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

Abstract

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.

Keywords: allergy; clinical trial; desensitization; milk immunotherapy; regulatory T cells; tolerance.