Despite the success of peanut oral immunotherapy (OIT), there is little insight into the safety, efficacy, and immune underpinnings of OIT to treat cashew or shrimp allergy. Cashew or shrimp allergic participants aged 7-55 years were enrolled in the phase 2 clinical trial - MOTIF (NCT03504774) and underwent cognate allergen-specific OIT to a maintenance dose of 1,000 mg until week 52. Oral food challenge (OFC) at weeks 52 and 58 evaluated desensitization and sustained unresponsiveness (SU) respectively to a cumulative allergen dose of 2043 mg. We examined immune changes in allergen-reactive CD4+ T cells and plasma at baseline, week 52, and week 58 using flow cytometry and Luminex. We enrolled 40 cashew and 12 shrimp allergic participants, of whom 25 cashew-treated (62.5%) and 7 shrimp-treated (58.3%) participants completed and passed week 58 OFC. Overall, cashew and shrimp OIT was safe with only mild (90%) or moderate (10%) adverse events, with gastrointestinal symptoms (31%) being the most common. OIT induced a significant increase in allergen-specific IgG4 at week 52 (q < 0.1). A trend of downregulation of Th2 and Th1 surrogate marker CRTH2 and CXCR3-expressing allergen-reactive CD4+ T cells was evident in cashew- and shrimp-treated participants respectively at week 52. Higher baseline plasma levels of TARC, EGF, and IP10 implied a lower likelihood of SU. Cashew and shrimp OIT could safely desensitize allergic study participants to respective allergen protein. Mechanistic analyses confirm readouts linked to successful desensitization and offer potential biomarkers for predicting SU that warrant further investigation.

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis.

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.

Background: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. Objectives: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. Methods: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4(+) T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. Results: IL-21 activated STATS and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21(lo)Tbet(+) B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. Conclusions: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

Individuals with multiple food allergies represent approximately 30% of all patients with food allergies. Desensitization can be achieved safely and rapidly with multi-food oral immunotherapy (OIT) in combination with Omalizumab; however the immune mechanism behind desensitization to multiple foods remains unclear. Mass cytometry was used to assess the immune profile of 12 participants from a published trial (Andorf et al., Lancet eClinicalMedicine, 2019) who were allergic to multiple foods including peanut and cashew and were successfully desensitized. PBMCs collected before and post-OIT were stimulated with peanut or cashew protein or medium. Immunophenotyping and functional analysis were performed through unsupervised clustering and manual gating. Wilcoxon signed-rank test were used to compare cell subset frequencies and functional marker expression before and post-OIT, adjusted for multiple comparisons. After treatment with anti-IgE and multi-OIT, the frequency of allergen specific CD25+CD40L+ T cells following peanut and cashew stimulations was significantly decreased. In contrast, an increased trend of IFN-gamma positive allergen specific cells and a significant increase of CCR4+CLA+ Th17 in CD4+ T cells were observed post-OIT. We also observed an increased frequency of CCR4+CLA+ Th2 effector memory cells (Th2EM) post-OIT with diminished activation markers (CD25/CD40L) expression under peanut and cashew stimulations. Overall, we observed a decrease of allergen specific T cells and a shift from Th2 immunity toward a Th1/Th17 response in response to both peanut and cashew stimulation after multi-OIT treatment. Increased frequency of CCR4+CLA+ Th2EM cells post-OIT suggests possible Th2 cell proliferation and tissue homing during OIT.

Food allergies are a leading cause of anaphylaxis, and cellular mechanisms involving antigen presentation likely play key roles in their pathogenesis. However, little is known about the response of specific antigen-presenting cell (APC) subsets to food allergens in the setting of food allergies. Here, we show that in peanut-allergic humans, peanut allergen drives the differentiation of CD209+ monocyte-derived dendritic cells (DCs) and CD23+ (FcєRII) myeloid dendritic cells through the action of allergen-specific CD4+ T cells. CD209+ DCs act reciprocally on the same peanut-specific CD4+ T cell population to reinforce Th2 cytokine expression in a positive feedback loop, which may explain the persistence of established food allergy. In support of this novel model, we show clinically that the initiation of oral immunotherapy (OIT) in peanut-allergic patients is associated with a decrease in CD209+ DCs, suggesting that breaking the cycle of positive feedback is associated with therapeutic effect.

