Correlation Between Defective NK Activity and Low Expression of CD16, NKp44 and NKG2D in Beta Thalassemia Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5425-5425
Author(s):  
Belkis Atasever ◽  
Serap Erdem Kuruca ◽  
Zeynep Karakas ◽  
Batu Erman ◽  
Arzu Ergen ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
K562 Cells ◽  
Tnf Alpha ◽  
Beta Thalassemia ◽  
Nk Activity ◽  
Ifn Gamma ◽  
Cytolytic Function

Abstract Beta thalassemia patients have a major global impact on health and mortality and are characterized by absence of beta globin chain production. In most patients, multiple blood transfusions can induce differences of immune response Therefore, they are often associated with bone marrow expansion and immunodeficiency in terms of lymphocyte subsets and cytokine levels in the peripheral blood and presence of alloantibodies. We have previously shown that children with beta thalassemia major have had decreased NK cells. Natural killer (NK) cells are lymphocyte subpopulations that are important effectors of innate immune responses against infectious pathogens and tumor cells. The cytotoxic activity of NK cells is regulated by the equilibrium between positive and negative signals from multiple receptors expressed on their cell surface; signals that can trigger the cytolytic machinery as well as cytokine or chemokine secretion. The activator receptors of NK cells are natural cytotoxicity receptors (NCR) and NKG2D. NCR are represented by NKp46, NKp44, and NKp30. These receptors, upon engagement by their specific ligands, induce a strong activation of NK-mediated cytotoxic activity. NKp44, a triggering receptor selectively expressed by activated NK cells. NK cells can make cytolytic function by regulating pro-inflammatory cytokines as IFN-gamma, TNF-alpha and IL10. This study was carried out to investigate details NK cell function of 27 transfusion-dependent children with beta thalassemia. Data from 18 age- and sex-balanced children served as controls. For this purpose, we analyzed their cytolytic function against K562 cells in both pure NK cells (CD56+CD16+CD3−) and PBMC. Before and after the assessment of NK activity, we have examined the levels of NK activating receptors expressed on NK cells. The expression levels of the activation receptors (NKp30, NKp44, NKG2D) on CD56+CD16+CD3− NK cells was quantified by multicolour immunofluorescent analysis using flow cytometry. In addition, supernatant IL2, IL12, IFN-gamma, TNF-alpha, TFG-beta, IL10 levels after induced K562 cells were measured by ELISA. We observed that beta thalassemia patients had lower NK activity than controls. Before the assessment of NK activity, we found that NKG2D (2064.03+/−638.64/molecule, p<0.04) and NKp44 (1057.03+/−211.21/molecule, p<0.01) surface density was reduced in a statistically significant manner in beta thalassemia patients. This phenotype correlated with low cytolytic activity. No statistically significant differences were found in the expression of NKp30. In our experimental setting where NK cells encountered K562 targets, samples from patients had significantly increased TGF-beta (544.25+/−521.5 pg/ml, p<0.03), IL10 (16.14+/−11.1 pg/ml p<0.04) when compared with controls. In addition, expression of CD16 of NK cells that induced against K562 only (12924.47+/−6913.37/molecule, p<0.006) significantly increased in controls. As a result, our findings demonstrate that environmental factors such as ineffective cytokine production and functionally defective monocytes, may cause low NK activity in beta thalassemia patients.

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.

scholarly journals Effects of β-adrenergic receptor activation, cholera toxin and forskolin on human natural killer cell function

1990 ◽  
Vol 272 (2) ◽  
pp. 327-331 ◽  
Author(s):  
M M Whalen ◽  
A D Bankhurst
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cholera Toxin ◽  
Cell Function ◽  
Nk Cell ◽  
Killer Cell ◽  
K562 Cells ◽  
Receptor Activation ◽  
Tumour Cells ◽  
Human Natural Killer Cell

Membranes from highly purified natural killer (NK) cells were ADP-ribosylated by treatment with cholera toxin (CTX). CTX resulted in a single band of specific 32P incorporation at Mr 43,600. CTX treatment of intact NK cells caused a 9-fold increase in cyclic AMP (cAMP) concentrations. Pretreatment of NK cells with CTX diminished their ability to lyse K562 tumour cells by up to 79%. Forskolin treatment elevated NK cell cAMP levels 8-fold and decreased lysis of K562 cells by up to 45%. Adrenaline and isoprenaline (isoproterenol) both inhibited lysis of K562 cells by approx. 35% and elevated cAMP by at least 2.5-fold, and their inhibition of lysis was reversed by propranolol. These data suggest that the stimulatory guanine-nucleotide-binding protein GS coupled to beta-adrenergic receptors is involved in transducing signals which inhibit NK cell lysis of tumour cells. CTX and forskolin also diminish the ability of NK cells to bind K562 cells (binding is necessary for lysis). This suggests that the NK-cell receptor(s) for the tumour cell may be altered as a consequence of cAMP-mediated events or by activation of GS.

