Development of a novel stimulation method to expand CIK cells containing high proportion of CD3⁺ CD56⁺ cells

摘要

Background: Cytokine-induced killer (CIK) cells are highly efficient cytotoxic effector cells generated by culturing PBMCs in the presence of anti- CD3 mAb, IL-2 and IFN-?, and they characteristically contain the “NK-like T cell” population expressing both T cell marker CD3 and NK cell marker CD56 (CD3+CD56+). Among CIK cells, NK-like T cells (CD3+CD56+) are the main effector cells and demonstrate the most potent cytolytic activity to various types of tumor cells. Therefore, it is suggested that the technology for increasing the NK-like T cells proportion in CIK cells is a key to success for enhancing the clinical effects. We have previously reported the efficient T-cell expansion method using immobilized anti-CD3mAb and RetroNectin? (RN). In this method, NK-like T cells are not so much in expanded T cells even though a large amount of naive T cells can be obtained. Here, we show a novel expansion method for producing CIK cells by two step stimulations, with initial anti-CD3mAb/RN and second “supplement X”. Results: By combining anti-CD3mAb/RN (1st) and supplement X (2nd) stimulations, PBMCs proliferated 250- 700 fold on day14-15, and the expanded cells contained higher proportion (40-60%) of CD3+CD56+ than ones proliferated without second stimulation by “supplement X” (10- 20%). Most of the expanded cells expressed both CD8 and NKG2D, and showed strong cytotoxicity and IFN- ? release. Next, we evaluated which population of CD3+CD56+ or CD3+CD56- cells showed strong cytotoxicity. As a result, CD3+CD56+ was more potent than CD3+CD56- population. Further, we established largescale culture system using gas-permeable culture bags, CultiLife?215 and CultiLife?Eva. With this system, we could obtain a large number of CIK cells (>1010) containing high proportion of NK-like T cells (CD3+CD56+) ( ? 50%) from 50mL of blood . Conclusion: We expect that this newly devised method for CIK expansion would be an effective tool for CIK-based immunotherapy.