Correlation Between Defective NK Activity and Low Expression of CD16, NKp44 and NKG2D in Beta Thalassemia Patients
Authors: Atasever Belkis , Kuruca Serap Erdem , Karakas Zeynep , Erman Batu , Ergen Arzu , Isbir Turgay 
Published: April 2020

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.