scholarly journals A simple method for in vitro preparation of natural killer cells from cord blood

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Yong Xu Mu ◽  
Yu Xia Zhao ◽  
Bing Yao Li ◽  
Hong Jing Bao ◽  
Hui Jiang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Sorting ◽  
Interleukin 2 ◽  
Feeder Cells ◽  
Simple Method ◽  
Economical Method ◽  
Large Numbers ◽  
Promising Source

Abstract Background Cord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines. Results The large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells. Conclusion We develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

scholarly journals Control of Cell Cycle Progression in Human Natural Killer Cells Through Redox Regulation of Expression and Phosphorylation of Retinoblastoma Gene Product Protein

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 4092-4099 ◽  
Author(s):  
Akira Yamauchi ◽  
Eda T. Bloom
Keyword(s):  
Cell Cycle ◽  
Nk Cells ◽  
Natural Killer ◽  
Gene Product ◽  
Redox Regulation ◽  
Interleukin 2 ◽  
Retinoblastoma Gene ◽  
Regulation Of Expression ◽  
Retinoblastoma Gene Product

Abstract Using thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2–dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-γ activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.

scholarly journals Ultrastructural analysis of human natural killer cell activation

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1725-1736 ◽  
Author(s):  
D Zarcone ◽  
EF Prasthofer ◽  
F Malavasi ◽  
V Pistoia ◽  
AF LoBuglio ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Nk Cells ◽  
Natural Killer ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Resting Cells ◽  
Human Natural Killer Cell ◽  
Dense Granules

In this study we describe characteristic ultrastructural changes of CD3- large granular lymphocytes (LGL), ie, natural killer (NK) cells, following stimulation with recombinant (r) interleukin 2 (IL 2) or r- gamma interferon (r-gamma IFN) and after interaction with K562 target cells (TC) or Sepharose-bound anti-Fc gamma receptor (FcR) monoclonal antibody (MoAb). When compared to resting cells the cytolytic activity of r-IL 2- and r-gamma IFN-stimulated cells against K562 TC was enhanced. The r-IL 2-stimulated LGL were larger and consistently displayed the shape and cytoskeletal rearrangement characteristic of activated cells. The Golgi apparatus was expanded, and the number of electron-dense granules and vesicles was increased. The ultrastructural changes in r-gamma IFN-stimulated LGL were markedly different from those observed following r-IL 2 activation. Cells did not exhibit changes in size, shape, cytoskeletal organization, or in the structure of the Golgi apparatus. However, r-gamma IFN-stimulated cells exhibited distinctive changes in the structure and content of electron-dense granules with deaggregation of the matrix and parallel tubular arrays (PTAs). Within organelles apparently derived from the electron-dense granules, vesicular and tubular structures were noted that may be the morphological equivalent of cytotoxic factors produced by cytolytic effector cells. These ultrastructural observations indicate that r-IL 2 and r-gamma IFN enhance the lytic ability of NK cells by acting on distinct cell machineries. The cytolytic ability was decreased when LGL were pretreated with K562 TC or immobilized anti-FcR antibody. In both experimental conditions cells displayed ultrastructural features indicating activation as well as loss of cytoplasmic granules and other Golgi-derived organelles. Stimulation of r-gamma IFN- or r-IL 2- activated LGL with K562 TC or Sepharose-bound anti-FcR antibody decreased their cytolytic ability, with cells depleted of granules at the ultrastructural level. Intracytoplasmic fusion of granules and a massive release of the granule content were found in r-IL 2-stimulated cells, reminiscent of the mechanism of basophil degranulation. These observations suggest that multiple activation signals involving distinct surface membrane molecules induce release of cytolytic factors by both resting and activated NK cells.

scholarly journals In Vivo Survival and Homeostatic Proliferation of Natural Killer Cells

