BioSample

Protocols: Five NSG and five NSG-HLA-A2/HHD mice were transplanted with PBMCs (unstimulated, PBMCs were transplantated immediately after their isolation) from 4 different donors (2 millions PBMCs iv, 24h after 2.5Gy TBI), 3 out of 5 mice receiving the PBMCs from a single donor for each mouse while the two remaining animals received the PBMCs from the same donor. PBMCs were isolated by Ficoll density centrifugation. One million of CD3+ cells were sorted from PBMCs of each donor by flow cytometry (FACS Aria III) and preserved at -80°C in Tripure (Roche) while another million of sorted T cells was stimulated in vitro with CD3/CD28 dynabeads (bead:cell ratio 1:1, Invitrogen, Waltham, MA) in X-VIVO 15 (Lonza, Verviers, Belgium). After 4 days of culture, 1 million of T cells was collected from the stimulated cells and cryopreserved in Tripure. At day 7 post-transplantation, mice were sacrificed and 1 million of human CD3+ T cells were sorted from their spleen (in all but one NSG-HLA-A2/HHD mice which died due to irradiation). All sorted T cells were preserved at -80°C in Tripure until the day of RNA extraction. Total RNA was isolated by using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands), following manufacturer’s instructions. Libraries were prepared with the Illlumina Truseq stranded mRNA sample prep kit, based on polyA selection of mRNAs