Skin suction blisters and biopsies

Skin suction blisters were induced on the antecubital fossa using a low-pressure device from Electronic Diversities (Ridge Road, Finksburg, MD), as described previously (20, 78). Cell-free blister fluid was used for proteomic multiplex assays, and cells pelleted from blister fluid and the enzymatically digested blister roof (i.e., the epidermis) were subjected to FACS sorting (FACSAria III, BD Biosciences, Franklin Lakes, NJ) (fig. S1) for enrichment of CD45⁺ leukocytes (50% of sample), to obtain comparable numbers between treated and untreated samples and allow meaningful comparisons even of small cell fractions. Leukocytes were then admixed to CD45⁻ cells in equal amounts and then analyzed using scRNA-seq (10x Genomics, Pleasanton, CA), as previously described (20). Six-millimeter full-thickness punch biopsies were performed from an independent cohort of patients with untreated AD, patients with AD treated for 1 year with dupilumab standard dosing, and healthy controls (table S1), according to standard procedures.