DNA methylation (DNAm) has been shown to play a role in mediating food allergy; however, the mechanism by which it does so is poorly understood. In this study, we used targeted next-generation bisulfite sequencing to evaluate DNAm levels in 125 targeted highly informative genomic regions containing 602 CpG sites on 70 immune-related genes to understand whether DNAm can differentiate peanut allergy (PA) versus nonallergy (NA). We found PA-associated DNAm signatures associated with 12 genes (7 potentially novel to food allergy, 3 associated with Th1/Th2, and 2 associated with innate immunity), as well as DNAm signature combinations with superior diagnostic potential compared with serum peanut–specific IgE for PA versus NA. Furthermore, we found that, following peanut protein stimulation, peripheral blood mononuclear cell (PBMCs) from PA participants showed increased production of cognate cytokines compared with NA participants. The varying responses between PA and NA participants may be associated with the interaction between the modification of DNAm and the interference of environment. Using Euclidean distance analysis, we found that the distances of methylation profile comprising 12 DNAm signatures between PA and NA pairs in monozygotic (MZ) twins were smaller than those in randomly paired genetically unrelated individuals, suggesting that PA-related DNAm signatures may be associated with genetic factors.

Oral Immunotherapy (OIT) is a promising intervention for food allergic individuals, and has been FDA-approved to treat peanut allergy. Various hitherto studies have probed the clinical efficacy and immune mechanism of OIT-induced desensitization to peanut, milk, and egg, although immunomodulatory effects of cashew and shrimp-OIT have not been investigated. Thus, we examined pre- vs. post-OIT PBMCs from 38 cashew (n=33)- or shrimp (n=5)-OIT treated participants.

Background Broadly, much of variance in immune system phenotype has been linked to the influence of non-heritable factors rather than genetics. In particular, two non-heritable factors: aging and human cytolomegavirus (CMV) infection, have been known to account for significant inter-individual immune variance. However, many specific relationships between them and immune composition remain unclear, especially between individuals over narrower age ranges. Further exploration of these relationships may be useful for informing personalized intervention development. Results To address this need, we evaluated 41 different cell type frequencies by mass cytometry and identified their relationships with aging and CMV seropositivity. Analyses were done using 60 healthy individuals, including 23 monozygotic twin pairs, categorized into young (12–31 years) and middle-aged (42–59 years). Aging and CMV discordance were associated with increased immune diversity between monozygotic twins overall, and particularly strongly in various T cell populations. Notably, we identified 17 and 11 cell subset frequencies as relatively influenced and uninfluenced by non-heritable factors, respectively, with results that largely matched those from studies on older-aged cohorts. Next, CD4+ T cell frequency was shown to diverge with age in twins, but with lower slope than in demographically similar non-twins, suggesting that much inter-individual variance in this cell type can be attributed to interactions between genetic and environmental factors. Several cell frequencies previously associated with memory inflation, such as CD27- CD8+ T cells and CD161+ CD4+ T cells, were positively correlated with CMV seropositivity, supporting findings that CMV infection may incur rapid aging of the immune system. Conclusions Our study confirms previous findings that aging, even within a relatively small age range and by mid-adulthood, and CMV seropositivity, both contribute significantly to inter-individual immune diversity. Notably, we identify several key immune cell subsets that vary considerably with aging, as well as others associated with memory inflation which correlate with CMV seropositivity.

