Successful Milk Oral Immunotherapy Promotes Generation of Casein-Specific CD137+ FOXP3+ Regulatory T Cells Detectable in Peripheral Blood

BackgroundOral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (T-REG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific T-REG in a cohort of subjects undergoing OIT. MethodsPediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4(+) T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4(+) T cell phenotypes were quantified by flow cytometry. ResultsOur culture system induced activated casein-specific FOXP3(+)Helios(+) T-REG cells and FOXP3(-) T-EFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific T-REG cells increased significantly with escalating doses of milk during OIT while casein-specific T-EFF cell frequencies remained constant. Moreover, expanded casein-specific T-REG cells expressed higher levels of FOXP3 compared to polyclonal T-REG cells, suggesting a more robust T-REG phenotype. The induction of casein-specific T-REG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific T-REG cells negatively correlated with the time required to reach the maintenance phase of desensitization. ConclusionsOverall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3(+) T-REG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro T-REG response to casein may correlate with the time to reach maintenance in CMP-OIT.