towards viral infections.

The Road Toward Transformative Treatments for Food Allergy.

A series of landmark studies have provided conclusive evidence that the early administration of food allergens dramatically prevents the emergence of food allergy. One of the greatest remaining challenges is whether patients with established food allergy can return to health. This challenge is particularly pressing in the case of allergies against peanut, tree nuts, fish, and shellfish which are lifelong in most patients and may elicit severe reactions. The standard of care for food allergy is allergen avoidance and the timely administration of epinephrine upon accidental exposure. Epinephrine, and other therapeutic options like antihistamines provide acute symptom relief but do not target the underlying pathology of the disease. In principle, any transformative treatment for established food allergy would require the restoration of a homeostatic immunological state. This may be attained through either an active, non-harmful immune response (immunological tolerance) or a lack of a harmful immune response (e.g., anergy), such that subsequent exposures to the allergen do not elicit a clinical reaction. Importantly, such a state must persist beyond the course of the treatment and exert its protective effects permanently. In this review, we will discuss the immunological mechanisms that maintain lifelong food allergies and are, consequently, those which must be dismantled or reprogrammed to instate a clinically non-reactive state. Arguably, the restoration of such a state in the context of an established food allergy would require a reprogramming of the immune response against a given food allergen. We will discuss existing and experimental therapeutic strategies to eliminate IgE reactivity and, lastly, will propose outstanding questions to pave the road to the development of novel, transformative therapeutics in food allergy.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

Peanut allergy: Beyond the oral immunotherapy plateau.

BACKGROUND: There are a lack of disease-modifying treatments for peanut allergy, which is lifelong in most instances. Oral immunotherapy has remained at the forefront of prospective treatments, though its efficacy is consistently undermined by the risk of adverse reactions and meager sustained effects. AIM: This review discusses the current state of oral immunotherapy, its strengths and limitations, and the future of therapeutics for the treatment of peanut allergy. CONCLUSION: The persistence of peanut allergy is currently attributed to reservoirs of peanut-specific memory B cells and Th2 cells, though the cellular and molecular interplay that facilitates the replenishment of peanut-specific IgE remains elusive. Uncovering these events will prove critical for identification of novel targets as we forge ahead to a new age of peanut allergy treatment with biotherapeutics.

Memory Generation and Re-Activation in Food Allergy.

Recent evidence has highlighted the critical role of memory cells in maintaining lifelong food allergies, thereby identifying these cells as therapeutic targets. IgG+ memory B cells replenish pools of IgE-secreting cells upon allergen exposure, which contract thereafter due to the short lifespan of tightly regulated IgE-expressing cells. Advances in the detection and highly dimensional analysis of allergen-specific B and T cells from allergic patients have provided insight on their phenotype and function. The newly identified Th2A and Tfh13 populations represent a leap in our understanding of allergen-specific T cell phenotypes, although how these populations contribute to IgE memory responses remains poorly understood. Within, we discuss the mechanisms by which memory B and T cells are activated, integrating knowledge from human systems and fundamental research. We then focus on memory reactivation, specifically, on the pathways of secondary IgE responses. Throughout, we identify areas of future research which will help identify immunotargets for a transformative therapy for food allergy.

Interrupting reactivation of immunologic memory diverts the allergic response and prevents anaphylaxis.

BACKGROUND: IgE production against innocuous food antigens can result in anaphylaxis, a severe life-threatening consequence of allergic reactions. The maintenance of IgE immunity is primarily facilitated by IgG+ memory B cells, as IgE+ memory B cells and IgE+ plasma cells are extremely scarce and short-lived, respectively. OBJECTIVE: Our aim was to investigate the critical requirements for an IgE recall response in peanut allergy. METHODS: We used a novel human PBMC culture platform, a mouse model of peanut allergy, and various experimental readouts to assess the IgE recall response in the presence and absence of IL-4Rα blockade. RESULTS: In human PBMCs, we have demonstrated that blockade of IL-4/IL-13 signaling aborted IgE production after activation of a recall response and skewed the cytokine response away from a dominant type 2 signature. TH2A cells, identified by single-cell RNA sequencing, expanded with peanut stimulation and maintained their pathogenic phenotype in spite of IL-4Rα blockade. In mice with allergy, anti-IL-4Rα provided long-lasting suppression of the IgE recall response beyond antibody treatment and fully protected against anaphylaxis. CONCLUSION: The findings reported here advance our understanding of events mediating the regeneration of IgE in food allergy.

