Figure 1.
Ex vivo expansion and purification of human invariant natural killer T (iNKT) cells. (A) Scheme of iNKT-cell expansion from donor peripheral blood mononuclear cells (PBMCs) by glycolipid stimulation. iNKT cells were restimulated after 7 and 14 days with irradiated and glycolipid-pulsed autologous PBMCs. (B) Representative dot plots indicating percentage of iNKT cells cultured with KRN7000, PBS44, and PBS57 at days 0, 7, 14, and 21. All events were gated on live lymphocytes and iNKT cells were identified by expression of CD3 and staining with the PBS57-loaded CD1d tetramer. (C) Absolute iNKT-cell numbers during 21 days of expansion each starting from 2 × 106 PBMCs. Results for all tested glycolipid analogs [KRN7000 (n = 9), PBS44 (n = 9), and PBS57 (n = 9)] are shown. Error bars indicate SEM. (D) Representative dot plots showing further purification of iNKT cells after 21 days of cell culture by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). FSC/SSC plots were gated on live lymphocytes and iNKT cells were identified by expression of CD3 and staining with the PBS57-loaded CD1d tetramer.