scholarly journals Ultrastructural and biochemical characterization of glycosaminoglycans in HNK-1-positive large granular lymphocytes

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 20-25 ◽  
Author(s):  
RT Parmley ◽  
F Rahemtulla ◽  
MD Cooper ◽  
L Roden
Keyword(s):  
Nk Cells ◽  
Nitrous Acid ◽  
Mononuclear Cells ◽  
Biochemical Characterization ◽  
Dermatan Sulfate ◽  
Nk Cell ◽  
Large Granular Lymphocytes ◽  
Specific Staining ◽  
Intense Staining ◽  
Granular Lymphocytes

Abstract Natural killer (NK) cells are large granular lymphocytes (LGLs) that contain distinct lysosomal granules. The present study was undertaken to determine if these lysosomes contain glycosaminoglycans (GAGs) similar to those previously described in myeloid cells. Mononuclear cells from human blood were stained with HNK-1 fluoresceinated monoclonal antibody, and the NK cell population reactive with this antibody were isolated with a fluorescence-activated cell sorter (FACS). Specific staining of sulfated macromolecules with the cationic reagent, high iron diamine, was observed in the lysosomal granules of 90% of the HNK-1 positive cells. Staining in the same location was also observed in the unsorted LGLs, presumed to be NK cells, and intense staining of the cell surface was also a prominent feature of these cells. Surface staining was not evident in the majority of the FACS- separated NK cells. Digestion with chondroitinase ABC or treatment with nitrous acid reduced the staining in both locations; after sequential treatment with both chondroitinase and nitrous acid, little or no staining was seen. The presence of chondroitin sulfate (and/or dermatan sulfate) and heparan sulfate was also shown by the finding that incubation of the isolated NK cells with 35S-sulfate yielded cell- associated radiolabeled macromolecules with the characteristics of these two groups of GAGs. Of the labeled GAG pool, 60% was degraded by chondroitinase and 40% was susceptible to nitrous acid treatment. LGLs of a patient with Chediak-Higashi syndrome was also stained, and intracellular sulfate staining was clearly localized to the enlarged granules, supporting the conclusion that the lysosomes are the major site of intracellular accumulation of GAGs in normal NK cells.

scholarly journals Ultrastructural and biochemical characterization of glycosaminoglycans in HNK-1-positive large granular lymphocytes

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 20-25 ◽  
Author(s):  
RT Parmley ◽  
F Rahemtulla ◽  
MD Cooper ◽  
L Roden
Keyword(s):  
Nk Cells ◽  
Nitrous Acid ◽  
Mononuclear Cells ◽  
Biochemical Characterization ◽  
Dermatan Sulfate ◽  
Nk Cell ◽  
Large Granular Lymphocytes ◽  
Specific Staining ◽  
Intense Staining ◽  
Granular Lymphocytes

Natural killer (NK) cells are large granular lymphocytes (LGLs) that contain distinct lysosomal granules. The present study was undertaken to determine if these lysosomes contain glycosaminoglycans (GAGs) similar to those previously described in myeloid cells. Mononuclear cells from human blood were stained with HNK-1 fluoresceinated monoclonal antibody, and the NK cell population reactive with this antibody were isolated with a fluorescence-activated cell sorter (FACS). Specific staining of sulfated macromolecules with the cationic reagent, high iron diamine, was observed in the lysosomal granules of 90% of the HNK-1 positive cells. Staining in the same location was also observed in the unsorted LGLs, presumed to be NK cells, and intense staining of the cell surface was also a prominent feature of these cells. Surface staining was not evident in the majority of the FACS- separated NK cells. Digestion with chondroitinase ABC or treatment with nitrous acid reduced the staining in both locations; after sequential treatment with both chondroitinase and nitrous acid, little or no staining was seen. The presence of chondroitin sulfate (and/or dermatan sulfate) and heparan sulfate was also shown by the finding that incubation of the isolated NK cells with 35S-sulfate yielded cell- associated radiolabeled macromolecules with the characteristics of these two groups of GAGs. Of the labeled GAG pool, 60% was degraded by chondroitinase and 40% was susceptible to nitrous acid treatment. LGLs of a patient with Chediak-Higashi syndrome was also stained, and intracellular sulfate staining was clearly localized to the enlarged granules, supporting the conclusion that the lysosomes are the major site of intracellular accumulation of GAGs in normal NK cells.

