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Identification And Characterization Of T cell Epitopes In Mouse Allergy
AB92 Abstracts
SATURDAY
291
Investigation of Peanut-Specific T Effector (Teff) and T Regulatory (Treg) Cells in the Blood of Allergic and Non-Allergic Individuals
Katherine Weissler1, Marjohn Rasooly1, Daly Cantave2, Hyejeong Bolan1, and Pamela A. Frischmeyer-Guerrerio, MD, PhD1; 1Laboratory of Allergic Diseases, NIAID, NIH, Bethesda, MD, 2Clinical Center, NIAID, NIH, Bethesda, MD. RATIONALE: Peanut allergy is potentially life-threatening and generally has a poor prognosis. Recent data suggests the skin may be an important route of initial sensitization to peanut, and that early oral exposure to peanut is protective. In mice, Tregs are central to the development of tolerance to food, although definitive data in humans is lacking. We sought to quantify and phenotype peanut-specific Teffs and Tregs in individuals with and without peanut allergy. METHODS: Whole blood was obtained from peanut allergic and nonsensitized/non-allergic individuals and stimulated with crude peanut extract or media alone. Peanut-specific Teffs and Tregs were identified based on upregulation of CD154 (CD40L) or CD137 (4-1BB), respectively. Flow cytometry was used to evaluate expression of cytokines and homing receptors. RESULTS: Differential upregulation of CD154 and CD137 efficiently distinguished peanut-specific Tregs (>90% Foxp3+) and T effs (<10% Foxp3+). Peanut-reactive Tregs did not produce cytokine and were found at a similar or greater frequency in the blood of peanut allergic individuals compared to non-allergic controls. Peanut-specific Teffs from allergic individuals produced predominantly IL-13 to peanut while non-allergic subjects expressed mostly IFN-g. No significant difference in expression of skin (CLA) homing receptors on peanut-specific T cells was identified, although greater numbers of peanut-specific Teffs expressed gut (a4b7) homing receptors in allergic versus non-allergic individuals. CONCLUSIONS: Established peanut allergy is not associated with a deficiency in or altered homing of peanut-specific Tregs in the blood. Peanut-specific Teff cells are Th2- rather than Th1-skewed in peanut allergic subjects, and may be more likely to traffic to the gut.
292
CD4+ T cell Responses In Cow's Milk allergy
Diego Archila, PhD1, Mary L. Farrington, MD2, David M. Robinson, MD2, Fatima S. Khan, MD, FAAAAI3, and William W. Kwok, PhD1; 1Benaroya Research Institute at Virginia Mason, Seattle, WA, 2Virginia Mason Medical Center, Seattle, WA, 3Altru, Grand Forks, ND. RATIONALE: Cow’s Milk Allergy (CMA) is the most common food allergy in children with b-lactoglobulin and casein proteins as the major milk allergens. Casein consists of several isoforms: aS1-casein, aS2casein, b-casein and k-casein. Little is known about specific T-cell responses toward these allergens in adults and children with CMA and non-allergic subjects. METHODS: A total of 23 milk allergic subjects, including 10 adults and 13 children, and 18 milk non-allergic subjects were recruited. T cell responses toward b-lactoglobulin, aS1-casein, aS2-casein, b-casein and k-casein were evaluated by CD154+ up-regulation assays and tetramer assays. RESULTS: T cell responses were significantly higher in allergic subjects compared to non-allergic subjects. No significant difference in frequencies between adults and children with CMA were observed. With tetramers, blactoglobulin, aS1-casein, aS2-casein, b-casein and k-casein-epitope specific memory T cells were detected in 30%, 80%, 40%, 65% and 45% of 20 allergic subjects. Majority of allergen-specific cells were CCR4+. Variable expressions of CCR6, CD27 and CRTH2 were observed in these cells. With CD154+ up-regulation assay, cytokine profiles of Bos d-specific cells were examined. Amongst the cytokines producers, majority of cells
J ALLERGY CLIN IMMUNOL FEBRUARY 2017
produced IL-4 and IL-13. A subgroup of children with CMA had cells that coproduced IL-4 and IL-17. CONCLUSIONS: aS1-casein was the major allergen in eliciting T cell responses. In adults with CMA, a committed TH2 response was observed. On the other hand, in children with CMA, TH2/TH17 responses were more prevalent, suggesting that these T-cell populations are not fully committed into the TH2 phenotype and could play a role in CMA outgrowth.