Anti-Thymocyte Globulin Increases Dramatically Natural Killer Cell Interferon Gamma Production While Respecting Other Functionnal Aspects of These Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4957-4957
Author(s):  
Jean-Hugues Dalle ◽  
José Menezes ◽  
Eric Wagner ◽  
Martin A. Champagne ◽  
Michel Duval
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Conditioning Regimen ◽  
Annexin V ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Ifn Gamma ◽  
Hematopoietic Stem ◽  
Hla Dr

Abstract Rational: The beneficial effect of NK cells after clinical hematopoietic transplantation has been seen when anti-thymocyte globulin (ATG) was used within the conditioning regimen. We therefore investigated the effect of ATG on human NK cells. Materials and methods: Percentages of CD3+ and CD3−/CD56+ among PBMC from either normal adult volunteers or cord blood were assessed by flow cytometry. Negatively purified NK cells were cultured for 2–4 days either alone or in the presence of rabbit ATG (Thymoglobuline, Sangstat) and/or IL-15, and then assessed for cell viability and functional properties: apoptosis were investigated by Annexin V staining. Cell proliferation was assessed by CFSE dye dilution. IFN-gamma secretion was quantified by ELISA. Cytotoxicity against K562 cells was studied by a 51Cr release assay. Binding of monoclonal antibodies to NK cells was assessed alone or after competition with whole ATG or ATG-Fab’2 fragments. Results: The percentage of CD3+ cells in PBMC was over 65% before or after culture without ATG. In contrast, no T cell was detected after culture with ATG, whereas the percentage of NK cells increased from less than 15% to more than 50%. Both whole ATG and Fab’2 fragments bound NK cells while rabbit Fc fragments did not. ATG did not modify apoptotic and necrotic NK cells percentages. Proliferation was not affected by ATG. IFN-gamma production was dramatically increased by ATG treatment (figure). ATG respected IL15 induced NK cell cytolytic activity. Competition assays showed that ATG did not alter CD2, CD11a, CD16, CD56, CD57, CD62L, CD94, CD158a, CD158b, NKG2a, NKG2d, HLA-DR expression. CD18 and NKp46 expression was slightly decreased. Conclusion: Our data show that ATG may play a role in the results of KIR mismatch hematopoietic stem cell transplantation. It may be possible that ATG confers functional advantages to NK cells versus T cells. Figure Figure

Tumoricidal Potential of Native Human Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 449-449
Author(s):  
Marc Schmitz ◽  
Senming Zhao ◽  
Yvonne Deuse ◽  
Knut Schaekel ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Human Blood ◽  
Nk Cell ◽  
Tnf Alpha ◽  
Tumor Cell Lines ◽  
Specific Lysis ◽  
Tumoricidal Activity ◽  
Ifn Gamma

Abstract Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (Immunity2002;17:289-301). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (Blood;2002;100:1502-1504). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (>93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (>90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

Differential Ability of the Notch Ligands Jagged2, Delta1, and Delta4 To Accelerate Development of Human CD34+ Hematopoietic Progenitor Cells (HPCs) into Killer Inhibitory Receptor (KIR)-Negative NK Cells with Cytotoxic Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1326-1326
Author(s):  
Rose Beck ◽  
Mallika Padival
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Development ◽  
Ifn Gamma ◽  
Cell Fate Decisions ◽  
Human Cd34 ◽  
Notch Receptors ◽  
Culture Development ◽  
Notch Ligands

