Insufficient Ex Vivo Expansion of Vα24+ Natural Killer T Cells in Cancer Patients May Be Related to the Suppressed Expression of CD1d Molecules on CD14+ Cells.
Authors: Imataki Osamu , Heike Yuji , Takaue Yoichi , Wakasugi Hiro 
Published: April 2020

Abstract
Background: Natural killer T (NKT) cells are primary effectors of innate immune systems, and also play an important role in effectively stimulating and regulating adaptive immune responses. Previously, we reported that while Vα24+ NKT cells proliferated more efficiently from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMC) than from steady-state blood cells, this was not observed in cancer patients.
Object: Therefore, we analyzed surface antigens on G-CSF-mobilized CD14+ cells from healthy donors for allogeneic stem cell transplantation (SCT) and cancer patients to identify factors that contributed to this insufficient ex vivo expansion of NKT cells in cancer patients.
Methods: PBMC were collected from patients with malignant lymphoma (n=6) who were undergoing autologous SCT and healthy donors for SCT (n=10) before and after G-CSF administration, and cultured in AIM-V medium supplemented with 5% auto-plasma, 100 ng/mL α-galactosylceramide (α-GalCer, Kirin Pharmaceutical Co., Tokyo) and 100 U/mL recombinant human (rh) IL-2, which was supplied every 3 days. After culture for 12 days, the expansion efficacy of Vα24+ NKT cells was calculated. To analyze the contribution of the expression of CD1d molecules to NKT expansion, we sorted CD14+ cells from G-CSF-mobilized healthy donors and co-cultured them with a CD14-negative fraction that included NKT cells obtained from cancer patients.
Results: Although the proportions of NKT cells after G-CSF mobilization were comparable between healthy donors (median, 0.0055%) and cancer patients (0.0092%, P=0.14), the fold-expansion of cultured NKT cells derived from cancer patients was less than that in healthy donors (median: 1935- vs 2-fold, P=0.037). In cancer patients, the expression of CD1d molecules on CD14+ cells after G-CSF mobilization was low. A strong correlation was observed between CD1d expression and the fold-expansion of NKT cells in both cancer patients and healthy donors (r2=0.68 and 0.89, respectively). An analysis of cell kinetics showed that CD14+ cells from cancer patients survived longer in cultures than those from healthy donors. Additionally, we tested whether CD14+ cells mobilized from healthy donors could support the expansion of cancer patient’s NKT cells. CD14-positive and -negative fractions from healthy donors and cancer patients were cultured with the respective counterpart fractions. The fold-expansion for NKT cells in the CD14-negative fraction of cancer patients, including an NKT cell source, was partially retained when cells were co-cultured with CD14-positive cells derived from mobilized healthy donors (median: 32.5-fold).
Conclusions: The expression of CD1d molecules on CD14+ cells of cancer patients was lower than that in healthy donors, and cells from cancer patients survived longer than those of healthy donors. Taken together, these results suggest that the low expression of CD1d molecules in cancer patients may be related to suppressive interaction between NKT cells and CD14+ cells, which results in the lower expansion of NKT cells and longer survival of CD14+ cells. The partial recovery of NKT cell expansion after co-culture with CD14+ cells from healthy donors suggests that the suppressed expression of CD1d may contribute to the lower potential of NKT cell expansion.