Turkish Journal of Hematology Volume: 37 Issue: 1 / 2020

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RESEARCH ARTICLEDOI: 10.4274/tjh.galenos.2019.2019.0256Turk J Hematol 2020;37:36-41Percentages of CD4+CD8+ Double-positive T Lymphocytes in thePeripheral Blood of Adults from a Blood Bank in Bogotá, ColombiaKolombiya Bogota Kan Bankası Yetişkin Periferik Kan CD4+CD8+ Çift Pozitif T-LenfositYüzdeleriMiguel S. Gonzalez-Mancera 1 , Natalia I. Bolaños 1 , Manuel Salamanca 1 †, Guillermo A. Orjuela 2 , Ayda N. Rodriguez 2 ,John M. Gonzalez 11University of los Andes, School of Medicine, Grupo de Ciencias Básicas Médicas, Bogotá, Colombia2National Blood Bank, Colombian Red Cross, Bogotá, ColombiaAbstractObjective: CD4+CD8+ double-positive T-cells (DPTs) have beenclassified as a separate T-cell subpopulation, with two mainphenotypes: CD4 high CD8 low and CD4 low CD8 high . In recent years,the relevance of DPTs in the pathogenesis of infections, tumors, andautoimmune diseases has been recognized. Reference values amonghealthy individuals remain unknown. Therefore, the aim of this studyis to provide a reference value for DPTs in peripheral blood fromhealthy donors in a blood bank in Bogotá, Colombia, and to determinethe activation status using a surface marker.Materials and Methods: One hundred healthy donors were enrolledin the study. Peripheral blood cells were stained for CD3, CD4,CD8, and CD154 (CD40L), and cellular viability was assessed with7-aminoactinomycin D and analyzed by flow cytometry.Results: The median value for DPTs was 2.6% (interquartilerange=1.70%-3.67%). Women had higher percentages of DPTs thanmen (3.3% vs. 2.1%). The subpopulation of CD4 low CD8 high showedhigher expression of CD154 than the other T-cell subpopulations.Conclusion: DPT reference values were obtained from bloodbank donors. A sex difference was found, and the CD4 low CD8 highsubpopulation had the highest activation marker expression.Keywords: Flow cytometry, Lymphocyte, Lymphocyte subpopulation,T lymphocytesÖzAmaç: CD4+CD8+ çift pozitif T hücreleri (ÇPT) ayrı bir T hücre altpopülasyonu olarak iki temel fenotip ile sınıflandırılmaktadır:CD4 yüksek CD8 düşük ve CD4 düşük CD8 yüksek . Son yıllarda, ÇPT’lerinenfeksiyonlar, tümörler ve otoimmün hastalıkların patogenezi ileilişkisi tanımlanmıştır. Sağlıklı bireyler arasında referans değerleribilinmemektedir. Bu nedenle, bu çalışmanın amacı, KolombiyaBogota’daki bir kan bankasındaki sağlıklı vericilerden alınan periferikkandaki ÇPT’ler için bir referans değeri sağlamak ve bir yüzey belirtecikullanarak aktivasyon durumunu belirlemektir.Gereç ve Yöntemler: Çalışmaya yüz sağlıklı verici dahil edilmiştir.Periferik kan hücreleri CD3, CD4, CD8 ve CD154 (CD40L) için işaretlendive hücresel canlılık 7-aminoactinomycin D kullanarak akan hücreölçer ile analiz edildi.Bulgular: ÇPT’ler için ortanca değer %2,6 (çeyrekler arasıaralık=%1,70-%3,67) olarak saptandı. Kadınlarda ÇPT yüzdesierkeklere göre daha yüksek bulundu (%3,3 karşı %2,1). CD4 düşükCD8 yüksek alt popülasyonu diğer T hücre alt popülasyonlarından dahayüksek CD154 ekspresyonu gösterdi.Sonuç: ÇPT referans değerleri kan bankası vericilerinden elde edilmiştir.Cinsiyetler arası fark ve CD4 düşük CD8 yüksek alt popülasyonunda da enyüksek aktivasyon belirteci ekspresyonu saptanmıştır.Anahtar Sözcükler: Hücre ölçer, Lenfosit, Lenfosit alt popülasyonu,T lenfositleriIntroductionClassically, T-cells have been classified according to the cellsurface markers CD4 and CD8. The expression of these proteinsis considered to be a mutually exclusive event reflecting thespecific functions of each major T-cell population in peripheralblood: CD4+ or helper T-cells and CD8+ or cytotoxic T-cells.However, with the use of multiparametric cellular analysismethods, a variety of minor T-cell subpopulations have beendescribed [1], such as mature CD4+CD8+ or double-positiveT-cells (DPTs) [2,3]. This T-cell phenotype was initially describedin the thymus, where more than 80% of thymocytes expressedboth CD4+CD8+, which later commit to one cell lineage (CD8+©Copyright 2020 by Turkish Society of HematologyTurkish Journal of Hematology, Published by Galenos Publishing HouseAddress for Correspondence/Yazışma Adresi: John M. González, M.D., University of los Andes,School of Medicine, Grupo de Ciencias Básicas Médicas, Bogotá, ColombiaE-mail : johgonza@uniandes.edu.co ORCID: orcid.org/0000-0001-7251-5984Received/Geliş tarihi: July 8, 2019Accepted/Kabul tarihi: October 15, 201936
