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* Department of Cell Therapy and Transplantation Medicine, University of Tokyo Hospital, Tokyo, Japan;
Division of Transfusion and Cell Therapy, Tokyo Metropolitan Cancer and Infectious Disease Center Komagome Hospital, Tokyo, Japan;
Department of Hematology and Oncology, Graduate School of Medicine,
Department of Transfusion Medicine, Graduate School of Medicine, and
¶ 21th Century Center of Excellence Program, University of Tokyo, Tokyo, Japan;
|| Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan;
# Tokyo Metropolitan Red Cross Blood Center, Tokyo, Japan;
** Department of Immunology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan;
Corporate R&D Laboratory, Asahi Kasei Corporation, Tokyo, Japan; and
* Department of Clinical and Experimental Hematology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan
The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34+ cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-
secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of -secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56+ cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56+ cells generated by culture with both Delta4 and IL-15 were CD7+ and cytoplasmic CD3+ from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by the Research on Pharmaceutical and Medical Safety, Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan (H16-Iyaku-32) and grants from the Astellas Foundation for Research on Metabolic Disorders, the Uehara Memorial Foundation, and the Sagawa Foundation for Promotion of Cancer Research (to S.C.). K.H. was supported by a fellowship from the Society of Japanese Pharmacopoeia.
2 Current address: Department of Hematology, Jichi Medical University, Tochigi, Japan.
3 Current address: Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
4 Current address: Department of Urology, Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan.
5 Current address: Department of Clinical and Experimental Hematology, University of Tsukuba, Ibaraki, Japan.
6 Current address: Division of Regenerative Medicine, Jichi Medical University, Tochigi, Japan.
7 Current address: Department of Transfusion Medicine, Yokohama City University Hospital, Kanagawa, Japan.
8 Address correspondence and reprint requests to Dr. Shigeru Chiba, Department of Clinical and Experimental Hematology, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki. E-mail address: schiba-tky{at}umin.net
9 Abbreviations used in this paper: CB, cord blood; cy, cytoplasmic; FL, Fms-like kinase 3 ligand; DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine tert-butyl ester; CMA, concanamycin A.
10 The online version of this article contains supplemental material.
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