Single-cell transcriptome analysis of CD3+ T lymphocytes from prurigo nodularis lesional skin reveals Th17 polarization

Prurigo nodularis (PN) is a chronic inflammatory skin disease characterized by symmetrically distributed, intensely pruritic nodules of unknown etiology and unmet therapeutic need. The lack of approved therapies and the limited efficacy of off-label treatments reflect the current poor understanding of its pathogenesis. We aimed to comprehensively characterize the phenotypic variation of CD3+ T cells and asses cell-specific gene expression in the lesional skin of patients with PN versus in patients with atopic dermatitis (AD) and in healthy controls (HC). To this end, single-cell RNA sequencing (scRNA-Seq) using 10x Genomics was carried out to compare CD3+ T cells transcriptomic heterogeneity between lesional samples of PN (n=6), AD (n=5) and HC (n=5).

Four millimetres punch skin biopsies were obtained from lesional skin of 11 patients (6 from PN and 5 from AD) and 5 from HC. CD3+ T cells were sorted by flow cytometry from cell suspension obtained after enzymatic and mechanic dissociation of lesional skin biopsy samples or healthy skin samples. Samples with at least 90% viability after lymphocytes sorting were used for further scRNA-Seq analysis. Cell suspensions were loaded onto separate chip inlet to perform cell encapsulation into droplets. Reverse transcription, cDNA generation and library preparation were achieve using the standard 10X Genomics single cell protocol. Raw sequencing data were demultiplexed, aligned to a reference genome (GrCh38) and counted using Cell Ranger (version 3.0.2, 10x Genomics).