TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Gastrointestinal immunopathology of food protein-induced enterocolitis syndrome and other non-immunoglobulin E-mediated food allergic diseases.

OBJECTIVE: To provide a concise summary of the current literature regarding gastrointestinal immunopathology of food protein-induced enterocolitis syndrome (FPIES) and other non-immunoglobulin E (IgE)-mediated food allergic diseases. DATA SOURCES: Data were extracted from PubMed, MEDLINE, and ScienceDirect databases. STUDY SELECTIONS: Original articles, review articles, and guidelines published in the past 5 years in peer-reviewed journals were first summarized. The original articles cited were then reviewed and relevant results were extracted. RESULTS: Patients with FPIES and non-IgE-mediated food allergic diseases developed vomiting, diarrhea, and food aversion expelled food allergen from their bodies. Aside from T helper type 2 (TH2) immunity, TH1, TH17, innate immunity, and epithelial mucosal barrier defect were also found to be important in the pathogenesis. Eosinophils, widely identified in the biopsy samples, were key players or were late-recruited cells for tissue repairs in those diseases. Intestinal dysbiosis and their metabolites stimulated enterochromaffin cells or enteroendocrine cells to produce serotonin, interfering with intestinal motility and subsequently affecting brain function. FPIES and non-IgE-mediated food allergic diseases were likely part of the atopic march. Allergic inflammation in intestinal mucosa might result in subsequent inflammation in the airway mucosa, suggesting the theory of "one mucosa, one disease." CONCLUSION: The immune responses of FPIES and non-IgE-mediated food allergic diseases were not limited to the gastrointestinal tract, but also trigger wider inflammatory responses beyond it. Further research will be required to determine the systemic effect and intestinal microbiome of those diseases.

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.

Novel vaccines: Technology and development.

The development and widespread use of vaccines, which are defined by the World Health Organization as "biological preparations that improve immunity to a particular disease," represents one of the most significant strides in medicine. Vaccination was first applied to reduce mortality and morbidity from infectious diseases. The World Health Organization estimates that vaccines prevent 2 to 3 million human deaths annually, and these numbers would increase by at least 6 million if all children received the recommended vaccination schedule. However, the origins of allergen immunotherapy share the same intellectual paradigm, and subsequent innovations in vaccine technology have been applied beyond the prevention of infection, including in the treatment of cancer and allergic diseases. This review will focus on how new and more rational approaches to vaccine development use novel biotechnology, target new mechanisms, and shape the immune system response, with an emphasis on discoveries that have direct translational relevance to the treatment of allergic diseases.

Longitudinal Perspective on Managing Refractory Eosinophilic Esophagitis.

BACKGROUND: One half to one third of the patients with eosinophilic esophagitis (EoE) do not achieve histological remission on initial treatment. We wondered whether these treatment failure patients are a distinct clinical subset. OBJECTIVE: To analyze EoE treatment outcomes in a predominantly pediatric population. METHODS: We reviewed 100 serial EoE cases at Massachusetts General Hospital starting from 2007. We defined histological remission as peak esophageal eosinophil count of less than 10/hpf. RESULTS: Ninety-seven patients with EoE underwent initial treatments: 54 of 81 (67%) responded to dietary therapy, and 9 of 16 (56%) responded to topical glucocorticoids. Of the 34 who failed initial treatment, 24 underwent various second treatment regimens and 54% (13 of 24) responded. Eight of the remaining 11 who failed second treatment underwent additional treatments and 2 ultimately responded. The overall response rate by intent-to-treat analysis increased from 65% (63 of 97) with initial treatment to 78% (76 of 97) with rescue treatment, and further to 80% (78 of 97) with multiple treatments. On a per-protocol basis, the overall response rate was 93% (78 of 84); however, patients who failed the first 2 rounds of therapy had only a 20% response rate. Patients who responded to initial treatment were found to have more symptoms and endoscopic abnormalities. Comparison of patients who failed both initial and rescue therapy with those who responded to rescue therapy did not identify any differentiating clinical features. CONCLUSIONS: More than half of the patients who failed initial EoE treatment could still achieve histological remission with individualized rescue treatments. No clinical features could predict response to rescue treatment.