Turk J Hematol 2020;37:36-41Gonzalez-Mancera MS, et al: Double-positive T-Cells in Blood Donorsor CD4+) after interaction with human leukocyte antigen (HLA)class I or II molecules, respectively [4]. The origin of DPTs in theperipheral blood of healthy individuals has been attributed tothe premature release of CD4+CD8+ T-cells from the thymusto the periphery [5,6,7]. However, additional studies havesuggested that DPTs could originate from the acquisition of thesecond marker by single-positive (either CD4+ or CD8+) T-cellsin the periphery [6,8]. Although several investigations supportthat mature CD4+ T-cells are the source of DPTs, there is alsoevidence that CD8+ T-cells could be the primary cellular type[6]. Unlike immature thymic DPTs, peripheral DPTs exhibit thefunctional properties of mature T-cells, including antigendependentcytokine production, cytolytic activity, and expressionof markers associated with the memory phenotype [9,10]. DPTsare divided into two main populations based on the differentialexpression of each marker (CD4 high CD8 low and CD4 low CD8 high )[1,2,3]. In healthy donors, CD4 high CD8 low cells have an effector ormemory phenotype (T EM), whereas CD4low CD8 high cells display acentral memory phenotype (T CM), which can switch to an effectorphenotype during viral infections such as hepatitis C virus (HCV)and human immunodeficiency virus (HIV) [9,10]. Little is knownabout the functionality of DPTs, though their function seems tobe disease-specific. DPTs exhibit cytotoxic potential in chronicviral infections, such as lymphocytic choriomeningitis virus [11]and HIV [12], and in certain types of cancer [13,14,15]. DPTs canhave a regulatory role in malignancies [13,14], systemic sclerosis[16], and inflammatory bowel disease [17]. In autoimmunediseases, DPTs can be found in different compartments; theyincrease in peripheral blood among patients with myastheniagravis [18] but are found infiltrating the affected tissues inautoimmune thyroid disease and rheumatoid arthritis [19,20]. Insystemic sclerosis and rheumatoid arthritis, DPTs secrete mainlyinterleukin-4 [16,19], whereas in tumors, such as melanoma andcutaneous lymphoma, the primary cytokine produced is tumornecrosis factor (TNF)-α [13,14]. In chronic parasitic infectionssuch as Chagas disease, DPTs are not only increased in peripheralblood [21] but are also found infiltrating the cardiac tissue inpatients with advanced chagasic cardiomyopathy [22,23].Due to the growing interest in the study of DPT subpopulationsand their potential roles in specific diseases, it seems essentialto determine reference values among healthy individuals.Therefore, the main goal of this study is to establish standardvalues of DPTs and to evaluate their functional profile bydetermining the presence of one specific activation marker insuitable donors from a blood bank in Bogotá, Colombia.Materials and MethodsStudy and DonorsThis is a descriptive and cross-sectional study of suitable donorswho volunteered for blood donation in 2017 at the NationalBlood Bank of the Colombian Red Cross in Bogotá, Colombia.The protocol and informed consent was approved by the EthicalCommittee of the University of los Andes (Act 209 of 2013).One hundred and three donors were enrolled in this study andprovided informed consent. The demographic characteristics ofour study population are shown in Table 1. Three individualswere excluded due to reactive serological tests for syphilis.The study population included 55 men and 45 women whofulfilled the donation requirements and had negative screeningtests (HIV, syphilis, hepatitis C virus, hepatitis B virus, Chagasdisease, and human T-cell lymphotropic virus). They rangedfrom 19 to 61 years of age. Samples were obtained from citratephosphate dextrose anticoagulated blood bags and transportedrefrigerated from the blood bank to the biomedical scienceslaboratory where the cellular analyses were conducted.Cell Labeling and Cytometry AcquisitionBlood samples of 100 µL were used for labeling. Antibodiesincluded anti-CD3 APC (clone UCHT1), anti-CD4 PerCP (SK3),anti-CD8 FITC (SK1), and anti-CD154 PE (TRAP1). All antibodieswere purchased from BD Pharmingen (BD, San Diego, CA, USA).Blood was stained in darkness for 30 min at 4°C and thenincubated with a cell lysis buffer (BD FACS Lysing Solution)for 15 min at room temperature. Subsequently, cells werewashed twice in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) (0.01 M, pH 7.4, PBS 1X) and gentlyresuspended. Viability was assessed with 7-aminoactinomycin Dstaining (7-AAD, BD). Samples were acquired and analyzed witha FACSCanto II flow cytometer with FACSDiva 6.1 software (BDBioscience, San Jose, CA, USA). At least 5x10 4 cells were acquiredin the CD3+ T lymphocyte population gate according to theirforward scatter (FSC) and side scatter (SSC) features. The gatingstrategy for CD3+ T-cells and DPTs is shown in Figure 1.Statistical AnalysisInformation about the donor characteristics is given inpercentages. The Shapiro-Wilk normality test was conducted forall data obtained from the cellular analysis. A nonparametricstatistical analysis was performed in the study. The Mann-Whitney U test was used to compare between two groups. TheKruskal-Wallis test was used to compare among multiple groups.The results are shown as medians and interquartile rangesTable 1. Characteristics of the population studied.Age groupsAges 18-29 30-49 50-65 TotalWomen 11 18 16 45Men 6 33 16 55No. 17 51 32 100Age average (± SD) 24.4 (3.5) 40.9 (6.4) 55.7 (3.0) 42.9 (11.8)Age in years, SD: Standard deviation.37
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