It is postulated that loss of immune tolerance to food can lead to food allergies. CD8+ T cells are known to participate in pathologic immune responses to food antigen, such as seen in eosinophilic esophagitis and celiac disease.1Yu W. Zhou X. Dunham D. Lyu S.C. Manohar M. Zhang W. et al.Allergen-specific CD8(+) T cells in peanut-allergic individuals.J Allergy Clin Immunol. 2019; 143: 1948-1952Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 2Mazzarella G. Stefanile R. Camarca A. Giliberti P. Cosentini E. Marano C. et al.Gliadin activates HLA class I-restricted CD8+ T cells in celiac disease intestinal mucosa and induces the enterocyte apoptosis.Gastroenterology. 2008; 134: 1017-1027Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 3Han A. Newell E.W. Glanville J. Fernandez-Becker N. Khosla C. Chien Y.H. et al.Dietary gluten triggers concomitant activation of CD4+ and CD8+ alphabeta T cells and gammadelta T cells in celiac disease.Proc Natl Acad Sci U S A. 2013; 110: 13073-13078Crossref PubMed Scopus (138) Google Scholar, 4Wen T. Aronow B.J. Rochman Y. Rochman M. Kc K. Dexheimer P.J. et al.Single-cell RNA sequencing identifies inflammatory tissue T cells in eosinophilic esophagitis.J Clin Invest. 2019; 129: 2014-2028Crossref PubMed Scopus (63) Google Scholar In light of this, we recently reported that peanut-specific CD8+ T cells are increased in the blood of peanut-allergic individuals and express the TH2-associated chemokine receptor CCR4.1Yu W. Zhou X. Dunham D. Lyu S.C. Manohar M. Zhang W. et al.Allergen-specific CD8(+) T cells in peanut-allergic individuals.J Allergy Clin Immunol. 2019; 143: 1948-1952Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar We also identified an Ara h 1–derived peptide that triggered the activation of nearly 1% of CD8+ T cells from 1 HLA-A∗02:01+ peanut-allergic individual, and derived Ara h 1–specific CD8+ T-cell clones from the same person by in vitro expansion.1Yu W. Zhou X. Dunham D. Lyu S.C. Manohar M. Zhang W. et al.Allergen-specific CD8(+) T cells in peanut-allergic individuals.J Allergy Clin Immunol. 2019; 143: 1948-1952Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar In this follow-up study, we first wanted to better characterize the phenotype of the peanut-specific CD8+ T-cell clones described above in the setting of food allergy. Five Ara h 1–specific CD8+ T-cell clones were incubated for 2 days with or without PHA, after which their cytokine expression profile was measured by BD cytokine bead array assay. Supernatants from all 5 clones contained increased levels of IL-5, IL-13, and GM-CSF after stimulation (see Fig E1, A, in this article's Online Repository at www.jacionline.org). The same cytokines were increased after a 5-day incubation with the Ara h 1 peanut peptide, but not a control peptide (Fig E1, B). Thus, peanut-specific CD8+ T cells may contribute to food allergy by becoming activated and expressing TH2 cytokines after exposure to allergen. GM-CSF enables antigen cross-presentation by dendritic cells, which would promote further allergen recognition by CD8+ T cells.5Dresch C. Leverrier Y. Marvel J. Shortman K. Development of antigen cross-presentation capacity in dendritic cells.Trends Immunol. 2012; 33: 381-388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar The identification of a peanut epitope recognized in food allergy opened the opportunity to study the behavior of food-specific CD8+ T cells in nonallergic individuals. Doing so is challenging not only because one must first identify a food epitope that is recognized in disease, but the frequency of cells specific for a given food epitope is also low, making detection difficult. We addressed this issue by using tetramer enrichment with HLA-A∗02:01 tetramers loaded with the Ara h 1 peptide, and conjugated to phycoerythrin.6Yu W. Jiang N. Ebert P.J. Kidd B.A. Muller S. Lund P.J. et al.Clonal deletion prunes but does not eliminate self-specific alpha beta CD8(+) T lymphocytes.Immunity. 2015; 42: 929-941Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar In this technique, tetramer+ cells are incubated with paramagnetic beads coated with antiphycoerythrin antibody, and then enriched using a magnetized column before detection by flow cytometry. Peptide-specific cells at frequencies less than 1 cell per a million total CD8+ T cells can be detected by flow cytometry in this way.6Yu W. Jiang N. Ebert P.J. Kidd B.A. Muller S. Lund P.J. et al.Clonal deletion prunes but does not eliminate self-specific alpha beta CD8(+) T lymphocytes.Immunity. 2015; 42: 929-941Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar To determine whether peanut-specific CD8+ T cells are present in nonallergic individuals, we performed tetramer enrichment on PBMCs from HLA-A∗02:01+ blood donors from the Stanford Blood Center. The prevalence of peanut allergy is approximately 1.8% among US adults, so it is unlikely that multiple donors are peanut-allergic.7Gupta R.S. Warren C.M. Smith B.M. Jiang J. Blumenstock J.A. Davis M.M. et al.Prevalence and severity of food allergies among US adults.JAMA Netw Open. 2019; 2e185630Crossref PubMed Scopus (308) Google Scholar We found that the frequency of Ara h 1 peptide-specific CD8+ T cells in 8 different blood donors is approximately 1 cell in 105 to 106 total CD8+ T cells (Fig 1, A). We conclude that peanut-specific CD8+ T cells are not deleted (as a mechanism of immune tolerance) in peanut-tolerant individuals and are found in peripheral blood at frequencies consistent with CD8+ T cells specific for other antigens, both foreign and self.6Yu W. Jiang N. Ebert P.J. Kidd B.A. Muller S. Lund P.J. et al.Clonal deletion prunes but does not eliminate self-specific alpha beta CD8(+) T lymphocytes.Immunity. 2015; 42: 929-941Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar To measure the activation of Ara h 1–specific CD8+ T cells in nonallergic individuals, we incubated tetramer-enriched cells with autologous feeder cells, anti-CD28 antibody, and peanut peptide—the same conditions that previously activated nearly 1% of CD8+ T cells from an HLA-A∗02:01+ peanut-allergic individual.1Yu W. Zhou X. Dunham D. Lyu S.C. Manohar M. Zhang W. et al.Allergen-specific CD8(+) T cells in peanut-allergic individuals.J Allergy Clin Immunol. 