Associations between serum folate and vitamin D levels and incident mouse sensitization in adults.

BACKGROUND: Although both folic acid intake and vitamin D levels are hypothesized to be contributors to the increased incidence of allergic diseases, prospective studies of these relationships have not been done in adults. OBJECTIVES: We sought to determine whether serum folate or vitamin D levels are associated with incident mouse sensitization among new workers at a mouse facility. METHODS: Subjects started employment at the Jackson Laboratory between June 2004 and July 2007. Skin testing to mouse and other allergens and collection of questionnaire data were performed at baseline and every 6 months. Serum folate and vitamin D levels were assessed on baseline samples stored at -80°C. Folate was categorized into tertiles (2.5-10.5, 10.5-16.2, and 16.2-78.4 ng/mL, respectively). Vitamin D was categorized as less than 20 ng/mL, 20 to 29 ng/mL, or 30 ng/mL or greater. This was a nested case-control study in which 5 control subjects were matched to each case on baseline atopy and type of employment. Multivariate analyses controlled for age, sex, education, smoking, season, personal mouse exposure, and serum folate and vitamin D levels. RESULTS: Thirty-five cases and 47 control subjects were included. The odds of incident mouse sensitization were higher in the intermediate and highest tertiles of serum folate compared with the lowest tertile of serum folate (odds ratio of 10.5 [95% CI, 1.8-61.5; P = .009] and odds ratio of 5.6 [95% CI, 1.8-31.3; P = .049], respectively, in the multivariate model). Serum vitamin D levels were not associated with incident mouse sensitization. CONCLUSIONS: These findings support a role for higher serum folate levels in increased risk of incident allergic disease, even during adulthood.

Identification of human CCR8 as a CCL18 receptor.

The CC chemokine ligand 18 (CCL18) is one of the most highly expressed chemokines in human chronic inflammatory diseases. An appreciation of the role of CCL18 in these diseases has been hampered by the lack of an identified chemokine receptor. We report that the human chemokine receptor CCR8 is a CCL18 receptor. CCL18 induced chemotaxis and calcium flux of human CCR8-transfected cells. CCL18 bound with high affinity to CCR8 and induced its internalization. Human CCL1, the known endogenous CCR8 ligand, and CCL18 competed for binding to CCR8-transfected cells. Further, CCL1 and CCL18 induced heterologous cross-desensitization of CCR8-transfected cells and human Th2 cells. CCL18 induced chemotaxis and calcium flux of human activated highly polarized Th2 cells through CCR8. Wild-type but not Ccr8-deficient activated mouse Th2 cells migrated in response to CCL18. CCL18 and CCR8 were coexpressed in esophageal biopsy tissue from individuals with active eosinophilic esophagitis (EoE) and were present at markedly higher levels compared with esophageal tissue isolated from EoE patients whose disease was in remission or in normal controls. Identifying CCR8 as a chemokine receptor for CCL18 will help clarify the biological role of this highly expressed chemokine in human disease.

Development of a novel peptide microarray for large-scale epitope mapping of food allergens.

BACKGROUND: The peptide microarray is a novel assay that facilitates high-throughput screening of peptides with a small quantity of sample. OBJECTIVE: We sought to use overlapping peptides of milk allergenic proteins as a model system to establish a reliable and sensitive peptide microarray-based immunoassay for large-scale epitope mapping of food allergens. METHODS: A milk peptide microarray was developed by using commercially synthesized peptides (20-mers, 3 offset) covering the primary sequences of alpha(s1)-casein, alpha(s2)-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Conditions for printing and immunolabeling were optimized using a serum pool of 5 patients with milk allergy. Reproducibility of the milk peptide microarray was evaluated using replicate arrays immunolabeled with the serum pool, whereas specificity and sensitivity were assessed by using serial dilution of the serum pool and a peptide inhibition assay. RESULTS: Our results show that epitopes identified by the peptide microarray were mostly consistent with those identified previously by SPOT membrane technology, but with specific binding to a few newly identified epitopes of milk allergens. Data from replicate arrays were reproducible (r > or = 0.92) regardless of printing lots, immunolabeling, and serum pool batches. Using the serially diluted serum pool, we confirmed that IgE antibody binding detected in the array was specific. Peptide inhibition of IgE binding to the same peptide and overlapping peptides further confirmed the specificity of the array. CONCLUSION: A reliable peptide microarray was established for large-scale IgE epitope mapping of milk allergens, and this robust technology could be applied for epitope mapping of other food allergens.

