scholarly journals In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors.

1991 ◽  
Vol 174 (1) ◽  
pp. 21-26 ◽  
Author(s):  
M C Mingari ◽  
A Poggi ◽  
R Biassoni ◽  
R Bellomo ◽  
E Ciccone ◽  
...  
Keyword(s):  
Nk Cells ◽  
Surface Antigen ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Leukemia Cell ◽  
Peripheral Blood Lymphocytes ◽  
Leukemia Cell Line ◽  
Target Cells ◽  
In Vitro Proliferation

Purified CD3-4- thymocytes were obtained by depletion of CD3+ and CD4+ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20-40% of thymic CD16+ cells coexpressed CD1, while approximately half were cyCD3+. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3-4- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3-4- or CD3-4-16- thymocytes in the presence of irradiated H9 cells resulted in large proportions (approximately 50%) of CD16+ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16+ cyCD3+; CD16+ cyCD3-; CD16- cyCD3+; and CD16- cyCD3-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-gamma and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16+ cells expressed transcripts for CD16 and for CD3 epsilon (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long, 1988. J. Immunol. 140:1685.) and zeta chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Lond.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3- CD16+ NK cells may arise from immature thymocytes.

A family with Papillon-Lefèvre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3665-3668 ◽  
Author(s):  
Josephine L. Meade ◽  
Erika A. de Wynter ◽  
Peter Brett ◽  
Saghira Malik Sharif ◽  
C. Geoffrey Woods ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Granzyme B ◽  
Interleukin 2 ◽  
Target Cells ◽  
Loss Of Function ◽  
Cathepsin C ◽  
Cytolytic Function

Activation of granzyme B, a key cytolytic effector molecule of natural killer (NK) cells, requires removal of an N-terminal pro-domain. In mice, cathepsin C is required for granzyme processing and normal NK cell cytolytic function, whereas in patients with Papillon-Lefèvre syndrome (PLS), loss-of-function mutations in cathepsin C do not affect lymphokine activated killer (LAK) cell function. Here we demonstrate that resting PLS NK cells do have a cytolytic defect and fail to induce the caspase cascade in target cells. NK cells from these patients contain inactive granzyme B, indicating that cathepsin C is required for granzyme B activation in unstimulated human NK cells. However, in vitro activation of PLS NK cells with interleukin-2 restores cytolytic function and granzyme B activity by a cathepsin C-independent mechanism. This is the first documented example of a human mutation affecting granzyme B activity and highlights the importance of cathepsin C in human NK cell function.

Lenalidomide Effects on NK Function in Patients with Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3437-3437 ◽  
Author(s):  
P.K. Epling-Burnette ◽  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Julie Y. Djeu ◽  
Alan F. List
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Response ◽  
Nk Cell ◽  
Leukemia Cell ◽  
T Test ◽  
Leukemia Cell Line ◽  
Immunomodulatory Effects