scholarly journals Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 577
Author(s):  
Adrián Fernández ◽  
Alfonso Navarro-Zapata ◽  
Adela Escudero ◽  
Nerea Matamala ◽  
Beatriz Ruz-Caracuel ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Fold Increase ◽  
Clinical Grade ◽  
Transcriptome Profile ◽  
Expansion Process ◽  
Peripheral Blood Mononuclear ◽  
Activation Status

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

scholarly journals Natural killer-mediated lysis of normal and malignant target cells, and its regulation by monocytes.

1985 ◽  
Vol 162 (2) ◽  
pp. 472-486 ◽  
Author(s):  
K Oshimi ◽  
Y Oshimi ◽  
M Satake ◽  
H Mizoguchi
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
B Lymphocytes ◽  
Leukemia Cells ◽  
Target Cells ◽  
Large Granular Lymphocytes ◽  
Percoll Density Gradient ◽  
Granular Lymphocytes ◽  
Density Gradient Sedimentation

After depletion of monocytes, natural killer (NK) cells were partially purified from peripheral blood by Percoll density gradient sedimentation. The NK cells were then cultured for 1 d and assayed for their cytotoxicity against various types of normal and malignant target cells. All types of target cells tested were found to be susceptible to NK cells. The susceptible targets were autologous T and B lymphocytes, mitogen-induced T and B blasts, monocytes, large granular lymphocytes, autologous or allogeneic lymphoma and leukemia cells isolated from patients, and cultured cell lines, including those resistant to interferon-activated lymphocytes. Such a broad spectrum of cytotoxicity was demonstrated in 1 d of culture, and freshly prepared NK cells were not cytotoxic, or, if anything, were less cytotoxic. Monocytes and their supernatants, added throughout the course of culture, markedly inhibited the development of their cytotoxicity. These results may suggest that, although NK cells having ability to lyse autologous normal and malignant target cells are present in vivo, their lytic activity is regulated by coexisting monocytes.

scholarly journals Large granular lymphocytes have a promoting activity on human peripheral blood erythroid burst-forming units

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 464-472 ◽  
Author(s):  
V Pistoia ◽  
R Ghio ◽  
A Nocera ◽  
A Leprini ◽  
A Perata ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Accessory Cell ◽  
Surface Markers ◽  
Large Granular Lymphocytes ◽  
Peripheral Blood Mononuclear ◽  
Hla Dr ◽  
Granular Lymphocytes

Abstract Peripheral blood mononuclear cells were fractionated according to the expression of a variety of surface markers, and the fractions obtained were tested for erythroid burst-forming unit (BFU-E) colony formation. BFU-Es were detected in the HLA-DR+ non-T cell fraction, but gave rise to optimum colony numbers only in the presence of a nonadherent, relatively radioresistant cell. This accessory cell was found among the HLA-DR- non-T, non-B cells, a fraction that was particularly enriched in large granular lymphocytes (LGLs). Experiments carried out to assess directly the surface markers of the accessory cell revealed an FcR+, OKM1+, Leu 7+, Leu 11+, OKT4-, OKT8- surface phenotype, which is consistent with that of the majority of LGLs. Peripheral blood LGLs, purified by Percoll density gradient, proved very efficient in promoting optimal BFU-E colony formation. All of these results indicate that LGLs have a potent erythroid burst-promoting activity. Such activity is probably mediated through the release of soluble factors, as shown by the observation that LGL culture supernatants were as effective as LGLs in sustaining colony formation.