Microarrayed allergen molecules for diagnostics of allergy.

With the evolution of peptide synthesis techniques and microarray technology, it is now possible to map and characterize allergenic epitopes on a microarray platform. The peptide microarray-based immunoassay allows simultaneous analysis of thousands of target peptides using small volumes of diluted serum samples, and has a promising future to become a superior testing tool for aspects of food allergy diagnosis and prognosis, as well as for designing recombinant allergens for safe immunotherapy. Characterization of allergenic epitopes provides fundamental knowledge for understanding mechanisms of food allergy. This chapter describes in detail the development of a sensitive and reliable peptide microarray-based immunoassay. The information includes a comparison of different slide substrates, effect of buffers on spot morphology, performance of printing pins, immunolabeling detection systems with different levels of sensitivity, and suggested approaches to data analysis.

Association of allergen-specific regulatory T cells with the onset of clinical tolerance to milk protein.

BACKGROUND: About 70% of children with milk allergy tolerate extensively heated milk (HM) products and outgrow their allergy earlier than those who react to HM. OBJECTIVE: To test the hypothesis that HM-tolerant children have a higher precursor frequency of adaptive allergen-specific regulatory T (Treg) cells. METHODS: Allergic, HM-tolerant, outgrown, or control subjects were defined by oral food challenge. PBMCs were cultured with purified caseins and controls for 7 days, and proliferating CD25(+)CD27(+) Treg cells were identified by flow cytometry. Proliferating cells were also characterized for their expression of FoxP3, CTLA 4, CD45RO, and CD127. Allergen-specific Treg cell origin and function were assessed by depletion of CD25(hi) cells before culture. RESULTS: There was a higher percentage (median [25th% to 75th%], 16.85% [7.1-31.7]) of proliferating allergen-specific CD25(+)CD27(+) T cells from cultures of HM-tolerant subjects (n = 18) than subjects with allergy (n = 8; 4.91% [2.6-7.5]; P < .01). Control subjects with no history of milk allergy (n = 7) also had low percentages of these cells (2.9% [2.4-6.0]), whereas outgrown subjects (n = 7) had intermediate percentages (9.0% [2.7-16.4]). There were no significant differences between the patient groups in the frequency of polyclonal Treg cells or allergen-specific effector T cells. Allergen-specific Treg cells were found to be FoxP3(+)CD25(hi)CD27(+), cytotoxic T lymphocyte-associated antigen 4(+), CD45RO(+)CD127(-) and were derived from circulating CD25(hi) T cells. Depletion of the CD25(hi) cells before in vitro culture significantly enhanced allergen-specific effector T-cell expansion. CONCLUSION: A higher frequency of milk allergen-specific Treg cells correlates with a phenotype of mild clinical disease and favorable prognosis.

Evaluation of basophil activation in food allergy: present and future applications.

PURPOSE OF REVIEW: The diagnosis of immediate hypersensitivity relies on specific IgE and history. Because of low specificity, however, provocation challenges are often necessary. Furthermore, IgE testing does not predict features such as reaction severity; nor can it discriminate cross-reactivity from multiple sensitizations. Direct and passive basophil activation tests may address these needs. In addition, measuring basophil activation ex vivo may be useful for monitoring patients with food allergies. RECENT FINDINGS: Several papers using basophil activation tests demonstrate comparable sensitivity and specificity to current testing for food allergy. Flow-based basophil activation tests have also been used to assess functional characteristics of patient IgE. Finally, several activation phenotypes have been identified as markers of allergic inflammation in vivo; these phenotypes appear to correspond to earlier reports of spontaneous histamine-releasing basophils in patients with active allergic inflammation. SUMMARY: Although in their early stages, direct basophil activation tests may prove to be useful in the clinic. Indirect basophil activation studies are useful when applied to compare functional aspects of IgE. Identification of basophil activation ex vivo is a promising approach for monitoring allergic inflammation.