2019; 143: 1948-1952Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar After 1 week, we measured the number of activated CD8+ T cells by the expression of CD25 and CD38. In 5 blood bank donor samples, the number of activated CD8+ T cells after stimulation was less than the original input of Ara h 1–specific CD8+ T cells (Fig 1, B). This poor activation is similar to what we have previously observed in CD8+ T cells specific for self antigen,6Yu W. Jiang N. Ebert P.J. Kidd B.A. Muller S. Lund P.J. et al.Clonal deletion prunes but does not eliminate self-specific alpha beta CD8(+) T lymphocytes.Immunity. 2015; 42: 929-941Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar and is consistent with the need for immune tolerance to both food antigens and endogenous antigens. We conclude that Ara h 1–specific CD8+ T cells are poorly activated by stimulation through the TCR and CD28 costimulatory receptor in nonallergic individuals, consistent with a mechanism of tolerance to food. We hypothesized that CD4+ regulatory T (Treg) cells might contribute to the suppression of Ara h 1– specific CD8+ T cells in nonallergic individuals. To test this, we mixed Ara h 1–specific CD8+ T cells with either whole autologous PBMCs (as before) or CD25-depleted autologous PBMCs before stimulating with peanut peptide and anti-CD28 antibody (Fig 2, A). Because FOXP3+ Treg cells are CD25hiCD127lo, removing CD25+ cells by bead depletion should lift suppression due to this cell type. An analysis of 8 blood donor samples showed 2 outcomes: in the case of 4 samples, there was a 3.4- to 10.5-fold increase in the number of Ara h 1–specific CD8+ T cells when CD25+ cells were depleted; in the case of the other samples, there was little change (Fig 2, A, middle and right). We conclude that in a proportion of individuals, CD25+ cells suppress the expansion of Ara h 1–specific CD8+ T cells. We sought to determine which intercellular signals could relieve the activation block in Ara h 1–specific CD8+ T cells. Treg cells use multiple mechanisms to suppress other cell types including IL-2 sequestration, the inhibitory coreceptor CTLA-4, and IL-10 secretion.8Sakaguchi S. Mikami N. Wong J.B. Tanaka A. Ichiyama K. Ohkura N. Regulatory T cells and human disease.Ann Rev Immunol. 2020; 38: 541-566Crossref PubMed Scopus (211) Google Scholar To test these mechanisms in food-specific CD8+ T cells, we added IL-2 (50-100 U/mL) as well as blocking antibodies against CTLA-4 (20 μg/mL) and IL-10 (40 μg/mL) to the standard in vitro assay of Ara h 1 CD8+ T cells, peanut peptide, anti-CD28 antibody, and autologous PBMCs (including CD25+ Treg cells). Blocking IL-10 and CTLA-4 signaling while adding IL-2 resulted in robust proliferation of Ara h 1–specific CD8+ T cells from 8 blood bank donor samples, again consistent with a model in which Treg cells contribute to the suppression of peanut-specific CD8+ T cells (Fig 2, B, left and middle, P = .0078). This 1-week treatment combination did not increase expression of IL-5 or IL-13 in Ara h 1-specific CD8+ T cells (see Fig E2 in this article's Online Repository at www.jacionline.org). Because previously we had shown that self-specific CD8+ T cells fail to expand after stimulation with peptide and anti-CD28 antibody,6Yu W. Jiang N. Ebert P.J. Kidd B.A. Muller S. Lund P.J. et al.Clonal deletion prunes but does not eliminate self-specific alpha beta CD8(+) T lymphocytes.Immunity. 2015; 42: 929-941Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar we tested whether self-specific CD8+ T cells would proliferate (as peanut-specific CD8+ T cells do) after the addition of IL-2 combined with blocking antibodies to CTLA-4 and IL-10. We used tetramer enrichment to isolate CD8+ T cells specific for endogenous peptides derived from human preproinsulin. An analysis of 5 blood bank samples showed minimal expansion of self-specific CD8+ T cells even with the addition of IL-2 combined with blocking antibodies to CTLA-4 and IL-10 (Fig 2, B, left and right). We conclude that despite the need to develop CD8+ T-cell tolerance to 2 parallel classes of antigens—food or endogenous—different mechanisms are used to achieve this goal. In summary, these initial results show that Ara h 1–specific CD8+ T cells from nonallergic individuals are not deleted, but expand poorly after stimulation through the TCR and CD28 coreceptor. Their expansion is augmented in some individuals by the deletion of CD25+ cells, and more generally by the addition of IL-2 combined with a block on CTLA-4 and IL-10. These interventions are consistent with a role for Treg cells. Interestingly, in a mouse food allergy model, blocking CTLA-4 during a longer 4-week sensitization with peanut protein and cholera toxin enhanced the IgE response against Ara h 1, whereas CTLA-4 blockade combined with peanut exposure resulted in increased TH2 cytokines.9van Wijk F. Hoeks S. Nierkens S. Koppelman S.J. van Kooten P. Boon L. et al.CTLA-4 signaling regulates the intensity of hypersensitivity responses to food antigens, but is not decisive in the induction of sensitization.J Immunol. 2005; 174: 174-179Crossref PubMed Scopus (52) Google Scholar We propose a model in which food antigen-specific CD8+ T cells are susceptible to suppression by Treg cells in an antigen-independent manner via a combination of IL-10, CTLA-4, and IL-2 signaling. A breakdown of this tolerance mechanism would be a step in enabling CD8+ T cells to augment an allergic response to food via the secretion of TH2 cytokines and by promoting the cross-presentation of food allergen. The expansion of this study to a larger population will provide additional confirmation of these mechanisms. Fig E2No increased expression of IL-5 or IL-13 by Ara h 1–specific CD8+ T cells from (nonallergic) HLA-A∗02:01+ blood bank donors after treatment with IL-2 and blocking antibodies to IL-10 and CTLA-4. Ara h 1–specific CD8+ T cells were isolated from 2 blood bank donors by tetramer enrichment. For each donor, Ara h 1 CD8+ T cells were divided equally and then incubated with Ara h 1 peptide, anti-CD28 antibody, and autologous PBMCs ± a mixture of IL-2 and blocking antibodies to IL-10 and CTLA-4. After 1 week, PBMCs were incubated for 4 hours with brefeldin A either with (left) or without (right) PHA/ionomycin. Flow cytometry panels are gated on CD8+ T cells, while histograms show relative IL-5 and IL-13 staining among Ara h 1–specific CD8+ T cells, based on the activation markers CD25 and CD38. Ab, Antibody; MFI, mean fluorescence intensity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

