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, 11 (1), 1091

Mass Cytometry Reveals Cellular Fingerprint Associated With IgE+ Peanut Tolerance and Allergy in Early Life

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Mass Cytometry Reveals Cellular Fingerprint Associated With IgE+ Peanut Tolerance and Allergy in Early Life

Melanie R Neeland et al. Nat Commun.

Abstract

IgE-mediated peanut allergic is common, often serious, and usually lifelong. Not all individuals who produce peanut-specific IgE will react upon consumption of peanut and can eat the food without adverse reactions, known as sensitized tolerance. Here, we employ high-dimensional mass cytometry to define the circulating immune cell signatures associated with sensitized tolerance and clinical allergy to peanut in the first year of life. Key features of clinical peanut allergic are increased frequency of activated B cells (CD19hiHLADRhi), overproduction of TNFα and increased frequency of peanut-specific memory CD4 T cells. Infants with sensitized tolerance display reduced frequency but hyper-responsive naive CD4 T cells and an increased frequency of plasmacytoid dendritic cells. This work demonstrates the utility and power of high-dimensional mass cytometry analysis to interrogate the cellular interactions that are associated with allergic sensitization and clinical food allergy in the first year of life.

Conflict of interest statement

K.C.N. reports grants and other from NIAID other from Novartis, personal fees and other from Regeneron, grants and other from FARE, grants from EAT, other from Sanofi, other from Astellas, other from Nestle, other from BeforeBrands, other from Alladapt, other from ForTra, other from Genentech, other from AImmune Therapeutics, other from DBV Technologies, personal fees from Astrazeneca, personal fees from ImmuneWorks, personal fees from Cour Pharmaceuticals, grants from Allergenis, grants from Ukko Pharma, other from AnaptysBio, other from Adare Pharmaceuticals, other from Stallergenes-Greer, other from NHLBI, other from NIEHS, other from EPA, other from WAO Center of Excellence, other from Iggenix, other from Probio, other from Vedanta, other from Centecor, other from Seed, from Immune Tolerance Network, from NIH, outside the submitted work. In addition, K.C.N. has a patent Inhibition of Allergic Reaction to Peanut Allergen using an IL-33 Inhibitor pending, a patent Special Oral Formula for Decreasing Food Allergy Risk and Treatment for Food Allergy pending, a patent Basophil Activation Based Diagnostic Allergy Test pending, a patent Granulocyte-based methods for detecting and monitoring immune system disorders pending, a patent Methods and Assays for Detecting and Quantifying Pure Subpopulations of White Blood Cells in Immune System Disorders pending, a patent Mixed Allergen Compositions and Methods for Using the Same pending, and a patent Microfluidic Device and Diagnostic Methods for Allergy Testing Based on Detection of Basophil Activation pending.

