Blood Cals, Molecules and Diseases 83 (2020) 102857 ‘Contents lists available at ScienceDirect Blood Cells, Molecules and Diseases journal homepage: www.elsevier.comilocatelbemd Increased expression of CD8 marker on T-cells in COVID-19 patients io Ali Ganji’, Iman Farahani", Behzad Khansarinejad", Ali Ghazavi’, Ghasem Mosayebi"** *woiclrond meine Research Ger, Dgarnant f Merely an rune. cho of Meee. Arak net of Medal Sls Arak ran "rave and Complononary Meine Reseach Cone CMC), earner Mroaoy alent, Sal of Mee Ara Urry of Mea ‘Sten Aaron tr Monde Naa ‘ockgrount Cell-medlted immunkty ining Tell CF helper and eyttoae) plays an essen cole nef ent antiviral responses agains coronviras disease. 2019 (COVID-19). Therefore in thi sty, we evalated Kowords Conan ‘the ratio and expression of CD# and CDS markers in COVID-19 patents to larity the immune characterizatons copie ‘f CD# and CDS Tals in COVID-19 patients. aoloecer [Methods Peripberal bleed samples of 25 COVID-10 patients and 25 normal inividals with iilar age ad sex Cot ype asthe contol group were elected. White blod cells, places and lymphocytes were counted and CDs and cos pate {C8 TIymphocytes were evaluated by Now cytometry. ‘esis: The number f white blood eels, ympboeyts, and patlts were reduced skgaiieanly in COVID-19 patients (P< 0.05) The dllerence in CD4:CDS ratio, CD4 Tal frequency, CBB Tell fequeney, and Dd ‘mean Morescence intensty (MFP wat no sigaeant beween COVID-19 patients and healthy Individuals ( > 005); however the CDS MF increase significantly in COVID-19 infected patents (P = 0.05) Concisn: Although, there is no significant difference in the ratio of CD4 to CDS betwen ewo groups, the ‘expression lve of CDS in COVID-19 patients was sigaiicandy higher than tbe normal individuals. This result saggzted thatthe cellular immune responses triggered by COVID-19 infection were developed through over ‘expreton of CDS and hyperactvation of eyttonle T lymphocytes 1. Introduction lymphocytes, including CD4 T lymphocytes and CDS TT lymphocytes, ‘The coronavirus disease 2019 (COVID-19) ea isolated for the frst time from a cluster of pneumonia patents samples in China {1]. The virus spread rapidly in numerous countries around the world [2]. On March 11, 2020, because of the alarming levels of spread and severity of the viru, the WHO characterized COVID-19 as a pandemic disease 0. Human coronavirus is characterized as the main pathogen of the respiratory system. There are two types of extremely pathogenic cor- fonaviruses, SARS-CoV and MERSCoV, which result in severe c= spiratory syndrome in humans and four other human coronaviruses, HCoVOCA3, HCoV229E, HCoVNL63, and HCoVHKU which cause mild upper resplratory diseases [1,5]. The clinical manifestation ofthe di case includes fever, cough, shortness of breath, muscle ache, confusion, headache, sore throat, rhinoees, chest pain, darrhea, nausea, and vomiting (51. The host responses tothe viral infections depend on the interactions Derween the Innate and adaptive immune systems of the body. The T Sciences, Ara, ‘Email dress: ghesemmosayebi@arakm. hps//dl.og/10.1016/.beme.2000,102437 (6 Mosayebi. playa critical role in effective antiviral responses ofthe immune system against viruses [7]. Therefore, changes in the immunological state ‘specially alterations in the normal CD4:CDS ratio, in viral infected pitients was abserved due to the antiviral immune responses, Zaunders et al. suggested that primary HIV-1 infection and in- fectious mononucleosis patients had inverted the CD#:cD8 ratio; thls {immune state was not observed in HIV-ninfected patients oF HIV non- convertase [8]. Also, Sainz and coworkers have reported thatthe im- mune responses triggered by CMV and HIV are accompanied by a lower CDA-CDS ratio resulting