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 Aberrant anti-viral response of natural killer cells in severe asthma

2020 ◽  
Vol 55 (5) ◽  
pp. 1802422
Author(s):  
Justine Devulder ◽  
Cécile Chenivesse ◽  
Valérie Ledroit ◽  
Stéphanie Fry ◽  
Pierre-Emmanuel Lobert ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Severe Asthma ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Asthma Patients ◽  
Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

CD69 Is Required for Activated NK Cell-Mediated Killing of Resistant Targets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3322-3322 ◽  
Author(s):  
Chloe M. Marden ◽  
Janet North ◽  
Robert Anderson ◽  
Ismail A. Bakhsh ◽  
Elena Addison ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Nk Cells ◽  
Myeloid Leukaemia ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Autologous Bone Marrow Transplantation ◽  
Target Cells ◽  
Monomeric Form ◽  
Normal Peripheral Blood ◽  
Raji Cells

Abstract The C-type lectin receptor CD69 is expressed on a range of haematopoietic cells following activation. On natural killer (NK) cells CD69 may play a direct role in mediating cytotoxicity of tumour targets. We have previously shown that remission following chemotherapy or autologous bone marrow transplantation (BMT) for acute myeloid leukaemia (AML) is dependent on NK cell cytotoxicity and have observed CD69 capping the immune synapse between autologous NK cells and conjugated AML cells (Lowdell et al, Br J Haematol, 2002; 117(4):821–7). Tumour cells which are resistant to NK-mediated lysis are often susceptible to lysis by activated NK cells (e.g. after stimulation with IL-2); thus we hypothesized that the interaction between CD69 on activated NK cells and its unidentified ligand (CD69L) on target cells is required for target cell lysis. Here we use soluble recombinant CD69 (rCD69) to investigate the role of CD69-CD69L interaction in mediating activated-NK cytotoxicity of NK-resistant and NK-sensitive target cells. The extracellular domain of CD69 (amino acids 65–199) fused with an N-terminal biotinylation sequence (Avidity) was expressed in Escherichia coli and a multivalent rCD69 reagent was created by binding biotinylated CD69 protein to avidin coated fluorescent beads (Spherotech Inc). Binding of rCD69 to NK-resistant Raji and Daudi Burkitt’s lymphoma cell line targets was determined by flow cytometry (11.9%, 12.4% positive respectively) and confocal microscopy; rCD69 did not bind to 293 kidney epithelial cells, K562 chronic myeloid leukaemia (an NK-sensitive target) or normal peripheral blood mononuclear cells. rCD69 was used to block the interaction between activated-NK cells and target cells; pre-incubation of Raji target cells with rCD69 reduced specific cytotoxicity to the level of unactivated NK cells (31.2 +/−1.6% to 8.0 +/−0.7%, Figure 1). Furthermore, activation of the intracellular tyrosine kinase Syk, which is selectively phosphorylated following CD69 signalling on activated NK cells (Pisegna et al, JI, 2002; 169: 68–74), was abrogated by rCD69 pre-incubation as determined by confocal microscopy. These data show that rCD69 binds NK-resistant target cells and blocks the killing of these cells by activated NK cells. We conclude that CD69 is required for activated NK-cell-mediated killing of resistant targets and that CD69L may be a tumour-restricted marker. Screening of primary tumours for CD69L is ongoing. Figure 1. rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only. Figure 1. rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only.

NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4879-4879
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Zimin Sun ◽  
Baolin Tang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Linear Regression Analysis ◽  
Cell Activity ◽  
Target Cells ◽  
Peripheral Blood Stem Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1865-1865
Author(s):  
Inger S. Nijhof ◽  
Michel de Weers ◽  
Pascale Andre ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Monoclonal Antibody ◽  
Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

scholarly journals Human natural killer cells

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 461-469 ◽  
Author(s):  
Michael A. Caligiuri
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Cell Receptor ◽  
Human Malignancy ◽  
Human Natural Killer Cells ◽  
Receptor Biology ◽  
Granular Lymphocytes

Abstract Natural killer (NK) cells were discovered more than 30 years ago. NK cells are large granular lymphocytes that belong to the innate immune system because unlike T or B lymphocytes of the adaptive or antigen-specific immune system, NK cells do not rearrange T-cell receptor or immunoglobulin genes from their germline configuration. During the past 2 decades there has been a substantial gain in our understanding of what and how NK-cells “see,” lending important insights into their functions and purpose in normal immune surveillance. The most recent discoveries in NK-cell receptor biology have fueled translational research that has led to remarkable results in treating human malignancy.

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