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Background Allergen-specific type 2 CD4+ T helper (Th2) cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the Th2 response has only recently been appreciated. Objective To characterize at the single-cell level the ex vivo phenotype, transcriptomic profile and 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 Th2-type. Th2 cells could be phenotypically further divided into three subsets, which consisted of Th2-like (CCR6-CXCR3-CRTH2-), Th2 (CCR6-CXCR3-CRTH2+CD161-) and Th2A (CCR6-CXCR3-CRTH2+CD161+CD27-) cells. All these subsets were non-existent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the Th2-biased signature in allergen-specific T cells from allergic subjects and revealed a Th1/17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to non-allergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between Th2-like, Th2 and Th2A cells, the last ones representing the most terminally differentiated and highly polarized subtype. Conclusions Our study demonstrates heterogeneity within allergen-specific Th2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.

Mouse allergy has become increasingly common, mainly affecting laboratory workers and inner-city households. To date, only one major allergen, namely Mus m 1, has been described. We sought to identify T cell targets in mouse allergic patients. PBMC from allergic donors were expanded with either murine urine or epithelial extract and subsequently screened for cytokine production (IL-5 and IFNγ) in response to overlapping peptides spanning the entire Mus m 1 sequence, peptides from various Mus m 1 isoforms [major urinary proteins (MUPs)], peptides from mouse orthologs of known allergens from other mammalian species and peptides from proteins identified by immunoproteomic analysis of IgE/IgG immunoblots of mouse urine and epithelial extracts. This approach let to the identification of 106 non-redundant T cell epitopes derived from 35 antigens. Three major T cell-activating regions were defined in Mus m 1 alone. Moreover, our data show that immunodominant epitopes were largely shared between Mus m 1 and other MUPs even from different species, suggesting that sequence conservation in different allergens is a determinant for immunodominance. We further identified several novel mouse T cell antigens based on their homology to known mammalian allergens. Analysis of cohort-specific T cell responses revealed that rhinitis and asthmatic patients recognized different epitope repertoires. Epitopes defined herein can be formulated into an epitope “megapool” used to diagnose mouse allergy and study mouse-specific T cell responses directly ex vivo. This analysis of T cell epitopes provides a good basis for future studies to increase our understanding of the immunopathology associated with MO-allergy and asthma.