Efficient Ex Vivo Expansion of Vα24⁺ NKT Cells from G-CSF-Mobilized PBMC.

Background:

Natural killer T (NKT) cells are one of the primary effectors of the innate immune systems, and also have an important role to initiate and regulate adaptive immune responses. Previously, we reported that Vα24⁺ NKT cells proliferated more efficiently from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMC) compared with steady-state blood cells. However, optimal culture conditions and characterization of ex vivo expanded NKT cells have not been fully evaluated.

Object:

We examined several different conditions including cell ratios, mediums, growth factors and incubation schedules to seek an optimal culture system for expansion of NKT cells.

Methods:

PBMC were collected from donors for hematopoietic stem cell transplantation before and after G-CSF administration, and were cultured in AIM-V medium supplemented with 10% auto-plasma, 100 ng/mL α-galactosylceramide (α-GalCer) and 100 U/mL recombinant human (rh) IL-2. IL-2 alone was repeatedly charged every 3 days to maintain its biological activity. After 12 days culture, we compared the expansion efficacy of Vα24⁺CD3⁺ NKT cells derived from PBMC with or without G-CSF. For depletion analysis, we used a magnetic cell sorting (MACS) system with labeling magnetic beads-conjugated monoclonal antibody against CD14, 56, 34, and TCR Vα24 chain.

Results:

The expansion fold of Vα24⁺CD3⁺ NKT cells were significantly higher with G-CSF (669 vs 182 fold, n=20). Among cell populations we tested, the proportion of CD14⁺CD16⁺ cells before cultures were associated with the efficacy of Vα24⁺CD3⁺ NKT cells expansion, and the proportion of CD34⁺, Vα24⁺, CD56⁺ and CD56⁺CD161⁺ cells were not. The magnitude of expansion of Vα24⁺CD3⁺ NKT cells was correlated with the percentage of CD14⁺ cells at the initiation of cultures. Proliferation of Vα24⁺CD3⁺ NKT cells was abrogated by the depletion of Vα24⁺cells, but notCD34⁺ cells. Depletion of CD56⁺ T cells induced higher expansion ratio of Vα24⁺CD3⁺ NKT, which was abolished when CD56⁺ and CD56⁻ cells were cultured separately using a 3 μm pored-membrane filter. Furthermore, co-culture of enriched Vα24⁺ cells and purified CD56⁺ cells inhibited the proliferation of Vα24⁺CD3⁺ NKT cells. It was hypothesized that the repeated IL-2 supplementation resulted in enhancement of CD56⁺ cells (NK cells) to suppress the proliferation of Vα24⁺CD3⁺ NKT cells. We tested different administration schedule of IL-2 as follow: on day 0 only, day 0 & 3, day 0, 3 & 6, day 0, 3, 6 & 9 (that is every 3 days), and we found that Vα24⁺CD3⁺ NKT cells expanded most effectively when IL-2 was supplemented on day 0 only. In order to modify the number of CD14⁺ cells in culture system, we added back CD14⁺ cells to CD14⁻ cells culture on day 0, 3, 6, 9 or every 3 days, but this did not result in significant enhancement of proliferation of Vα24⁺CD3⁺ NKT cells.

Conclusions:

For efficient ex vivo culture of Vα24+ NKT cells, the presence of Vα24⁺ NKT cells and CD14⁺ cells are critical. The NK cells may interfere the interaction between antigen presenting cells (APC) and NKT cells by hindering a function of antigen presentation or yielding direct cytotoxicity against APC. These findings are helpful to develop an efficient expansion system of NKT cells in feature adaptive immunotherapy.