In Vitro Generation of Highly-Purified Functional Invariant NKT Cells: A Strategy for Immunotherapy in Multiple Myeloma.

Invariant NKT (iNKT) cells are characterized by the expression of invariant T-cell receptor encoded by Vα24-JαQ and NK receptors. iNKT cells recognize glycolipid antigens with CD1d restriction, and play an important immunoregulatory role in innate immunity. Through the production of IFN-γ, iNKT cells can contribute to immune surveillance in malignancy. However, in progressive multiple myeloma (MM), as well as in other advanced cancers, iNKT cells have a marked deficiency of ligand-dependent IFN-γ production. Thus, the development of effective iNKT cells is a novel strategy for the immunotherapy of MM. In this study, we report the establishment of highly purified primary iNKT cell lines from healthy donors and MM patients. iNKT cells derived from peripheral blood or bone marrow mononuclear cells were enriched with anti-TCRVα 24 mAb or anti-6B11 mAb and further expanded by several rounds of stimulation with α-GalCer-loaded dendritic cells. Phenotype analysis confirmed 95% purity in expanded iNKT cell lines. No significant phenotypic difference was observed in iNKT cells between healthy donors and MM patients. Most of iNKT cell lines are CD4+ cell lines (CD4⁺ cells > 90%, with less than 2% were CD8⁺ cells). Majority of iNKT cells expressed CD161 and CD28, whereas CD56 expression was at very low level. Following anti-CD3 or α-GalCer-loaded dendritic cells stimulation, iNKT cells showed strong proliferative activity as measured by ³H-TdR incorporation assay. These cells expressed high level of CD25 and produced high levels of IFN-γ as well as IL-2 as measured by ELISA assay. These results provide the preclinical feasibility of producing large volume of highly-enriched functional iNKT cells from myeloma patients and rationale to clinically evaluate the efficacy of adoptive transfer of iNKT cells in MM. Based on these results a clinical study is under development.