Abstract The evolutionarily conserved Notch receptors play important roles in cell fate decisions. Ligation of Notch-1 causes differentiation of T/NK cell precursors from HPCs and is critical for development of T cells in the thymic microenvironment. Five known Notch ligands exist in mammals (Delta1, Delta3, Delta4, Jagged1, and Jagged2), and Delta4 in particular has a greater capacity to support T cell development than Delta1. Notch ligation through Delta1 has also been shown to potentiate CD56+ NK cell differentiation from human HPCs in the presence of IL-15, although the phenotype and functionality of these cells has not been extensively described. We compared the ability of the 5 mammalian Notch ligands to induce the development of functional NK cells from human CD34+ HPCs derived from umbilical cord blood (CB). CD34+ cells isolated from CB were cultured in RPMI + 10% FBS on a murine stromal cell line, OP-9, expressing one of the five mammalian Notch receptors (Jagged1, Jagged2, Delta1, Delta3, or Delta4) or OP-9 cells transfected with vector alone, in the presence of IL-7, Flt3 ligand (FL), and IL-15. After three weeks of culture, development of CD56+CD3− cells was greatly accelerated by the ligands Jagged2 (53.4 +/− 5.5% CD56+CD3− cells), Delta-1 (38.6 +/− 5.7%), and Delta-4 (65.0 +/− 3.9%) versus culture in the absence of ligand (17.6 +/−10.3%, p = < 0.02) or in the presence of Jagged1 or Delta3. By 5 weeks, the percentage of NK cells seen in cultures containing Jagged2, Delta1, or Delta4 reached 80–90%. These NK cells expressed CD117 but only partially expressed CD94, with positivity ranging from 12.1 to 34.1% of NK cells derived from these 3 ligands after 5 weeks in culture; similarly, few CD16+ NK cells were seen in these cultures (0 to 12.1% of NK cells after 5 weeks). KIR expression in more than 1% of NK cells was not identified under any culture condition. In preliminary experiments, the addition of IL-2 or IL-21, both of which have been shown to induce KIR expression in non-Notch mediated models of NK cell development, did not significantly alter the percentages of NK cells expressing CD94, CD16, or KIR. Because the ligands Jagged2 and Delta4 induced the highest percentages of NK cells in culture, we examined the cytotoxic activity of these cells. NK cells derived from Jagged2 or Delta4 ligation expressed perforin and displayed in vitro cytotoxic activity against the human leukemia cell lines K562 (34.1% or 40.8% target cell lysis, respectively, at an E:T of 10:1) and HL-60 (14.1% or 31.6%, respectively). These cells also produced IFN-gamma, with Delta4 cultures producing higher levels of IFN-gamma versus Jagged2 cultures (1112 vs. 163.9 pg/ml, respectively). These data demonstrate that the Notch ligands vary in their ability to induce differentiation of NK cells from human CD34+ HPCs. Jagged2 and Delta4 in particular have greater capacity to generate functional NK cells which have cytolytic activity and can secrete IFN-gamma, while at the same time lacking a majority of inhibitory NK receptors (KIR and the NKG family of receptors which dimerize with CD94). The generation of cytolytic KIR-negative NK cells is of interest for cellular therapy against malignancies that are susceptible to NK cell killing.

Human Cytokine-Induced Memory-like (CIML) NK Cells Are Active Against Myeloid Leukemia in Vitro and in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1117-1117 ◽  
Author(s):  
Maximillian Rosario ◽  
Rizwan Romee ◽  
Stephanie E Schneider ◽  
Jeffrey W Leong ◽  
Ryan P Sullivan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Low Dose ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Lymphoid Cells ◽  
Ifn Gamma

Abstract NK cells are innate lymphoid cells that mediate anti-leukemia responses. The ability of MHC-haploidentical NK cells to recognize and eliminate AML blasts have been established in the setting of stem cell transplantation and early phase adoptive NK cell immunotherapy trials. However, the optimal approach to prepare human NK cells for maximal anti-leukemia capacity is unclear. As one form of innate NK cell memory, cytokine-induced memory-like (CIML) NK cells are induced by a brief (16 hour) pre-activation of human NK cells with the combination of IL-12, IL-15, and IL-18, while control NK cells from the same donor are activated by IL-15 only. In published work, this combined IL-12, IL-15, and IL-18 pre-activation results in enhanced proliferation and augmented IFN-gamma responses to cytokine or activating receptor-based re-stimulation following a rest period of 1 – 6 weeks. We hypothesized that CIML NK cells exhibit improved anti-leukemia properties compared to control NK cells from the same individual. Purified primary human CIML NK cells [both CD56bright and CD56dim subsets] produce more IFN-gamma, compared to control NK cells, upon re-stimulation with K562 cells or primary AML blasts after 7 days of rest (p<0.05 and p<0.001, N=5). CIML NK cells also exhibit higher granzyme B protein expression (p<0.01; N=8), and increased cytotoxicity against K562 leukemia targets in vitro (p<0.001, 2.5:1 and 5:1 E:T ratios). We next established a NOD-SCID-gamma-c-/- (NSG) xenograft model to investigate primary human CIML NK cell responses in vivo, with survival supported by low dose IL-2 administered every other day. Seven days following injection of 4 million NK cells / mouse, human CIML NK cells traffic to the bone marrow, spleen, liver and blood, and exhibited better in vivo expansion and persistence, compared to control NK cells (p=0.05 in the blood and bone marrow). Further, the characteristic enhanced functionality of CIML compared to control NK cells when restimulated with K562 targets was retained when assessed ex vivo 7 days post-transfer (p<0.05). Next, we investigated the ability of CIML versus control NK cells from the same donor to clear K562 AML cells in vivo. First, luciferase expressing K562 cells (1 million / mouse) were engrafted into sub-lethally irradiated (250 cGy) NSG mice. On day 3 after K562 challenge, primary human CIML or control NK cells from the same donor (4 million / mouse) were injected, which were supported in vivo using low dose IL-2. CIML NK cells exhibited significantly improved in vivo leukemia clearance as evidenced by whole mouse bioluminescence imaging (see Figure, P=0.03, N=7 mice per group). Thus, human CIML NK cells exhibit enhanced in vitro and in vivo anti-leukemia effects, compared to control NK cells. Based on these findings, a first-in-human phase 1 study of CIML NK cells in relapsed/refractory AML is currently underway. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Chediak-Higashi gene in humans. I. Impairment of natural - killer function

