Shreffler:Research

Clinical Reactivity or Tolerance to Mouse Allergen
There is poor understanding of how natural allergen exposure relates to the ensuing allergen-specific immune responses, and how atopic disposition modifies allergen exposure-immune response relationships. In collaboration with Drs. Elizabeth Matsui at The Johns Hopkins School of Medicine and Beverly Paigen at the Jackson Laboratory, we are using an occupational model of allergen exposure to test the hypothesis that high levels of natural exposure to mouse allergen influences the subsequent immune responses to the major mouse allergen, Mus m 1, and ultimately, the clinical allergy phenotype, in a way that favors tolerance.

We are following workers at The Jackson Laboratory (TJL) who have a wide range of allergen exposure and whose exposure can be carefully measured over time. The specific aims are as follows:
 * 1) To continue to repeatedly assess mouse allergen exposure, mouse allergen-specific humoral responses, and clinical allergy phenotype in currently and newly enrolled participants;
 * 2) To assess the diversity of IgG, IgG4, and IgE mouse allergen epitope repertoires among current and newly enrolled participants;
 * 3) To assess mouse-specific Tr1 cell frequency in newly enrolled participants; and
 * 4) To assess IgE-mediated function by assessing basophil phenotypes and Mus m 1 uptake by antigen presenting cells in newly IgE-sensitized cohort members. We are examining relationships between allergen exposure levels and these allergen-specific immune responses and clinical allergy phenotype.

We believe that this prospective cohort study, in a setting in which repeated assessments of personal allergen exposure are feasible, will allow us to better understand the complex, time- and exposure-dependent relationships and ultimately provide a foundation for the development of immunomodulatory strategies aimed at prevention and treatment of allergic diseases.

Mechanisms of Peanut Adjuvanticity
Allergy to peanut (Arachis hypogaea) tends to be persistent and severe, and it represents an increasingly prevalent public health problem. More than 3 million Americans have peanut and/or tree nut allergy. To help account for the severity and prevalence of peanut allergy we hypothesize that peanut glycans function as Th2 adjuvants, directly activating innate immune cells to promote a strong and persistent allergic response in susceptible individuals. Most of the characterization of allergens has focused on structural properties such as size, solubility, stability, etc, that contribute to their allergenicity. There has been less attention directed at whether these proteins, or associated molecules, possess innate immunostimulatory function. Mammalian innate immune cells have evolved to recognize molecules like LPS as pathogen-associated molecular patterns (PAMPs) and in response promote a Th1 immune response to associated microbial proteins. Similarly, there are molecular patterns that have Th2 adjuvant function. Among the few well-studied Th2 PAMPs are glycans from Schistosoma mansoni egg antigens. Some of the core fucosylated and xylosylated complex mannose glycans implicated from schistosomes are also common on plant, arthropod, and helminth but not mammalian glycoproteins. Therefore these glycans make attractive candidates as non-self signals recognized by the innate immune system to induce Th2-skewed responses in susceptible individuals and contribute to clinical allergy. An identified peanut glycoprotein allergen, Arah1, is known to bear N-linked high mannose xylosylated glycans. We have shown that purified, endotoxin-free Arah1 can directly activate human dendritic cells (DC) to induce Th2-skewed T cell response and that Arah1 is a ligand of the C-type lectin pathogen receptor, DC-SIGN. The current aims of this project are:
 * 1) To define signaling pathways induced by the peanut allergen, Ara h 1, that are necessary for its Th2-skewing effect.
 * 2) To characterize the dendritic cell phenotype induced by Ara h 1 and other Th2 adjuvants that is necessary for priming Th2-polarized T cells.
 * 3) To characterize the adjuvant capacity of Ara h 1 in vivo.

Basophil Activation and Regulation
Effective allergen-specific immunotherapy is known to induce several immunological changes that correlate well with clinical efficacy, including increased levels of allergen-specific IgG and decreased effector cell (basophil, mast cell) responsiveness. These changes precede any decrease in specific IgE and this correlation has led to the hypothesis that the induced IgG inhibits IgE-mediated effector cell activation. Two mechanisms of IgG-mediated inhibition, which are not mutually exclusive, have been proposed: blocking of IgE binding by direct competition for allergen epitopes and IgG-allergen complex engagement of the inhibitory FcγRIIb receptor on effector cells leading to down regulation of FcεRI signaling. Murine studies have suggested that IgG-mediated inhibition of anaphylaxis involves both mechanisms. A third mechanism of effector cell hypo-responsiveness, termed ‘desensitization’, may also be clinically important, especially during the early phases of immunotherapy during which doses of allergen – initially below the threshold for eliciting reactions – are rapidly increased to an effective dose that is above that threshold. The induction of a transient refractory or ‘desensitized’ state by suboptimal doses of allergen has been well studied in vitro using both basophils and mast cells and this is thought to directly relate to the mechanism of in vivo desensitization. Basophils are ideal for the study of immunotherapy mechanisms for several reasons. They are predominantly present in the peripheral blood, can be readily stimulated ex vivo, share expression of both receptor and signaling pathway molecules with mast cells, and can be measured by flow cytometry with minimal manipulation. We have the opportunity to conduct a small, self-contained research project that complements an already funded large prospective clinical study of sublingual immunotherapy for food allergy. This project has the potential both to give new and unique insight into the mechanisms of sublingual immunotherapy and to establish innovative methods for the assessment of basophil activation by direct measurement of intracellular phosphorylation of signaling molecules. Our central hypothesis is that immunotherapy down-regulates effector cell function by inhibiting FcεRI signaling.

Mechanisms of Immune Tolerance in Children with Food Allergy
The majority of infants and toddlers diagnosed with milk allergy will go on to outgrow the condition on their own by the time they reach school age. We are collaborating with Anna Nowak-Wegrzyn, M.D. and Hugh A. Sampson, M.D. to study mechanisms for the resolution of milk and egg allergy. Two putative mechanisms of food allergy resolution are currently being investigated:
 * 1) As food allergy may be driven by a failure of normal immune tolerance, we hypothesize that its resolution will be associated with an increase of allergen-specific regulatory T cells. Using CFSE to identify allergen-specific cells by proliferation, we are currently measuring the frequency of CD4 T cells expressing a CD25+ CD127- FoxP3+ phenotype in the peripheral blood from food-allergic children over multiple time points as they become tolerant.
 * 2) Basophils are the largest population of allergen-specific cells in peripheral blood. We are studying basophil activation both directly ex vivo and in response to in vitro allergen stimulation in patients as they become tolerant. We hypothesize that basophil hyporesponsiveness will predict clinical tolerance.

Clonal Diversity of B cell Response to Food Antigens
Individuals with immediate hypersensitivity to food have a pathological immune response to ubiquitous dietary antigens characterized by the failure of immune tolerance and the production of specific IgE. There is a spectrum of immunity to food proteins from non-sensitized, to sensitized, transiently allergic, or persistently allergic individuals. The natural history of this immune response and how it varies between these groups is poorly understood. We hypothesize that increased allergen-specific antibody diversity, avidity, and IgE/ IgG4 ratio are markers of Th2 immune progression and will correlate with reaction severity and allergy persistence. We have developed a microarray-based immunoassay for the analysis of peanut-specific IgE clonal diversity and shown in a retrospective study a correlation between diversity and reaction severity. We have now developed such an assay for milk allergens as well. This study aims to follow a cohort of milk-sensitized children and age-matched controls prospectively using these markers to evaluate longitudinal changes in the humoral immune responses between and within clinical groups in order to better understand the natural history of milk allergy and the potential prognostic usefulness of these disease markers.