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

Yi Zhang,Nils Pavey,Duncan Lejtenyi,Bruce Mazer,Geneviève Genest,Lei Li,Bahar Torabi,Sabrina Bartolucci,Wei Zhao,Tho-Alfakar Al-Aubodah,Ciriaco A Piccirillo,Dan Ke,Moshe Ben-Shoshan

doi:10.3389/fimmu.2021.705615

Abstract

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 (TREG) 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 TREG 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+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG 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+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.

Highlights

Cow’s milk allergy (CMA) affects close to 0.6% of children under 2-years of age [1, 2]
We aimed to evaluate whether CM-Oral immunotherapy (OIT) induced casein-specific, stablysuppressive FOXP3+Helios+ TREG cells and whether this cellular response correlated with successful OIT
The details of the global trial design were recently published and is depicted in Figure 1A [4]. Seven children from this cohort who successfully achieved cow’s milk protein (CMP)-OIT maintenance dosing were randomly selected for this study

Summary

Introduction

Cow’s milk allergy (CMA) affects close to 0.6% of children under 2-years of age [1, 2]. Cow’s milk oral immunotherapy (CMOIT) is emerging as an effective experimental approach to induce tolerance to milk protein, with up to 75% of patients successfully achieving desensitization [4,5,6,7]. Successful CM-OIT requires rigorous patient compliance, any deviation in protocol may prolong the length of time required to reach maintenance or increase the risk of developing an allergic reaction the scheduled CMP doses [9]. Since the clinical response to CM-OIT is highly variable, developing biomarkers that successfully predict ability to achieve desensitization, time to reach maintenance or risk of developing adverse events during therapy would enable the individualization of CM-OIT and increase safety of the procedure. Oral immunotherapy (OIT) is an emerging treatment for cow’s milk protein (CMP) allergy in children. We sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT

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