GolgiPlug (BD Biosciences, San Jose, CA) was added 4 hours prior to harvesting. T cell phenotypic analysis. Harvested PBMCs were stained for viability (Live/Lifeless Fixable stain, Invitrogen), washed and stained for surface markers, and washed for fixation and permeabilization. IL-10 or IFN-, as well as presence of egg-responsive Foxp3+CD25+CD127low Tregs. Egg-responsive T cells expressed CCR4, CCR6, and CXCR5, indicating capacity for homing to skin, mucosa, and B cell follicles. However, neither the frequency nor phenotype of egg-responsive T cells were different in those with tolerance or reactivity to BE. Conclusions: Egg-specific antibody and basophil responses, but not T cell responses, are higher in those with reactivity versus tolerance to BE. The egg-specific antibody and ELN484228 T cell responses were highly heterogeneous in this cohort. The clinical implications of this immune heterogeneity will need to be analyzed longitudinally. Keywords: Egg allergy, food allergy, IgE, IgG4, basophil, CD4+ T lymphocyte, Treg, Th2 Capsule Summary In a study of 129 children challenged to baked and unheated forms of egg, significantly lower egg-specific IgE and basophil activation, but no differences in egg-specific Th2 cells or Tregs, were associated with tolerance to BE. Introduction Over the past decade, it has become increasingly clear that a large subset of children with egg allergy are able to tolerate egg protein after it has been extensively heated, i.e. heat-denatured.1-4 Specific predictors of those patients most likely to tolerate baked egg (BE) have been identified, including prick skin screening, egg-specific IgE and ovomucoid-specific IgE.5, 6 Children with tolerance to heated egg are more likely to outgrow their egg allergy by 2 years of life 7. Longitudinal studies have also exhibited that exposure to heated egg enhances eventual tolerance to less heated forms of egg. 8, 9 In addition to these clinical outcomes, studies have also helped to define the immunologic changes that occur with regular exposure to BE in egg-allergic patients.4, 8 Despite these improvements, there is a lack of information about detailed immunologic characteristics that may differentiate egg allergic patients who do or do not tolerate extensively heated egg. In this study, we had the opportunity to conduct detailed studies profiling basophil and T cell responsiveness as well as serologic steps at baseline in a large cohort of children who underwent double blind placebo controlled challenges to baked or unheated egg. Methods Participants. ELN484228 Baseline blood ELN484228 samples were obtained from children aged 3-16 years at the time of enrollment (prior to egg exposure through diet or immunotherapy) in a multi-center CoFAR intervention trial comparing ELN484228 the efficacy of BE diet or egg oral immunotherapy (OIT) in the treatment of egg allergy (CoFAR7, “type”:”clinical-trial”,”attrs”:”text”:”NCT01846208″,”term_id”:”NCT01846208″NCT01846208). These participants included children with an egg-specific IgE > 5 kUA/L who were avoiding all forms of egg in the diet. Participants underwent an initial double-blind placebo controlled food challenge (DBPCFC) to BE in the form of a muffin. Those who reacted during the BE challenge were categorized as BE reactive. Those who tolerated the BE challenge then underwent a DBPCFC to unheated egg white protein to confirm reactivity to unheated egg. This group was categorized as BE tolerant. After enrollment of the BE reactive group was total (40 participants per the protocol), the baseline blood sample was drawn post-BE challenge from the remaining BE tolerant participants (to determine their qualification for the study before conducting immune profiling). The National Institute of Allergy and Infectious Diseases Allergy and Asthma Data and Security Monitoring Table, and the Institutional Review Boards at Icahn School of Medicine at Mount Sinai, National Jewish Health, Johns Hopkins Medical School, University or college of Arkansas for KIAA0901 Medical Sciences, and University or college of North Carolina, Chapel Hill approved study procedures. Written informed consent was obtained from the parent or guardian. Blood processing and PBMC isolation. Blood samples were obtained in 10 ml sodium-heparin Vacutainer tubes at the 5 clinical sites. An aliquot was retained for basophil.