293
Identification And Characterization Of T cell Epitopes In Mouse Allergy
Luise Westernberg1, Sinu Paul2, Bjoern Peters3, Alessandro Sette, PhD4, and Veronique Schulten, PhD2; 1La Jolla Institute for Allergy and Immunology, La Jolla, 2La Jolla Institute for Allergy and Immunology, La Jolla, CA, 3La Jolla Institute for Allergy and Immunology, San Diego, CA, 4La Jolla Institute for Allergy & Immunology, La Jolla, CA. RATIONALE: Mice (Mus musculus) are ubiquitous, exiting in every climate all over the world, both indoors and out. Mouse allergy has become a prevalent disease affecting mostly laboratory workers and inner-city households. Although several allergenic proteins has been identified in mouse epithelium and urine, little is know about their immunogenicity. METHODS: Using a combination of proteomics and bioinformatics, we sought to map T cell epitopes using PBMC from mouse allergic patients. Peptides derived from the major allergen Mus m 1, its variants, other proteins derived from mouse urine and epithelial extract as well as homologs to other mammalian allergens were screened for T cell reactivity in PBMCs from mouse allergic patients by ELISPOT. RESULTS: Screening of ;1200 peptides lead to the identification of 59 different T cell epitopes from 18 different proteins that elicited cytokine production in PBMC from 2 or more allergic donors. Ninety-two percent of the total response targeted murine urinary proteins. CONCLUSIONS: To the best of our knowledge, this is the first study to identify T cell epitopes from mouse proteins. The set of peptides characterized herein will serve as a tool to study mouse-specific T cells in different contexts. Mouse allergy in has been described as a correlate of asthma disease. Studying antigen-specific T cell responses in allergic, asthmatic and healthy but exposed individuals may give us great insight into what immunological events are associated with onset of disease. Furthermore, clearly defined T cell epitopes holds great value for the development of an immunotherapeutic approach,
SATURDAY
291
Investigation of Peanut-Specific T Effector (Teff) and T Regulatory (Treg) Cells in the Blood of Allergic and Non-Allergic Individuals
Katherine Weissler1, Marjohn Rasooly1, Daly Cantave2, Hyejeong Bolan1, and Pamela A. Frischmeyer-Guerrerio, MD, PhD1; 1Laboratory of Allergic Diseases, NIAID, NIH, Bethesda, MD, 2Clinical Center, NIAID, NIH, Bethesda, MD. RATIONALE: Peanut allergy is potentially life-threatening and generally has a poor prognosis. Recent data suggests the skin may be an important route of initial sensitization to peanut, and that early oral exposure to peanut is protective. In mice, Tregs are central to the development of tolerance to food, although definitive data in humans is lacking. We sought to quantify and phenotype peanut-specific Teffs and Tregs in individuals with and without peanut allergy. METHODS: Whole blood was obtained from peanut allergic and nonsensitized/non-allergic individuals and stimulated with crude peanut extract or media alone. Peanut-specific Teffs and Tregs were identified based on upregulation of CD154 (CD40L) or CD137 (4-1BB), respectively. Flow cytometry was used to evaluate expression of cytokines and homing receptors. RESULTS: Differential upregulation of CD154 and CD137 efficiently distinguished peanut-specific Tregs (>90% Foxp3+) and T effs (<10% Foxp3+). Peanut-reactive Tregs did not produce cytokine and were found at a similar or greater frequency in the blood of peanut allergic individuals compared to non-allergic controls. Peanut-specific Teffs from allergic individuals produced predominantly IL-13 to peanut while non-allergic subjects expressed mostly IFN-g. No significant difference in expression of skin (CLA) homing receptors on peanut-specific T cells was identified, although greater numbers of peanut-specific Teffs expressed gut (a4b7) homing receptors in allergic versus non-allergic individuals. CONCLUSIONS: Established peanut allergy is not associated with a deficiency in or altered homing of peanut-specific Tregs in the blood. Peanut-specific Teff cells are Th2- rather than Th1-skewed in peanut allergic subjects, and may be more likely to traffic to the gut.
