Single-cell characterization of dog allergen-specific T cells reveals TH2 heterogeneity in allergic individuals

Background: Allergen-specific type 2 CD4(+) T(H)2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the T(H)2 response has only recently been appreciated. Objective: 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. Methods: 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. Results: Dog allergen-specific T-cell responses in allergic subjects were dominantly of T(H)2 type. T(H)2 cells could be phenotypically further divided into 3 subsets, which consisted of T(H)2-like (CCR6(-) CXCR3(-) CRTH2(-)), T(H)2 (CCR6(-)CXCR3(-)CRTH2(+)CD161(-)), and T(H)2A (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(H)2-biased signature in allergen-specific T cells from allergic subjects and revealed a T(H)1/T(H)17 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(H)2-like, T(H)2, and T(H)2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. Conclusions: Our study demonstrates heterogeneity within allergen-specific T(H)2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.