1980 ◽  
Vol 151 (5) ◽  
pp. 1039-1048 ◽  
Author(s):  
T Haliotis ◽  
J Roder ◽  
M Klein ◽  
J Ortaldo ◽  
AS Fauci ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Suppressor Cells ◽  
Delayed Response ◽  
Nk Activity ◽  
Fetal Fibroblasts

Natural-killer (NK)-cell function was profoundly depressed in donors homozygous for the Chediak-Steinbrinck-Higashi syndrome (C-HS) gene when compared with age- and sex-matched normals. This apparent defect was not simply a result of a delayed response because little cytolysis was evident in kinetics experiments even after 24 h of incubation. NK cells from C-HS donors failed to lyse adherent (MDA, CEM, and Alab) or nonadherent (K562 and Molt-4) tumor cell lines or nontransformed human fetal fibroblasts. Therefore, the apparent C-HS defect was not a result of a shift in target selectivities. In addition, the depressed reactivity did not appear to be a result of suppressor cells or factors because: (a) enriched NK populations (nonadherent lymphocytes bearing receptors for the Fc portion of IgG) from C-HS donors were low in NK-cell function, (b) C-HS mononuclear cells did not inhibit the cytotoxicity of normal cells in coculture experiments, and (c) cells from the C-HS donors remained poorly reactive even after culture for up to 7 d. The nature of the defective NK activity in C-HS patients is not clear but may lie within the lytic mechanism rather than at the level of the recognition structure or population size because the frequency of target-binding cells was normal. In vitro NK activity could be partially restored by interferon treatment. Combined with the results presented in the following paper (4), these observations suggest that the C-HS gene causes a selective immunodeficiency disorder, mainly involving NK cells. This finding, in conjunction with the high incidence of spontaneous possibly malignant, lymphoproliferative disorders in these patients, may have important implications regarding the theory of immune surveillance mediated by NK cells.

Natural Killer Cell Activities Against iPSCs-Derived HLA-Knockout Platelets and Megakaryocytes Reveal Perfect Rejection Profiles for Allotransfusion

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3841-3841 ◽  
Author(s):  
Daisuke Suzuki ◽  
Naoshi Sugimoto ◽  
Norihide Yoshikawa ◽  
Hiroshi Endo ◽  
Sou Nakamura ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
Annexin V ◽  
Hla Class I ◽  
K562 Cells ◽  
Class I ◽  
Target Cells