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We implemented the BD Rhapsody Single-Cell Analysis System to assess cellular heterogeneity present in twins by capturing and sequencing over 40000 single cells across 10 samples from 5 twins patients with asthma or without asthma using oligos-conjugated antibodies and the whole blood immune targeted gene panel that utilizes multiplex PCR for detection of over 500 genes for immune cells subtyping, immunophenotyping, cell-type identification and markers correlated with stimulations. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We demonstrate the ability of the BD Rhapsody targeted panel to distinguish six different cell types and 18 distinct gene-expression biomarkers, including CLEC4E, CSF2, IL1RN, TPSAB1, CCL19, CCL22, CXCL2, LAMP3, SPP1, and VMO etc.. The HLA-DRA, HLA-DQB1, SPP1, and IL1RN had allelic polymorphism related to asthma. The BD Rhapsody targeted panel yields more sequencing depth with much fewer (<2%) sequencing reads per cell. The measurement of specific proteins and transcripts in individual cells is critical for understanding the role of cellular diversity in development, health, and disease.

Oral immunotherapy (OIT) is currently under investigation for the treatment of peanut allergy and long-term safety is unknown. We evaluated OIT-related adverse events (AEs) during multi-year peanut OIT to better understand the long-term safety of OIT. 120 participants aged 7-55 years with confirmed peanut allergy were enrolled in a double-blind, placebo-controlled, phase 2 study of peanut OIT. Participants were randomized to escalate to and maintain 4000 mg peanut protein (n=95) or placebo (n=25) daily over 104 weeks. The per-person AE rate was calculated by dividing the number of AEs per year by the number of doses taken each year. Differences in per-person median AE rates between groups of interest were evaluated using Kruskal-Wallis rank sum test. The overall AE rate significantly decreased from 0.50 in year 1 to 0.14 in year 2 (p<0.0001), with a significantly greater reduction in AE rates from year 1 to year 2 in the peanut arm compared to placebo (-0.22 vs 0.00, p=0.0043). The rate of moderately severe AEs significantly declined from year 1 to year 2 in the peanut arm (0.09 vs 0.00, p=0.093). Participants reporting AEs related to accidental peanut ingestion decreased between year 1 (9%) and 2 (2%) in the peanut arm, while placebo did not change (12% to 16%). Participants requiring the use of epinephrine declined within the peanut arms with longer duration on peanut (19% year 1, 13% year 2). These findings show that the safety profile of peanut OIT improves as time on therapy increases.

