Single-cell profiling of peanut-responsive T cells in patients with peanut allergy reveals heterogeneous effector TH2 subsets

Abstract

Background: The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood.

Objectives: Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of subjects with and without peanut allergy (PA).

Methods: We obtained samples from patients with PA, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (Consortium of Food Allergy Research [CoFAR] 6). Subjects were confirmed as having PA, or if they passed a 1-g peanut challenge, they were termed high-threshold subjects. Healthy control (HC) subjects were also recruited. Peanut-responsive T cells were identified based on CD154 expression after 6 to 18 hours of stimulation with peanut extract. Cells were analyzed by using flow cytometry and single-cell RNA sequencing.

Results: Patients with PA had tissue- and follicle-homing peanut-responsive CD4+ T cells with a heterogeneous pattern of TH2 differentiation, whereas control subjects had undetectable T-cell responses to peanut. The PA group had a delayed and IL-2-dependent upregulation of CD154 on cells expressing regulatory T (Treg) cell markers, which was absent in HC or high-threshold subjects. Depletion of Treg cells enhanced cytokine production in HC subjects and patients with PA in vitro, but cytokines associated with highly differentiated TH2 cells were more resistant to Treg cell suppression in patients with PA. Analysis of gene expression by means of single-cell RNA sequencing identified T cells with highly correlated expression of IL4, IL5, IL9, IL13, and the IL-25 receptor IL17RB.

Conclusions: These results demonstrate the presence of highly differentiated TH2 cells producing TH2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional TH2 response was more evident than a Treg cell deficit among peanut-responsive T cells.

Trial registration: ClinicalTrials.gov NCT01904604.

Keywords: Food allergy; T(H)2; peanut allergy; regulatory T; tolerance.

Copyright © 2018 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Figures

Figure 1. Immunologic responses to peanut stimulation in allergic and control subjects

A. Representative dot plot of CD154 response to peanut in CD3+CD4+ cells. B. Quantification of CD154+ T cells in peanut allergic (PA, n=69, CoFAR cohort), healthy control (HC, n=7), and high threshold (HT, n=9) subjects after culture with peanut (+) or media (−). C. Quantification of total cytokine+CD154+ T cells in PA, HC, and HT subjects for IFNγ, IL-10, IL-4, and IL-13 in response to peanut stimulation. Statistics calculated by Kruskal-Wallis with Dunn’s multiple comparison test. As illustrated in D, total CD3+CD4+ T cells, CD154+ T cells (gated rectangle), and Th2 cells (IL-4+CD154+ T cells, gated circle) were evaluated for CXCR5 (n=60), CCR4 (n=61), and CCR6 (n=61) expression (calculated as % positive within indicated populations) after stimulation for 6h with peanut extract (E) or anti-CD3/CD28 (F). Statistics calculated by Friedman’s test with Dunn’s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2. Phenotypic heterogeneity of peanut-responsive Th2 cells

A. Frequency of expression of memory and differentiation markers on CD154+ cells after stimulation with peanut (CPE) or anti-CD3/CD28. Each symbol represents one subject (n=6–13, MSSM PA cohort). B. Representative flow cytometry plot showing co-expression of IL-5 and IL-9 in peanut-responsive Th2 (CD154+IL-4+) cells. C. Percent frequency of CD154+ T cells co-expressing indicated cytokines after stimulation with peanut (CPE) or anti-CD3/CD28. Each symbol represents an individual subject (n=6–11, MSSM PA Cohort). D. Memory marker expression on each cell subset. Each bar represents the mean and SEM of 8 subjects (MSSM PA cohort). E. Quantification of secreted cytokines after peanut stimulation of PBMCs from PA (n=33), HC (n=5), and HT (n=3) subjects for 5 days. Statistics calculated by Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. Identification and phenotypic analysis of peanut-responsive Tregs

A. Quantification of CD154+FoxP3+CD25+CD127lowCD4+ T cells after stimulation with peanut (+) for 18 h in PA (n=62, CoFAR cohort), HC (n=6), and HT (n=3) subjects. B. Expression of CCR4 and CCR6 (n=57, CoFAR PA cohort) on CD4+ T cells, CD154+CD4+ T cells (CD154 T), FoxP3+CD25+CD127low Tregs (Treg), and CD154+FoxP3+CD25+CD127− cells (CD154 Treg) after peanut stimulation. C. Representative dot plots showing the impact of rhIL2 on CD154 expression in CD4+ T cells or Tregs after 18h. D. Impact of IL-2 neutralization on CD154 expression on CD4+ T cells or Tregs after 18h of peanut stimulation (n = 4 PA subjects). E. Impact of Treg depletion (removal of CD3+CD4+CD25highCD127low by FACS) on peanut-induced cytokine secretion. Individual values are shown for PA (MSSM cohort, n=10) or HC (n=9) subjects. *p<0.05, **p<0.01 ***p<0.001 **** p<0.0001. Statistics calculated with Wilcoxon matched pairs signed rank test (A,E) or Friedman’s test with Dunn’s post-test correction (B).

Figure 4. Single-Cell Sequencing Analysis of CD154+ T cells and Tregs

A. Minimum spanning tree showing clustering and ordering of peanut-activated T cells, polyclonally-activated T cells, and Tregs from PA or HC into 3 states, and the distribution of cells by phenotype within those states. B. Heat maps showing genes differentially expressed between peanut-activated T cells in the 3 states. C. Pearson correlation of genes significantly upregulated in State 3. The x and y axes depict a hierarchy of genes that exhibit similar correlation values and are thus co-expressed. Only genes with a correlation >0.5 with another significantly upregulated gene are shown. D. TCR analysis of peanut-activated cells from a peanut allergic donor (Subject ID 1401) indicating shared TCR α and/or β chains. Each circle indicates one cell, with red line indicating a shared α chain and blue line indicating shared β chain.

Figure 5. Proposed model of the contribution of peanut-responsive T cells to peanut allergy

Stimulation of PBMCs with peanut leads to early (6h) activation of memory Th2 cells expressing IL-4 and IL-13, and memory Th2+ cells co-expressing IL-4 and IL-13 with IL-5 +/− IL-9. Th1 and Th17 cells remain quiescent. Th2 cells express molecules to facilitate homing to skin, lung, intestine, or B cell follicles, where we hypothesize that they may promote allergic effector cell expansion (skin, mucosa) or IgE-class switch (follicle). The heterogeneous T cell effector T cell response to peanut also includes IL-3 and Csf2 that may also contribute to tissue inflammation through actions on allergic effector cells and DCs, respectively. These distal effects on effector cells and IgE production would be expected to contribute to symptoms on peanut re-exposure. Release of IL-2 promotes the subsequent activation of Tregs. Tregs completely suppress (red arrows) Th1 and Th17 responses, partially suppress Th2 responses, but do not significantly suppress Th2+ responses. We speculate that targeting the heterogeneous Th2 response, including the Th2+ cells through molecules such as IL17RB, may be effective in the treatment of peanut allergy.