Expansion and Isolation of Ara h 1-Specific CD4+ T Cells

Cryopreserved PBMCs from 1 of the 27 individuals (patient 107) were thawed and cultured in RPMI 1640 supplemental with 2 mM Glutamax (both from Gibco), 10% human serum (MilliporeSigma), and 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher) (complete RPMI), with 50 μg/ml natural Ara h 1 (Indoor Biotechnologies), at a density of 6 × 10⁶ cells in 1 ml medium per well in 24-well plates. Complete RPMI + 10 U/mL IL-2 (R&D Systems) was added after 5 days, and cells were cultured for a total of 14 days to expand Ara h 1–specific CD4⁺ T cells. After harvesting, memory CD4⁺ T cells were isolated with the EasySep human memory CD4⁺ T cell enrichment kit (Stemcell Technologies) and labeled with APC-conjugated Ara h 1 (DRB1*03:01, amino acid 415–425) tetramer (32 (link)) (made in-house), at a concentration of 10 nM for 1 hour at room temperature. After washing off excess tetramer, the cells were labeled with BUV395-conjugated anti-CD3 (clone UCHT1; BD Biosciences), APC-Cy7–conjugated anti-CD4, FITC-conjugated anti-CD45RA, and Live/Dead Fixable Blue stain (L23105; Thermo Fisher) for 30 minutes at 4°C. Live CD3⁺CD4⁺CD45RA^– tetramer⁺ and tetramer^– T cells were sorted with a FACSAria Fusion instrument (BD Biosciences), and genomic DNA was isolated using the AllPrep DNA/RNA Micro Kit.