Background Dietary avoidance is recommended for peanut allergies. We evaluated the sustained effects of peanut allergy oral immunotherapy (OIT) in a randomised long-term study in adults and children. Methods In this randomised, double-blind, placebo-controlled, phase 2 study, we enrolled participants at the Sean N Parker Center for Allergy and Asthma Research at Stanford University (Stanford, CA, USA) with peanut allergy aged 7-55 years with a positive result from a double-blind, placebo-controlled, food challenge (DBPCFC; <= 500 mg of peanut protein), a positive skin-prick test (SPT) result (>= 5 mm wheal diameter above the negative control), and peanut-specific immunoglobulin (Ig)E concentration of more than 4 kU/L. Participants were randomly assigned (2.4:1.4:1) in a two-by-two block design via a computerised system to be built up and maintained on 4000 mg peanut protein through to week 104 then discontinued on peanut (peanut-0 group), to be built up and maintained on 4000 mg peanut protein through to week 104 then to ingest 300 mg peanut protein daily (peanut-300 group) for 52 weeks, or to receive oat flour (placebo group). DBPCFCs to 4000 mg peanut protein were done at baseline and weeks 104, 117, 130, 143, and 156. The pharmacist assigned treatment on the basis of a randomised computer list. Peanut or placebo (oat) flour was administered orally and participants and the study team were masked throughout by use of oat flour that was similar in look and feel to the peanut flour and nose clips, as tolerated, to mask taste. The statistician was also masked. The primary endpoint was the proportion of participants who passed DBPCFCs to a cumulative dose of 4000 mg at both 104 and 117 weeks. The primary efficacy analysis was done in the intention-to-treat population. Safety was assessed in the intention-to-treat population. This trial is registered at ClinicalTrials.gov, NCT02103270. Findings Between April 15, 2014, and March 2, 2016, of 152 individuals assessed, we enrolled 120 participants, who were randomly assigned to the peanut-0 (n=60), peanut-300 (n=35), and placebo groups (n=25). 21 (35%) of peanut-0 group participants and one (4%) placebo group participant passed the 4000 mg challenge at both 104 and 117 weeks (odds ratio [OR] 12.7, 95% CI 1.8-554.8; p=0.0024). Over the entire study, the most common adverse events were mild gastrointestinal symptoms, which were seen in 90 of 120 patients (50/60 in the peanut-0 group, 29/35 in the peanut-300 group, and 11/25 in the placebo group) and skin disorders, which were seen in 50/120 patients (26/60 in the peanut-0 group, 15/35 in the peanut-300 group, and 9/25 in the placebo group). Adverse events decreased over time in all groups. Two participants in the peanut groups had serious adverse events during the 3-year study. In the peanut-0 group, in which eight (13%) of 60 participants passed DBPCFCs at week 156, higher baseline peanut-specific IgG4 to IgE ratio and lower Ara h 2 IgE and basophil activation responses were associated with sustained unresponsiveness. No treatment-related deaths occurred. Interpretation Our study suggests that peanut OIT could desensitise individuals with peanut allergy to 4000 mg peanut protein but discontinuation, or even reduction to 300 mg daily, could increase the likelihood of regaining clinical reactivity to peanut. Since baseline blood tests correlated with week 117 treatment outcomes, this study might aid in optimal patient selection for this therapy. Copyright (C) 2019 Elsevier Ltd. All rights reserved.

