Determination of Immunophenotypic Changes by CyTOF, Epigenetics and Component Resolved Diagnostics During Successful Desensitization in Multi-food Oral Immunotherapy

Abstract

Participants (n=44, age 4-15 yrs) with double-blind, placebo-controlled food challenge proven food allergy to multiple foods, were administered omalizumab (anti-IgE, n=40) or placebo (n=4) for 16 weeks with oral immunotherapy (OIT) for 2-5 foods, starting 8 weeks after the beginning of omalizumab or placebo (clinical outcomes of this trial in \citeANDORF2018 ). To better understand the immunophenotypical changes leading to successful desensitization, we interrogated changes in immune cell subtypes in PBMCs before and after successful OIT using mass cytometry (CyTOF) on unstimulated as well as PMA/Ionomycin stimulated samples. The first step in this analysis was an unsupervised clustering across the markers within the CyTOF panel used for cell type identification (lineage markers) of a pooled dataset of all cells of the samples of the two time points. This was done through FlowSOM \citeVanGassen2015, using self-organizing maps followed by hierarchical consensus meta-clustering. The immune cell subtype of each cluster was determined based on the expression level of the lineage markers of the cells within that cluster. The median level of various functional markers within each cluster were individually determined for each sample. Subsequently we tested whether the median level for each functional marker in each cell type (cluster) was significantly different between baseline and post-OIT. Further mechanistic experiments included epigenetics (pyrosequencing of bisulfite treated genomic DNA purified from participant's PBMCs) and component resolved diagnostics (ThermoFisher). Our preliminary results indicated a significant decrease (FDR-adjusted P < 0.01) of CD28 and GPR15 levels in effector memory CD4+ T cells after successful OIT compared to baseline. A significant increase (FDR-adjusted P < 0.01) in IL-10 was detected in the Treg and gamma-delta T cell populations. Epigenetic data demonstrated hypermethylation of the -48 CpG site in the IL-4 promoter region post-OIT (FDR-adjusted P < 0.01). The IgG4/IgE ratio of antibodies to most of the whole foods in the participant's OIT and to the corresponding storage proteins showed a significant increase (FDR-adjusted P < 0.01) between baseline and post-OIT. Our data thus imply that T cell anergy induced through OIT might contribute to successful desensitization.