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

BACKGROUND: Oral 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. METHODS: Pediatric 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. RESULTS: Our 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. CONCLUSIONS: Overall, 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.