2003 ◽  
Vol 197 (8) ◽  
pp. 967-976 ◽  
Author(s):  
Martin Prlic ◽  
Bruce R. Blazar ◽  
Michael A. Farrar ◽  
Stephen C. Jameson
Keyword(s):  
T Cells ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Sorting ◽  
Functional Activity ◽  
Homeostatic Proliferation ◽  
Killer Cells ◽  
Bystander Cells

While the specificity and development of natural killer (NK) cells have been intensely studied, little is known about homeostasis of the mature NK population. Here we show that mouse NK cells undergo homeostatic proliferation when transferred into NK-deficient Rag−/− γC−/− hosts. Normal NK functional activity is maintained during this process, although there are some changes in NK phenotype. Using cell sorting, we demonstrate that mature (Mac-1hi) NK cells undergo homeostatic proliferation in an NK-deficient environment, yet immature (Mac-1lo) NK cells also proliferate in such hosts. We find that mature NK cells survive but do not proliferate in hosts which possess an endogenous NK pool. However, we go on to show that mature NK survival is critically dependent on interleukin (IL)-15. Surprisingly, NK survival is also compromised after transfer of cells into IL-15Rα−/− mice, implying that IL-15 responsiveness by bystander cells is critical for NK maintenance. These data imply that, similar to T cells, homeostasis of the NK pool is much more dynamic than previously appreciated and this may be relevant to manipulation of NK cells for therapeutic purposes.

scholarly journals Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2594-2601 ◽  
Author(s):  
JS Miller ◽  
KA Alley ◽  
P McGlave
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Culture System ◽  
Interleukin 2 ◽  
Early Event ◽  
Hematopoietic Progenitors ◽  
Long Term Culture ◽  
Lineage Differentiation ◽  
Marrow Stroma

Abstract We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34–7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.

scholarly journals Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 841-846 ◽  
Author(s):  
MR Silva ◽  
R Hoffman ◽  
EF Srour ◽  
JL Ascensao
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Mononuclear Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Specific Lysis ◽  
Human Natural Killer Cells

Abstract Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

scholarly journals Molecules and structures involved in the adhesion of natural killer cells to vascular endothelium.

1991 ◽  
Vol 173 (2) ◽  
pp. 439-448 ◽  
Author(s):  
P Allavena ◽  
C Paganin ◽  
I Martin-Padura ◽  
G Peri ◽  
M Gaboli ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adhesion Molecule ◽  
Vascular Endothelium ◽  
Nk Cell ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Interleukin 2 ◽  
Intercellular Adhesion Molecule

The present study was designed to define molecules and structures involved in the interaction of natural killer (NK) cells with the vascular endothelium in vitro. Resting and interleukin 2 (IL-2)-activated NK cells were studied for their capacity to adhere to resting and IL-1-treated human umbilical vein endothelial cells (EC). In the absence of stimuli, NK cells showed appreciable adhesion to EC, with levels of binding intermediate between polymorphs and monocytes. The binding ability was increased by pretreatment of NK cells with IL-2. Using the appropriate monoclonal antibody, the beta 2 leukocyte integrin CD18/CD11a was identified as the major adhesion pathway of NK cells to unstimulated EC. Activation of EC with IL-1 increased the binding of NK cells. In addition to the CD18-CD11a/intercellular adhesion molecule pathway, the interaction of resting or IL-2-activated NK cells to IL-1-activated EC involved the VLA-4 (alpha 4 beta 1)-vascular cell adhesion molecule 1 receptor/counter-receptor pair. No evidence for appreciable involvement of endothelial-leukocyte adhesion molecule was obtained. Often, NK cells interacted either with the culture substrate or with the EC surface via dot-shaped adhesion structures (podosomes) protruding from the ventral surface and consisting of a core of F-actin surrounded by a ring of vinculin and talin. The identification of molecules and microanatomical structures involved in the interaction of NK cells with EC may provide a better understanding of the regulation of NK cell recruitment from blood, their extravasation, and their migration to tissues.

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