Perturbations to Homeostasis in Experimental Models Revealed Innate Pathways Driving Food Allergy.

While type 2 immunity has been conventionally viewed as beneficial against helminths, venoms, and poisons, and harmful in allergy, contemporary research has uncovered its critical role in the maintenance of homeostasis. The initiation of a type 2 immune response involves an intricate crosstalk between structural and immune cells. Structural cells react to physical and chemical tissue perturbations by secreting alarmins, which signal the innate immune system to restore homeostasis. This pathway acts autonomously in the context of sterile injury and in the presence of foreign antigen initiates an adaptive Th2 response that is beneficial in the context of venoms, toxins, and helminths, but not food allergens. The investigation of the triggers and mechanisms underlying food allergic sensitization in humans is elusive because sensitization is a silent process. Therefore, the central construct driving food allergy modeling is based on introducing perturbations of tissue homeostasis along with an allergen which will result in an immunological and clinical phenotype that is consistent with that observed in humans. The collective evidence from multiple models has revealed the pre-eminent role of innate cells and molecules in the elicitation of allergic sensitization. We posit that, with the expanding use of technologies capable of producing formidable datasets, models of food allergy will continue to have an indispensable role to delineate mechanisms and establish causal relationships.

Microbial Regulation of Enteric Eosinophils and Its Impact on Tissue Remodeling and Th2 Immunity.

Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the impact of the microbiota on their frequency and function at mucosal sites remains unclear. Here, we investigated the role of the microbiota in the regulation of enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This was associated with changes in the production of enteric signals that regulate eosinophil attraction and survival, and was fully reversed by complex colonization. Additionally, SI eosinophils of GF mice exhibited more cytoplasmic protrusions and less granule content than SPF controls. Lastly, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate, and adaptive immune processes in health and disease.

IL-17 Production by γδ+ T Cells Is Critical for Inducing Th17 Responses in the Female Genital Tract and Regulated by Estradiol and Microbiota.

IL-17 can be produced by adaptive immune cells such as Th17 cells and by immune cells that produce IL-17 without prior priming. This latter category, which we will refer to as "innate," includes innate cells such as NK cells and innate lymphoid cells and innate-like T cell populations such as NKT cells and γδ+ T cells. Studies in mucosal tissues have shown that the induction of Th17 immunity is amplified by innate IL-17 produced within those tissues. However, the role of innate IL-17 and its effect on Th17 induction in the female genital tract (FGT) is largely unknown. In this study, we characterize the primary source of IL-17-secreting vaginal cells and show that innate IL-17 plays a critical role in priming adaptive Th17 responses in the FGT. Under homeostatic conditions, γδ+ T cells were the predominant source of innate IL-17 in the murine FGT, and this population was modulated by both the sex hormone estradiol and the presence of commensal microbiota. Compared with wild-type C57BL/6 mice, vaginal APCs isolated from IL-17A-deficient (IL-17A-/- ) mice were severely impaired at priming Th17 responses in APC-T cell cocultures. Furthermore, the defect in Th17 induction in the absence of innate IL-17 was associated with impairment of IL-1ß production by vaginal CD11c+ dendritic cells. Overall, our study describes a novel role for IL-17 in the FGT and further demonstrates the importance of factors in the vaginal microenvironment that can influence adaptive immune responses.

Kaiso-induced intestinal inflammation is preceded by diminished E-cadherin expression and intestinal integrity.

