Expansion of α-galactosylceramide-stimulated Vα24⁺ NKT cells cultured in the absence of animal materials

Vα24⁺ NKT is an innate lymphocyte with potential antitumor activity. Clinical applications of Vα24⁺ natural killer (NK) T cells, which are innate lymphocytes with potential antitumor activity, require their in vitro expansion. To avoid the potential dangers posed to patients by fetal bovine serum (FBS), the authors evaluated non-FBS culture conditions for the selective and efficient expansion of human Vα24⁺ NKT cells. Mononuclear cells (MNCs) and plasma from the peripheral blood of normal healthy donors were used before and after G-CSF mobilization. MNCs and plasma separated from apheresis products were also used. MNCs were cultured for 12 days in AIM-V medium containing α-galactosylceramide (α-GalCer) (100 ng/mL) and IL-2 (100 U/mL) supplemented with FBS, autologous plasma, or autologous serum. The cultured cells were collected and their surface markers, intracellular cytokines, and cytotoxicity were evaluated. The highest expansion ratio for Vα24⁺ NKT cells was obtained from G-CSF-mobilized MNCs cultured in medium containing 5% autologous plasma. Cultures containing MNCs and autologous plasma obtained before and after G-CSF mobilization had approximately 350-fold and 2,000-fold expansion ratios, respectively. These results suggest that G-CSF mobilization conferred a proliferative advantage to Vα24 ⁺ NKT cells by modifying the biology of cells and plasma factors. Expanded Vα24⁺ NKT cells retained their surface antigen expression and production of IFN-γ and exhibited CD1d-independent cytotoxicity against tumor cells. Vα24⁺ NKT cells can be efficiently expanded from G-CSF-mobilized peripheral blood MNCs in non-FBS culture conditions with α-GalCer and IL-2.