Abstract Lenalidomide, which is a 4-amino-glutarimide analogue of thalidomide, has significant erythropoietic activity in patients with lower-risk MDS (List et al, NEJM, 351:26,2004). Although its precise target of action in MDS is not known, lenalidomide modulates cellular response to varied stimuli including inhibition of angiogenic response and endotoxin induction of inflammatory cytokines and enhancement of antigen-induced immunologic response and erythropoietin receptor signaling. Clinical investigations in multiple myeloma indicate that the immunomodulatory effects of thalidomide and lenalidomide extends to the expansion of natural killer (NK) cells. We recently found that MDS patients have defective NK function, araising in part to reduced expression of activating NK receptors (NKRs) NKp30, CD244 (2B4), and NKG2D. To determine if lenalidomide may restore NK function in MDS, we investigated the effects of in vitro treatment with lenalidomide on NK function and phenotype. Lytic function was studied using peripheral blood mononuclear cells (PBMCs) as effector cells and the leukemia cell line, K562, as a target in standard 4-hr 51Cr-release assays at 12:1 and 25:1 effector:target (E:T) ratios. Among eight MDS patient’s specimens evaluated, five patients had significant increase in tumor lysis after treatment with 1 μM lenalidomide for 72 hours (p ≤ 0.01, T-test). In similar experiments using PBMCs from normal donors, we found that NK lysis of K562 was 42% ± 15 (25:1 E:T ratio) pre-treatment which increased to 71% ± 17 (25:1 E:T ratio) after treatment, which was statistically significant (p ≤ 0.01, T-test). We also examined the in vitro effects of lenalidomide on lytic activity by the NK cell lines, NK92 and NKL, which was significantly increased after drug treatment. To discern the mechanisms of lenalidomide action in NK cell lines and normal NK cells, we evaluated NKR display by flow cytometry, and NKR function by antibody redirected cytotoxicity using the FcγR+ murine mastocytoma (P815) target cell line. Using NK92 and NKL cells, treatment with lenalidomide 1 μM for 72 hrs increased lysis by anti-NKG2D and anti-CD244 activating antibodies. We also found that NKG2D surface expression was increased on normal NK cells after lenalidomide treatment in vitro. These results suggest that some MDS patients may have improved NK function through the immunomodulatory effects of lenalidomide. The relationship between in vitro NK responsiveness to lenalidomide and in vivo hematological response warrants investigation in patients with MDS.

scholarly journals Exploitation of natural killer cells for the treatment of acute leukemia

Blood ◽  
2016 ◽  
Vol 127 (26) ◽  
pp. 3341-3349 ◽  
Author(s):  
Rupert Handgretinger ◽  
Peter Lang ◽  
Maya C. André
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Target Cells

Abstract Natural killer (NK) cells play an important role in surveillance and elimination of malignant cells. Their spontaneous cytotoxicity was first demonstrated in vitro against leukemia cell lines, and NK cells might play a crucial role in the therapy of leukemia. NK cell activity is controlled by an array of germ line–encoded activating and inhibitory receptors, as well as modulating coreceptors. This biologic feature can be exploited in allogeneic cell therapy, and the recognition of “missing-self” on target cells is crucial for promoting NK cell–mediated graft-versus-leukemia effects. In this regard, NK cells that express an inhibitory killer immunoglobulin-like receptor (iKIR) for which the respective major histocompatibility complex class I ligand is absent on leukemic target cells can exert alloreactivity in vitro and in vivo. Several models regarding potential donor–patient constellations have been described that have demonstrated the clinical benefit of such alloreactivity of the donor-derived NK cell system in patients with adult acute myeloid leukemia and pediatric B-cell precursor acute lymphoblastic leukemia after allogeneic stem cell transplantation. Moreover, adoptive transfer of mature allogeneic NK cells in the nontransplant or transplant setting has been shown to be safe and feasible, whereas its effectivity needs further evaluation. NK cell therapy can be further improved by optimal donor selection based on phenotypic and genotypic properties, by adoptive transfer of NK cells with ex vivo or in vivo cytokine stimulation, by the use of antibodies to induce antibody-dependent cellular cytotoxicity or to block iKIRs, or by transduction of chimeric antigen receptors.

scholarly journals Identification of an unusual Fc gamma receptor IIIa (CD16) on natural killer cells in a patient with recurrent infections

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 3022-3027 ◽  
Author(s):  
E de Vries ◽  
HR Koene ◽  
JM Vossen ◽  
JW Gratama ◽  
AE von dem Borne ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Peripheral Blood Lymphocytes ◽  
Target Cells ◽  
Fc Gamma Receptor ◽  
Gamma Receptor ◽  
Tract Infections

