title: In vitro differentiation of natural killer T cells from human cord blood CD34+ cells.
abstract: Natural killer T (NKT) cells are involved in innate immune defence and also in the regulation of adaptive immune responses. However, the development of NKT cells in vitro has not been fully characterized and culture conditions have not been fully optimized. In the present study, we found that an NKT cell fraction developed during the in vitro culture of cord blood (CB) CD34+ cells, and this was subsequently characterized both phenotypically and morphologically. CD34+ cells purified from 10 human CB were cultured in the presence of several cytokines and analysed by flow cytometry, light microscopy and electron microscopy. The NKT cell fraction, defined phenotypically (CD3+CD16+CD56+CD94+) as expressing the invariant T-cell receptor Valpha24 and Vbeta11, appeared in the CD56hi fractions. Intracytoplasmic staining demonstrated that interferon-gamma and interleukin 4 (IL-4) were detected in the CD56hi fractions. IL-15 was essential and, in combination with either flt3-ligand (FL) or stem cell factor (SCF), was sufficient to induce the development of NKT cells. The phenotype of the NKT cell fraction was CD45RO+CD45RA- and CD4+CD8alpha+. Morphologically, they were very large, with either round or oval nuclei, moderately condensed chromatins, voluminous weakly basophilic cytoplasm and various cytoplasmic granules such as dense core granules, multivesicular bodies, and intermediate form granules. When CD34+ cells purified from bone marrow (BM) were compared with those from CB, the latter were consistently more efficient at generating CD56hi NKT cell fractions. In conclusion, IL-15 in combination with FL and/or SCF can induce the differentiation of NKT cells from human CB CD34+ cells.
authors.affiliation: Department of Microbiology, College of Medicine, Ewha Womans University, Mok-6-Dong 911-1, Yangchon-Gu, Seoul 158-710, Korea.
authors.forename: So-Youn
authors.initials: SY
authors.lastname: Woo
authors.forename: Yu-Jin
authors.initials: YJ
authors.lastname: Jung
authors.forename: Kyung-Ha
authors.initials: KH
authors.lastname: Ryu
authors.forename: Hae-Young
authors.initials: HY
authors.lastname: Park
authors.forename: Jeong-Hae
authors.initials: JH
authors.lastname: Kie
authors.forename: Seok-Ah
authors.initials: SA
authors.lastname: Im
authors.forename: Wha-Soon
authors.initials: WS
authors.lastname: Chung
authors.forename: Ho-Seong
authors.initials: HS
authors.lastname: Han
authors.forename: Ju-Young
authors.initials: JY
authors.lastname: Seoh
mesh_terms: D015703:Antigens, CD; D017252:CD3 Complex; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D019002:CD56 Antigen; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D005312:Fetal Blood / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D019409:Interleukin-15; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology / Q000648:ultrastructure; D037181:Lectins, C-Type; D008854:Microscopy, Electron; D051920:NK Cell Lectin-Like Receptor Subfamily D; D017452:Receptors, IgG; D019089:Stem Cell Factor
chemical_list: D015703:Antigens, CD; D017252:CD3 Complex; D019002:CD56 Antigen; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D019409:Interleukin-15; C497869:KLRD1 protein, human; D037181:Lectins, C-Type; D051920:NK Cell Lectin-Like Receptor Subfamily D; D017452:Receptors, IgG; D019089:Stem Cell Factor