Single-cell characterization of dog allergen-specific T cells reveals T_H2 heterogeneity in allergic individuals.

Abstract

Allergen-specific type 2 CD4⁺ T_H2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the T_H2 response has only recently been appreciated. We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4⁺ T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy. Dog allergen-specific memory CD4⁺ T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing. Dog allergen-specific T-cell responses in allergic subjects were dominantly of T_H2 type. T_H2 cells could be phenotypically further divided into 3 subsets, which consisted of T_H2-like (CCR6^-CXCR3^-CRTH2^-), T_H2 (CCR6^-CXCR3^-CRTH2⁺CD161^-), and T_H2A (CCR6^-CXCR3^-CRTH2⁺CD161⁺CD27^-) cells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the T_H2-biased signature in allergen-specific T cells from allergic subjects and revealed a T_H1/T_H17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between T_H2-like, T_H2, and T_H2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. Our study demonstrates heterogeneity within allergen-specific T_H2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.