Chronic intestinal inflammation contributes to pathologies such as inflammatory bowel disease (IBD) and colon cancer. While the precise etiology remains controversial, IBD is believed to manifest as a result of various factors. We previously reported that intestinal-specific overexpression of the transcription factor Kaiso results in an intestinal inflammatory response; however, the cause of this inflammation is unknown. To elucidate the underlying mechanism(s) of the Kaiso-mediated intestinal inflammatory phenotype, we evaluated two independent transgenic mouse lines that express varying levels of Kaiso (KaisoTg). Histological analyses of KaisoTg mice revealed intestinal damage including thickening of the mucosa, intestinal "lesions" and crypt abscesses, which are reminiscent of IBD pathology. Additionally, higher Kaiso levels induced intestinal neutrophilia as early as 12 weeks, which worsened as the mice aged. Notably, the Kaiso-induced intestinal inflammation correlated with a leaky intestinal barrier and mis-regulation of E-cadherin expression and localization. Interestingly, Kaiso overexpression resulted in reduced proliferation but enhanced migration of intestinal epithelial cells prior to the onset of inflammation. Collectively, these data suggest that Kaiso plays a role in regulating intestinal epithelial cell integrity and function, dysregulation of which contributes to a chronic inflammatory phenotype as mice age.

Oral immunotherapy for peanut allergy (PACE): a systematic review and meta-analysis of efficacy and safety.

BACKGROUND: Oral immunotherapy is an emerging experimental treatment for peanut allergy, but its benefits and harms are unclear. We systematically reviewed the efficacy and safety of oral immunotherapy versus allergen avoidance or placebo (no oral immunotherapy) for peanut allergy. METHODS: In the Peanut Allergen immunotherapy, Clarifying the Evidence (PACE) systematic review and meta-analysis, we searched MEDLINE, EMBASE, Cochrane Controlled Register of Trials, Latin American & Caribbean Health Sciences Literature, China National Knowledge Infrastructure, WHO's Clinical Trials Registry Platform, US Food and Drug Administration, and European Medicines Agency databases from inception to Dec 6, 2018, for randomised controlled trials comparing oral immunotherapy versus no oral immunotherapy for peanut allergy, without language restrictions. We screened studies, extracted data, and assessed risk of bias independently in duplicate. Main outcomes included anaphylaxis, allergic or adverse reactions, epinephrine use, and quality of life, meta-analysed by random effects. We assessed certainty (quality) of evidence by the GRADE approach. This study is registered with PROSPERO, number CRD42019117930. RESULTS: 12 trials (n=1041; median age across trials 8·7 years [IQR 5·9-11·2]) showed that oral immunotherapy versus no oral immunotherapy increased anaphylaxis risk (risk ratio [RR] 3·12 [95% CI 1·76-5·55], I2=0%, risk difference [RD] 15·1%, high-certainty), anaphylaxis frequency (incidence rate ratio [IRR] 2·72 [1·57-4·72], I2=0%, RD 12·2%, high-certainty), and epinephrine use (RR 2·21 [1·27-3·83], I2=0%, RD 4·5%, high-certainty) similarly during build-up and maintenance (pinteraction=0·92). Oral immunotherapy increased serious adverse events (RR 1·92 [1·00-3·66], I2=0%, RD 5·7%, moderate-certainty), and non-anaphylactic reactions (vomiting: RR 1·79 [95%CI 1·35-2·38], I2=0%, high-certainty; angioedema: 2·25 [1·13-4·47], I2=0%, high-certainty; upper tract respiratory reactions: 1·36 [1·02-1·81], I2=0%, moderate-certainty; lower tract respiratory reactions: 1·55 [0·96-2·50], I2=28%, moderate-certainty). Passing a supervised challenge, a surrogate for preventing out-of-clinic reactions, was more likely with oral immunotherapy (RR 12·42 [95% CI 6·82-22·61], I2=0%, RD 36·5%, high-certainty). Quality of life was not different between groups (combined parents and self report RR 1·21 [0·87-1·69], I2=0%, RD 0·03%, low-certainty). Findings were robust to IRR, trial sequential, subgroup, and sensitivity analyses. INTERPRETATION: In patients with peanut allergy, high-certainty evidence shows that available peanut oral immunotherapy regimens considerably increase allergic and anaphylactic reactions over avoidance or placebo, despite effectively inducing desensitisation. Safer peanut allergy treatment approaches and rigorous randomised controlled trials that evaluate patient-important outcomes are needed. FUNDING: None.