292
CD4+ T cell Responses In Cow's Milk allergy
Diego Archila, PhD1, Mary L. Farrington, MD2, David M. Robinson, MD2, Fatima S. Khan, MD, FAAAAI3, and William W. Kwok, PhD1; 1Benaroya Research Institute at Virginia Mason, Seattle, WA, 2Virginia Mason Medical Center, Seattle, WA, 3Altru, Grand Forks, ND. RATIONALE: Cow’s Milk Allergy (CMA) is the most common food allergy in children with b-lactoglobulin and casein proteins as the major milk allergens. Casein consists of several isoforms: aS1-casein, aS2casein, b-casein and k-casein. Little is known about specific T-cell responses toward these allergens in adults and children with CMA and non-allergic subjects. METHODS: A total of 23 milk allergic subjects, including 10 adults and 13 children, and 18 milk non-allergic subjects were recruited. T cell responses toward b-lactoglobulin, aS1-casein, aS2-casein, b-casein and k-casein were evaluated by CD154+ up-regulation assays and tetramer assays. RESULTS: T cell responses were significantly higher in allergic subjects compared to non-allergic subjects. No significant difference in frequencies between adults and children with CMA were observed. With tetramers, blactoglobulin, aS1-casein, aS2-casein, b-casein and k-casein-epitope specific memory T cells were detected in 30%, 80%, 40%, 65% and 45% of 20 allergic subjects. Majority of allergen-specific cells were CCR4+. Variable expressions of CCR6, CD27 and CRTH2 were observed in these cells. With CD154+ up-regulation assay, cytokine profiles of Bos d-specific cells were examined. Amongst the cytokines producers, majority of cells
J ALLERGY CLIN IMMUNOL FEBRUARY 2017
produced IL-4 and IL-13. A subgroup of children with CMA had cells that coproduced IL-4 and IL-17. CONCLUSIONS: aS1-casein was the major allergen in eliciting T cell responses. In adults with CMA, a committed TH2 response was observed. On the other hand, in children with CMA, TH2/TH17 responses were more prevalent, suggesting that these T-cell populations are not fully committed into the TH2 phenotype and could play a role in CMA outgrowth.
293
Identification And Characterization Of T cell Epitopes In Mouse Allergy
Luise Westernberg1, Sinu Paul2, Bjoern Peters3, Alessandro Sette, PhD4, and Veronique Schulten, PhD2; 1La Jolla Institute for Allergy and Immunology, La Jolla, 2La Jolla Institute for Allergy and Immunology, La Jolla, CA, 3La Jolla Institute for Allergy and Immunology, San Diego, CA, 4La Jolla Institute for Allergy & Immunology, La Jolla, CA. RATIONALE: Mice (Mus musculus) are ubiquitous, exiting in every climate all over the world, both indoors and out. Mouse allergy has become a prevalent disease affecting mostly laboratory workers and inner-city households. Although several allergenic proteins has been identified in mouse epithelium and urine, little is know about their immunogenicity. METHODS: Using a combination of proteomics and bioinformatics, we sought to map T cell epitopes using PBMC from mouse allergic patients. Peptides derived from the major allergen Mus m 1, its variants, other proteins derived from mouse urine and epithelial extract as well as homologs to other mammalian allergens were screened for T cell reactivity in PBMCs from mouse allergic patients by ELISPOT. RESULTS: Screening of ;1200 peptides lead to the identification of 59 different T cell epitopes from 18 different proteins that elicited cytokine production in PBMC from 2 or more allergic donors. Ninety-two percent of the total response targeted murine urinary proteins. CONCLUSIONS: To the best of our knowledge, this is the first study to identify T cell epitopes from mouse proteins. The set of peptides characterized herein will serve as a tool to study mouse-specific T cells in different contexts. Mouse allergy in has been described as a correlate of asthma disease. Studying antigen-specific T cell responses in allergic, asthmatic and healthy but exposed individuals may give us great insight into what immunological events are associated with onset of disease. Furthermore, clearly defined T cell epitopes holds great value for the development of an immunotherapeutic approach,