Shrimp proteins are highly allergenic and the protein composition can be affected by methods of manufacturing. Understanding the proteins in shrimp powder is important due to use in skin tests and food challenges. Samples of brown shrimp, (Farfante) Penaeus aztecus, were processed under the following conditions: 1) desiccated only, 2) desiccated followed by heat for 15 minutes at 350°F as an end step to minimize microbial contaminants, 3) microwaved for 20 seconds, and 4) raw. These samples were compared to shrimp skin prick extract (Stallergenes Greer®, North Carolina, USA). All samples were prepared and analyzed by SDS-PAGE gel analysis using standard procedures followed by Coomassie Blue staining and densitometry readings. The major shrimp allergens: Arginine kinase (40 kDa), Tropomyosin (34-38 kDa), Myosin light chain (20 kDa) and Sarcoplasmic calcium-binding protein (20 kDa) bands differ in intensity (i.e. concentration) depending on the preparation method. Removing the heat as an end step preserves all proteins and is more comparable to the skin prick test extract (which is manufactured without cooking/heating). Cooking methods alter protein profiles in brown shrimp. Skin prick test extract most closely mimics raw shrimp; however, if shrimp powders are used for food challenges, we recommend performing studies to ensure all proteins are represented to avoid false negative food challenges.

IgE-mediated peanut allergic is common, often serious, and usually lifelong. Not all individuals who produce peanut-specific IgE will react upon consumption of peanut and can eat the food without adverse reactions, known as sensitized tolerance. Here, we employ high-dimensional mass cytometry to define the circulating immune cell signatures associated with sensitized tolerance and clinical allergy to peanut in the first year of life. Key features of clinical peanut allergic are increased frequency of activated B cells (CD19hiHLADRhi), overproduction of TNFα and increased frequency of peanut-specific memory CD4 T cells. Infants with sensitized tolerance display reduced frequency but hyper-responsive naive CD4 T cells and an increased frequency of plasmacytoid dendritic cells. This work demonstrates the utility and power of high-dimensional mass cytometry analysis to interrogate the cellular interactions that are associated with allergic sensitization and clinical food allergy in the first year of life.

BACKGROUNDIL-33, found in high levels in participants with allergic disorders, is thought to mediate allergic reactions. Etokimab, an anti-IL-33 biologic, has previously demonstrated a good safety profile and favorable pharmacodynamic properties in many clinical studies.METHODSIn this 6-week placebo-controlled phase 2a study, we evaluated the safety and the ability of a single dose of etokimab to desensitize peanut-allergic adults. Participants received either etokimab (n = 15) or blinded placebo (n = 5). Clinical tests included oral food challenges and skin prick tests at days 15 and 45. Blood samples were collected for IgE levels and measurement of ex vivo peanut-stimulated T cell cytokine production.RESULTSEfficacy measurements for active vs. placebo participants at the day 15 and 45 food challenge (tolerating a cumulative 275 mg of peanut protein, which was the food challenge outcome defined in this paper) demonstrated, respectively, 73% vs. 0% (P = 0.008) to 57% vs. 0% (ns). The etokimab group had fewer adverse events compared with placebo. IL-4, IL-5, IL-9, IL-13, and ST2 levels in CD4+ T cells were reduced in the active vs. placebo arm upon peanut-induced T cell activation (P = 0.036 for IL-13 and IL-9 at day 15), and peanut-specific IgE was reduced in active vs. placebo (P = 0.014 at day 15).CONCLUSIONThe phase 2a results suggest etokimab is safe and well tolerated and that a single dose of etokimab could have the potential to desensitize peanut-allergic participants and possibly reduce atopy-related adverse events.TRIAL REGISTRATIONClinicalTrials.gov NCT02920021.FUNDINGThis work was supported by NIH grant R01AI140134, AnaptysBio, the Hartman Vaccine Fund, and the Sean N. Parker Center for Allergy and Asthma Research at Stanford University.

CD8+ T cells are uncommonly thought to be involved in IgE-mediated food allergy, but there are reasons to reconsider their role in this condition.1 CD8+ T cells are present in skin, and in the respiratory and gut mucosae, where they may modulate the immune response in atopic conditions such as asthma and atopic dermatitis.2-4 In mice, CD8+ T cells attenuate food allergy in some experimental models, whereas in humans, CD8+ T cells have been shown to expand in response to wheat ingestion in celiac disease.

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.