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

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). Subjectswere 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. PeanutresponsiveTcellswere identified based onCD154 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 peanutresponsive CD4 1 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 T(H)2 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 T(H)2 cells producing T(H)2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional T(H)2 response was more evident than a Treg cell deficit among peanut-responsive T cells.