C3 Receptors Are Rapidly Increased on NK Cells upon Stimulation with PMA, CD16 Antibodies and K562 Cells

2015 ◽  
pp. 103-107
Author(s):  
Thomas Werfel ◽  
Wolfgang Witter ◽  
Otto G�tze
Keyword(s):  
Nk Cells ◽  
K562 Cells ◽  
C3 Receptors

scholarly journals Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 156-164
Author(s):  
V Pistoia ◽  
S Zupo ◽  
A Corcione ◽  
S Roncella ◽  
L Matera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Suspensions ◽  
Normal Peripheral Blood ◽  
Colony Stimulating Activity ◽  
Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

scholarly journals A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 659-659 ◽  
Author(s):  
Jan Davidson-Moncada ◽  
Noriko Sato ◽  
Robert F Hoyt ◽  
Robert N Reger ◽  
Marvin Thomas ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Raji Cell ◽  
K562 Cells ◽  
Ct Imaging ◽  
Pet Ct ◽  
Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicle Conditioning Has an Immunosuppressive Effect on NK Cells

2021 ◽  
Author(s):  
G. Dostert ◽  
V. Jouan-Hureaux ◽  
H. Louis ◽  
É. Velot
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Communication ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity

Background: In peripheral blood, human natural killer (NK) cells are immunological cells that nearly don’t express the ectonucleotidase CD73 on their plasma membrane. When exposed to mesenchymal stem cells (MSCs), NK cells are able to acquire CD73. MSCs are known to be CD73-positive (CD73+) and also to modulate the immune system, e.g. through adenosynergic pathway by ectonucleosidases, such as CD73. Extracellular vesicles (EVs) are involved in cell-to-cell communication. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as paracrine mediators that are part of MSC immunomodulatory effects including immunosuppressive properties and immune privilege. Objective: The aim of our work was to study if CD73 could be acquired by NK cells through cell-to-cell communication with MSC-EVs as cell culture additives. We also hypothesised that MSC-EVs would act as tolerance inducers to attenuate NK cell cytotoxicity. Methods: Cell isolation was made from human umbilical cords for MSCs and from human peripheral blood for NK cells. MSC-EVs were isolated by ultracentrifugation and filtration, then characterized by nanoparticle tracking assay and flow cytometry (CD9, 63, 81 and 73). MSC-EV interaction with NK cells was monitored by PKH67 staining. NK cell activation was followed by measuring the expression of CD73 and NK-activating receptor natural-killer group 2, member D (NKG2D) by flow cytometry. The cytotoxicity of NK cells or EV-conditioned NK cells was evaluated after co-culture with K562 cells. Results: We showed that MSC-EVs are nanoparticles able to express CD73 and interact with NK cells. MSC-EV conditioned NK cells seem to increase CD73 and decrease NKG2D through an EV-mediated mechanism. MSC-EVs have an immunosuppressive effect on NK cells by preventing NK cell activation and NK cell cytotoxicity towards K562 cells. Conclusions: Our results demonstrate that MSC-EVs could influence NK cell behaviour and act as immunosuppressant cell-based products.

scholarly journals Donor and host coexpressing KIR ligands promote NK education after allogeneic hematopoietic stem cell transplantation

2019 ◽  
Vol 3 (24) ◽  
pp. 4312-4325 ◽  
Author(s):  
Xiang-Yu Zhao ◽  
Xing-Xing Yu ◽  
Zheng-Li Xu ◽  
Xun-Hong Cao ◽  
Ming-Rui Huo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Nk Cell ◽  
K562 Cells ◽  
Inhibitory Receptors ◽  
Hematopoietic Stem

Abstract The rate and extent of natural killer (NK)–cell education after hematopoietic cell transplantation correlates with leukemia control. To study the effect of donor and host HLA on NK-cell reconstitution, single killer-cell immunoglobulin-like receptor (KIR)+ NK cells (exhibiting KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) were grouped into 4 groups based on the interaction between donor/host HLA and donor inhibitory KIR in 2 cohorts (n = 114 and n = 276, respectively). On days 90 to 180 after transplantation, the absolute number and responsiveness against K562 cells (CD107a or interferon-γ expression) of single-KIR+ NK cells were higher in pairs where donor and host HLA both expressed ligands for donor inhibitory KIRs than in pairs where 1 or both of the donor and recipient HLA lacked at least 1 KIR ligand. NK-cell responsiveness was tuned commensurate with the number of inhibitory receptors from the donor. When both donor and host expressed the 3 major KIR ligands (HLA-C1, HLA-C2, and HLA-Bw4), NK cells expressing 3 inhibitory receptors (KIR2DL1/2DL3/3DL1) reached the maximum responsiveness against K562 cells compared with those NK cells expressing only 1 or 2 inhibitory receptors. When donor and host HLA both expressed all ligands for donor inhibitory KIRs, patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) showed the lowest recurrence rate after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). In conclusion, this study demonstrates that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells achieve better functional education and contribute to least relapse among patients. This observation study was registered at www.clinicaltrials.gov as #NCT02978274.

Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP

Blood ◽  
2006 ◽  
Vol 107 (3) ◽  
pp. 1184-1191 ◽  
Author(s):  
Nobuyoshi Hanaoka ◽  
Tatsuya Kawaguchi ◽  
Kentaro Horikawa ◽  
Shoichi Nagakura ◽  
Hiroaki Mitsuya ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nk Cells ◽  
Natural Killer ◽  
Blood Cells ◽  
Cell Injury ◽  
Nk Cell ◽  
K562 Cells ◽  
Cytotoxic Cells ◽  
Immune Mediated

AbstractThe mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of GPI-linked membrane proteins. GPI-deficient blood cells often expand in patients with aplastic anemia who sustain immune-mediated marrow injury putatively induced by cytotoxic cells, hence suggesting that the injury allows PNH clones to expand selectively. We previously reported that leukemic K562 cells preferentially survived natural killer (NK) cell-mediated cytotoxicity in vitro when they acquired PIGA mutations. We herein show that the survival is ascribable to the deficiency of stress-inducible GPI-linked membrane proteins ULBP1 and ULBP2, which activate NK and T cells. The ULBPs were detected on GPI-expressing but not on GPI-deficient K562 cells. In the presence of antibodies to either the ULBPs or their receptor NKG2D on NK cells, GPI-expressing cells were as less NK sensitive as GPI-deficient cells. NK cells therefore spared ULBP-deficient cells in vitro. The ULBPs were identified only on GPI-expressing blood cells of a proportion of patients with PNH but none of healthy individuals. Granulocytes of the patients partly underwent killing by autologous cytotoxic cells, implying ULBP-associated blood cell injury. In this setting, the lack of ULBPs may allow immunoselection of PNH clones.

Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3244-3244
Author(s):  
Gabriele Multhoff ◽  
Catharina Gross ◽  
Anne Dickinson ◽  
Ernst Holler
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Hla Class I ◽  
K562 Cells ◽  
Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

Using the NOD/SCID Mice Model of Human Erythroleukemia for Evaluating the Cytotoxicity Activity of CB-CIK/NK Cells Stimulated by K562-DCs Fusion Vaccines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5240-5240
Author(s):  
Yang Li ◽  
Shaoliang Huang ◽  
Jianpei Fang ◽  
Xuchao Zhang ◽  
Jing Wei ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
K562 Cells ◽  
Scid Mice ◽  
Tumour Mass ◽  
Mice Model ◽  
Cytotoxicity Activity ◽  
Group A ◽  
Group D

Abstract CD3+CD56+ cytokine-induced killer (CIK) cells are prospective effectors for adoptive immunotherapy, CIK/NK cells incubated with K562-dendritic cells (DCs) fusion vaccines have more higher cytotoxicity activity. In this study, the efficacy and the safety of application of cord blood (CB) derived CIK/NK cells stimulated by K562-dendritic cells (DCs) fusion vaccines were evaluated in vivo by the NOD/SCID mice model of human erythroleukemia (K562 line, CD13+). DCs and CIK/NK cells were inducted by combination of cytokines from CB MNCs, DCs were fused with inactivated K562 tumor line by PEG (mw1500). 5 days before the harvest of CIK/NK cells, 1×105 K562-DCs fusion vaccines were co-cultured with 1×106 CB-CIK/NK cells to prepare for the K562-DCs fusion vaccines stimulated CIK/NK cells. NOD/SCID mice divided into six groups, eight in one group. Mice in A,B and C groups were inoculated with 1×106 K562 cells by tail vein. 24 hours later, 1×107 K562-DCs fusion vaccines stimulated CIK/NK cells and 1×107 unstimulated CIK/NK cells were transfued into the mice of group A and B, respectively. Group D and E were K562-DCs fusion vaccines stimulated CIK/NK cells and no-stimulated CIK/NK cells control, transfued by 1×107 stimulated CIK/NK cells and 1×107 no-stimulated CIK/NK cells, respectively. Group F is a normal control that no any inoculation were taken. None of the NOD/SCID mice in group C that inoculated with 1×106 K562 cells survived longer than 39 days, hepatosplenomegalic mass was seen in five mice. Death in group A and B were only one and two, respectively, at day 65 and day 56, 62. There was no tumour mass can be seen in group A and B, and the survial were more than 70 days. Both survival time of group A(69.38±1.77 days) and B(67.25±5.34 days) were longer than that of group C(30.38±4.57 days) significantly (P<0.01). The tumor marker (CD13) in periperal blood, live and lung of CIK/NK cells treated NOD/SCID mice (group A and B) significantly less than group C(P<0.01). There was no difference of CD56 positive in periperal blood of group A and B survival mice between that of the control (group D and E). These result indicated that K562-DCs fusion vaccines stimulating CB-CIK/NK cells have a potent anti-tumour activity in vivo and without any side-effect.

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3666-3666
Author(s):  
Tarun K. Garg ◽  
Susann Szmania ◽  
Jumei Shi ◽  
Katie Stone ◽  
Amberly Moreno-Bost ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Scid Mice ◽  
Target Cells ◽  
Recent Trial ◽  
Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

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&lt;0.04) and NKp44 (1057.03+/−211.21/molecule, p&lt;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&lt;0.03), IL10 (16.14+/−11.1 pg/ml p&lt;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&lt;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.

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