Single-cell Transcriptional Analysis Reveals Allergen-specific Signatures in Human ?dT Cells

The role of gamma-delta T (?dT) cells in immune responses to common allergens is poorly understood. Here, we utilized single-cell (sc) transcriptomic analysis of allergen-reactive ?dT cells in humans to characterize the transcriptional landscapes and TCR repertoires in response to cockroach (CR) and mouse (MO) allergens. Using a novel Activation-Induced Marker (AIM) assay that allows detection of ?dT cells combined with scRNA sequencing and TCR repertoire analysis, we identified both shared and allergen-specific ?dT cell activation patterns and gene expression profiles. While CR extract activated both Vd1 and Vd2 subsets, MO extract primarily stimulated Vd2 cells. Our analysis revealed allergen-specific clusters with distinct functional signatures, including enhanced inflammatory responses and cytotoxic effector functions in MO-specific ?dT cells and natural killer cell-mediated immunity and IFN? signaling in CR-specific populations. Comparison of allergic and non-allergic individuals highlighted differences in gene expression and TCR repertoires, including a higher IFNG expression in the CR-allergic compared to non-allergic cohorts, suggesting that phenotypic and functional differences are associated with ?dT allergen responses. This study provides insights into the cellular and molecular heterogeneity and functionality of allergen-reactive ?dT cells, offering a foundation for understanding their role in allergic diseases and potential therapeutic interventions. Overall design: PBMCs were isolated from 53 donors, of which 37 were sensitized to either mouse (MO, n=17) or cockroach (CR, n=20), 1 donor was sensitized to both MO and CR, and 15 were non-allergic healthy control subjects with undetectable IgE titers for both MO and CR allergens. Briefly, after overnight resting PBMCs were stimulated with extracts (10 µg/ml) or HDMAPP (10 µg/ml), or medium alone as negative control, and incubated for 24h at 37ºC. AIM+ (CD69+CD137+) Vd1 and Vd2 cells were captured by fluorescence-activated cell sorting and underwent 10x single-cell RNA sequencing.