Efficient ex vivo expansion of Valpha24+ NKT cells derived from G-CSF-mobilized blood cells.

Natural killer T (NKT) cells are involved in the function of innate immune systems and also play an important role in regulating acquired immune responses. In previous reports, we showed that Valpha24+ NKT cells proliferated more efficiently from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMC) than from non-mobilized PBMC. However, the mechanism of this enhanced NKT cell expansion is not yet clear. The goal of this research was to develop culture conditions for the more efficient ex vivo expansion of NKT cells. G-CSF-mobilized PBMC was cultured in AIM-V medium supplemented with 10% auto-plasma, 100 ng/mL alpha-galactosylceramide (alpha-GalCer) and 100 IU/mL recombinant human (rh) interleukin (IL)-2. The efficiency of the expansion of Valpha24+ NKT cells was evaluated on day 12. The expansion-fold of Valpha24+ NKT cells was augmented depending on the proportion of CD14+ cells at the beginning of culture. The depletion of Valpha24+ NKT cells abrogated the expansion of Valpha24+ NKT cells. Depletion of CD56+ NK cells from mobilized PBMC enhanced, and add-back of purified CD56+ NK cells suppressed the expansion of Valpha24+ NKT cells. Experiments with different timings for the addition of cells, IL-2 and alpha-GalCer suggested that follow-up supplementation with IL-2 or CD14+ cells should be avoided for the efficient expansion of Valpha24+ NKT cells. These results should be useful for the development of an efficient and practical expansion protocol for adoptive immunotherapy with Valpha24+ NKT cells.

Expansion of alpha-galactosylceramide-stimulated Valpha24+ NKT cells cultured in the absence of animal materials.

Valpha24+ NKT is an innate lymphocyte with potential antitumor activity. Clinical applications of Valpha24+ 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 Valpha24+ 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 alpha-galactosylceramide (alpha-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 Valpha24+ 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 Valpha24+ NKT cells by modifying the biology of cells and plasma factors. Expanded Valpha24+ NKT cells retained their surface antigen expression and production of IFN-gamma and exhibited CD1d-independent cytotoxicity against tumor cells. Valpha24+ NKT cells can be efficiently expanded from G-CSF-mobilized peripheral blood MNCs in non-FBS culture conditions with alpha-GalCer and IL-2.