Figures

Fig. 1
Fig. 1. Experimental workflow for mass cytometry analysis of PBMCs from 1-year-old infants.
Cryopreserved PBMCs from n = 12 peanut-allergic (PA), n = 12 peanut-sensitized tolerant (PST), and n = 12 non-allergic (NA) healthy 1-year-old infants were thawed and underwent a 24 h stimulation with media (control), pure peanut protein (specific stimulation), or PMA/ionomycin (nonspecific stimulation). Samples were barcoded and stained with a panel of antibodies against 24 surface markers and 8 intracellular markers, and analyzed with a Helios Mass Cytometer. For data analysis, unsupervised computational analyses (clustering and visualization) and manual gating analyses were performed in parallel, along with statistical analyses to identify immune signatures significantly different between the clinical groups.
Fig. 2
Fig. 2. Immune cell profiling of unstimulated PBMCs from 1-year-old infants by manual gating.
a The six major immune cell populations identified in unstimulated PBMC expressed as percentage of live cells in n = 12 healthy 1-year-old infants. b Stacked bar graph representing the major immune cell populations in each individual, stratified by clinical outcome for non-allergic (NA) (n = 12), peanut-sensitized tolerant (PST) (n = 12), and peanut-allergic (PA) (n = 12) infants. c Each major immune cell population was subtyped into further populations in unstimulated PBMC from n = 12 healthy infants. d Percentage of CD4 T cells, CD8 T cells, B cells, NK cells, monocytes, and dendritic cells (DCs) producing IFNγ, TNFα, or IL-2 following 24 h stimulation of PBMC with PMA/ionomycin in n = 12 healthy infants. In the boxplots, the medians are shown. The “hinges” represent the first and third quartile. The whiskers are the smallest and largest values after exclusion of outliers (greater than the 75th percentile plus 1.5 times the IQR or less than 25th percentile minus 1.5 times the IQR). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Unsupervised analysis using FlowSOM reveals distinct immune cell clusters in PBMC from 1-year-old infants.
a Clustering analysis with FlowSOM revealed 16 cell clusters, shown here as a heatmap of the median expression of 18 lineage markers along with a bar graph representing each cluster as percentage of live cells and the cell phenotype that each cluster was assigned based on lineage marker expression pattern. b Stacked bar graph representing all clusters identified in each individual, stratified by clinical outcome for non-allergic (NA) (n = 12), peanut-sensitized tolerant (PST) (n = 12), and peanut-allergic (PA) (n = 12) infants. Clusters with <1% of the analyzed cells were excluded from all other analyses and are represented here as “below cluster size cutoff”. c Uniform Manifold Approximation and Projection (UMAP) representation of 100,008 randomly selected cells (2778 per file) with clusters from the FlowSOM analysis overlaid. The same color is used for each cluster/cell type across all plots. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Unsupervised and manual gating analysis of resting and PMA/ionomycin-stimulated PBMCs reveals differences in multiple immune signatures between non-allergic (NA), peanut-sensitized tolerant (PST), and peanut-allergic (PA) infants.
Frequency (as % of live cells) of (a) naive CD4 T-cell cluster and (b) B-cell cluster 2 (CD19++HLADR++) identified by unsupervised analysis in unstimulated PBMCs from NA, PST, and PA infants. c, d Frequency (as % of live cells) of resting plasmacytoid DCs (CD123+CD11c) and TNF-α+ cells as identified by manual gating from NA, PST, and PA infants. Median expression of (e) IL-2 in the naive CD4 T-cell cluster and (f) IFNγ in effector memory HLADR + CD4 T-cell cluster as identified by unsupervised analysis in NA, PST, and PA infants. P-values by χ2-tests in mixed-effects model analyses, between the three groups and post-hoc pairwise. Median expression values were adjusted for batch before plotting. Blue dots represent values determined by manual gating, whereas red dots represent values derived from the unsupervised analysis. In the boxplots, the medians are shown. The “hinges” represent the first and third quartile. The whiskers are the smallest and largest values after exclusion of outliers (greater than the 75th percentile plus 1.5 times the IQR or less than 25th percentile minus 1.5 times the IQR). Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Analysis of manually gated peanut-specific CD4 T cells shows significant differences between peanut-sensitized tolerant (PST) and peanut-allergic (PA) infants.
a Peanut-specific CD4 T cells were identified in peanut-stimulated cultures based on upregulation and co-expression of T-cell activation markers CD40L and CD69. The positive control, PMA/ionomycin stimulation, was used to set the gates to identify these peanut-activated T cells in all groups. Peanut-specific CD4 T cells were identified in PA infants (n = 11), PST infants (n = 12), and NA infants (n = 12), and expressed as (b) proportion of CD4 T cells and (c) fold change after peanut stimulation over unstimulated (media) conditions. P-values by F-tests in linear models. In the boxplots, the medians are shown. The “hinges” represent the first and third quartile. The whiskers are the smallest and largest values after exclusion of outliers (greater than the 75th percentile plus 1.5 times the IQR, or less than 25th percentile minus 1.5 times the IQR). Source data are provided as a Source Data file.

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