We found an unusual fc gamma receptor IIIa (CD16) phenotype on the natural killer (NK) cells of a 3-year-old boy, who suffered from recurrent viral respiratory tract infections since birth. He also had severe clinical problems after Bacille Calmette-Geerin (BCG) vaccination and following Epstein-Barr virus and Varicella Zoster virus infections. His peripheral blood lymphocytes contained a normal percentage and absolute number of CD3-CD7+ cells, which were positively stained with the CD16 monoclonal antibodies (MoAbs) 3G8 and CLBFcRgran1, but did marginally stain with the CD16 MoAb Lau11c/B73.1. Fc gamma RillIb expression on granulocytes appeared to be normal. NK cell function, analyzed in vitro by direct cytotoxicity on K562 target cells and ADCC-activity on P815 target cells, was normal compared with an age-matched healthy control. Sequence analysis of the Fc gamma RIIIA gene, encoding CD16 on NK cells and macrophages, showed a T to A nucleotide substitution at position 230 on both alleles, predicting a leucine (L) to histidine (H) amino acid change position 48 in the first extracellular lg-like domain of Fc gamma RIIIa, which contains the Leu11c/B73.1 epitope. The combined use of CD16 and CD56 MoAbs labeled with the same fluorescent dye, as often applied in routine immunophenotyping procedures, will leave these homozygotes undiagnosed. The pattern of infections in this patient is in agreement with the postulated function of NK cells in the immunological defense against viruses and other intracellular microorganisms. Further analysis of the NK cell function in vitro and follow-up of the clinical course of Fc gamma RIIIA-48H/H homozygotes is required to ascertain whether this genotype is causally related to an NK cell immunodeficiency.

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 Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3647-3653 ◽  
Author(s):  
Todd A. Fehniger ◽  
William E. Carson ◽  
Ewa Mrózek ◽  
Michael A. Caligiuri
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Stem Cell Factor ◽  
Low Dose ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Innate Immune ◽  
Ifn Γ

Abstract The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.

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.

scholarly journals Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 670-677 ◽  
Author(s):  
WJ Murphy ◽  
JR Keller ◽  
CL Harrison ◽  
HA Young ◽  
DL Longo
Keyword(s):  
Growth Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Hematopoietic Growth Factors ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Growth Promoting

Abstract Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

scholarly journals Human natural killer cells can inhibit clonogenic growth of fresh leukemic cells

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 596-599 ◽  
Author(s):  
M Beran ◽  
M Hansson ◽  
R Kiessling
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Colony Growth ◽  
Inhibitory Effect ◽  
Leukemic Cells ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Release Assay

Abstract The effect of allogenic human natural killer (NK) cells on fresh leukemic cells from three patients was investigated. The low levels of leukemic target cell lysis in the conventional 51Cr-release assay contrasted with a pronounced inhibitory effect on the colony growth of the clonogeneic leukemic target cells (L-CFC). The ability of allogeneic lymphocytes to inhibit L-CFC increased if they were pretreated with interferon (IFN), which also increased their NK activity, monitored in parallel cytotoxicity assay, against K562. Furthermore, cell separation procedures, based on differences in density among nonadherent lymphocytes, revealed that only NK cell containing fractions were inhibitory. We have also compared the susceptibility to NK-mediated L-CFC inhibition of IFN pretreated leukemic target cells with that of nontreated target cells. As in the case of NK lysis in general, this pretreatment of target cells abolished the presumably NK-mediated L-CFC inhibition. In conclusion, these data provide the first indication that NK cells can inhibit the in vitro growth of fresh clonogenic leukemia cells from patients with nonlymphocytic leukemia. The identity of NK cells as effector is strongly suggested by Percoll separation and responsiveness to interferon; the final proof awaits more sophisticated purification of these cells.

Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 436-443 ◽  
Author(s):  
Angela Gismondi ◽  
Loredana Cifaldi ◽  
Cinzia Mazza ◽  
Silvia Giliani ◽  
Silvia Parolini ◽  
...  
Keyword(s):  
Nk Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Target Cells ◽  
Cell Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Functional Defect ◽  
Nk Cell Cytotoxicity ◽  
Syndrome Protein

Abstract In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or β2-integrin engagement on NK cells. (Blood. 2004;104:436-443)

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