Abstract Background Platelet transfusion refractoriness (PTR) due to immune factors occurs in 5-15% of thrombocytopenic patients who have received transfusions. The dominant cause of immune PTR is the production of allo-antibodies to human leukocyte antigen (HLA) class I, which is expressed on platelets. In current clinical settings, transfusion of HLA-compatible platelets is the only practical strategy, but their supply is weak due to limited donor source, gives excessive burden on specific donors, and requires increased efforts and costs. To overcome these issues, we plan to produce HLA-knockout platelets from iPSCs-derived megakaryocytes (MKs) as an alternative solution, applicable to all HLA types. However, whether they would be attacked by natural killer (NK) cells has not been well-studied. NK cells are known to show cytotoxic activity against cells downregulated for HLA class I ("missing self" theory). Therefore we assessed the interaction between HLA-knockout platelets derived from induced pluripotent stem cells (iPSCs) and NK cells in allogeneic settings. Methods and Results Immortalized megakaryocyte progenitor cell lines (imMKCLs) were previously established from iPSCs as a source of platelet production with a robust proliferation potential (Nakamura, 2014). Beta 2-microglobulin gene was knocked-out by CRISPR/Cas9 system to obtain HLA-knockout imMKCLs and platelets. NK cells were prepared from peripheral blood of eleven healthy donors. After co-cultures of NK cells and target cells for 6 hours with IL-2, we examined the NK cell cytolytic activity marker CD107, and target cell damage marker Annexin V using flow cytometry. Positive rates of both markers were not enhanced by co-culture with either HLA-expressed or HLA-knockout platelets for all donors. Furthermore, addition of platelets showed minimal effect on high cytotoxic activity of NK cells against K562 cells. In contrast, coculture of imMKCLs with NK cells resulted in higher detection of CD107 and Annexin V staining in some NK cell donors. These data suggested that platelets are immunologically inert for NK cells irrespective of class I HLA expression, while imMKCLs can be potentially attacked. Accordingly, platelets did not express NK cell activating ligands, which were expressed on imMKCLs and K562 cells. To confirm the above-mentioned results in vivo, mice were transfused with NK cells and platelets and MKs together. In our preliminary data, the circulation of platelets was not different between HLA-expressed or HLA-knockout type. In contrast, MKs were shown to be attacked in some cases. Conclusion HLA-knockout platelets evaded attacked from NK cells, while imMKCLs possessed immunogenicity to NK cells. This study provides extended experimental evidence that HLA-knockout platelets produced from a single imMKCL clone are immunologically applicable to all HLA types including majority of patients with PTR. On the other hand, contaminating imMKCLs in imMKCL-derived platelet products can be rejected by NK cells, contributing to their enhanced safety profiles. Taken together, stage of HLA-deficiency in imMKCLs as a starting material of platelet supply shall lead to industrial production of HLA universal platelets. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of hematopoiesis in vitro by alloreactive natural killer cell clones.

1993 ◽  
Vol 177 (4) ◽  
pp. 1117-1125 ◽  
Author(s):  
G Bellone ◽  
N M Valiante ◽  
O Viale ◽  
E Ciccone ◽  
L Moretta ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Nk Cell ◽  
Tnf Alpha ◽  
Target Cells ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Cell Clones

Natural killer (NK) cells lyse autologous and allogeneic target cells even in the absence of major histocompatibility complex (MHC) class I antigens on the target cells. Recently, however, human allospecific NK cell clones have been generated that recognize at least five distinct specificities inherited recessively and controlled by genes linked to the MHC. Because the genetic specificity of these alloreactive NK cells in vitro appears analogous to that of in vivo NK cell-mediated murine hybrid resistance, i.e., the rejection of parental bone marrow in irradiated F1 animals, we tested the ability of human alloreactive NK clones to recognize allogeneic hematopoietic progenitor cells. NK cells from two specificity 1 alloreactive NK clones, ES9 and ES10, significantly and often completely suppressed colony formation by purified peripheral blood hematopoietic progenitor cells from specificity 1-susceptible donors, but had no significant effect on the cells of specificity 1-resistant donors. Activated polyclonal NK cells were less efficient than the NK clones in inhibiting colony formation and had a similar effect on cells from both specificity 1-susceptible and -resistant donors. The alloreactive NK clones produced cytokines with a suppressive effect on in vitro hematopoiesis, such as interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), when exposed to phytohemagglutinin blasts from specificity 1-susceptible, but not -resistant donors. However, the mechanism by which alloreactive NK cells inhibit colony formation is more consistent with a direct cytotoxic effect than with the production of inhibitory cytokines because antibodies (anti-IFN-gamma, alpha-TNF-alpha, and -lymphotoxin) that completely blocked the inhibition by polyclonal NK cells had only a minimal effect on the inhibition by the alloreactive clones. Moreover, the alloreactive clones were directly cytolytic in a 51Cr release assay against enriched preparations of peripheral blood progenitor cells from specificity 1-susceptible donors. These data indicate that the alloreactive NK cells are likely the human counterpart of the cells mediating murine hybrid resistance and that these cells might play clinically important roles in rejection or in graft-versus-leukemia reactions after allogeneic bone marrow transplantation.

scholarly journals PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Dante Miranda ◽  
Claudia Jara ◽  
Jorge Ibañez ◽  
Viviana Ahumada ◽  
Claudio Acuña-Castillo ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Dependent Manner ◽  
Killer Activity ◽  
Mitochondrial Mass ◽  
Mitochondrial Adaptation ◽  
Lak Activity

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γfrom NK cells with LAK activity was also partially dependent on PGC-1αexpression. These results indicate that PGC-1αplays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

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