in a progressive decrease in immunity of the patients and suggesting a potential marker of immunosenescence [9]. Paltriniri etal, in a stady on the Bieman cats with feline coronavirus, reported higher Cb4+ T cell count and €D4&CDR ratio compared to other breeds [10]. Several studies have suggested that the total number ‘of Iymphocytes has decreased signifieantly in Covid-19_patlents [13 According to previous studies, viral infections initiate Tell re sponses, and this may impose some alterations in the Immunologleal ng author a: Molecular and Medicine Research Center, Department of Microbslogy and Immunology, School of Medicine, Ask University of Medial Received 2 April 2020; Received in revised form 7 April 2020; Accepted 7 April 2020 Aallable online 13 Apel 2020 1070.9706/ © 2020 Published by Elsevier le.WBC Count (#10 Lymphocyte (%) + COVID-19 infected patents Bledel, Mle nd Dees 882020) 12487 Platelet Count (*10°) + Healthy individuals Fig, 1. Blood cells analyzing in COVID-19 infected patients, (A) WDC, (B) Iympocyt, and (G) platelet in COVID-19 infected patients compared to healthy in dividoals P< 005, P< O01, **4; P< 0.001 A % B # D8 MFI . D4 MFI * D4 MFI ” a 4a Fe Healthy individuals COVID-19 infected patient Fig 2. Flow cytometry analysis ruts (A) Lymphocyte gating based on side ae forward scatter and CD4 and CDS counting, (B) CDS and CDS MI analysis of hasty Individuals, (€) CD8 and CD4 MFIs alysis of COVID-19 Infected paints state in infected patients. Therefore, in the present study, after blood counting, we investigated the CD4:CD8 ratio and assessed the protein expression of CD4 and CDE ofthe T cells through mean flor fescence intensity (MEI) evaluations in COVID-19 infected patients. The findings of this study may enhance our knowledge of the im- ‘munological responses to the COVID-19 infection and may have later ‘medical and diagnostic implications 2, Materials and methods 21. Suuly population ‘A.number of 25 patimts diagnosed with COVID-19 were included in the study. The patients were hospitalized in ICU with a serious condi tions. All of the patients were initially diagnosed based on the clinical symptoms and later confirmed by quantitative RT-PCR (qRT-PCR) analysis of throat swab samples. Also, 25 healthy individuals with s- milar age and sex were selected as the control group. Patients with a history of previous chronic diseases especially who treated with im- :munosuppressve therapies before the onset of COVID-19 infection, as well a those wh died ofthe disease, were excluded from the study. All ofthe subjects were informed of the objectives of the study and com: pleted the consent forms. Al of the samples were collected according to the laboratory testing of human suspected cases of novel coronaviris (oCoV) Infection guideline [16] and approved by the Human Ethies Committce of Arak University of Medical Sciences, Arak, Iran [IR-AR- AKMU.REC.1398.334]CDAICDS Count ratio D8 MFI Frequency of CDE T-cells + COVIDA9 infected patents . Bledel, Mle nd Dees 882020) 12487 c D4 MEL Healthy individuals Fig, 8. How cytometry sesults. (A) CDA:CDS count ratio, (8) CDS MII, and (C) CD4 MPL between COVID-19 infect putons and healthy individuals. NS; not significant, =< 0001 2.2. How cytometry analysis Venous blood sample (3 al) was analyzed for counting total white blood cells (WBCS), platelets (PID, and lymphocytes by hematology auto analyzer (Sysmex, KX-21N). For flow eytomeuy analysis, 50 ul of the samples were relocated into the round: botiom tubes to determine the CDi-+, and CD8+ T cells count by flow eytomeuy (BD FACSCalibur, USA) according to the instructions. Cell surface anti bodies were including peridinin chlorophyl protein (PesCP)-conjugated Aans-C and PerCP-conjugated ati-Chs (BD Biosciences, MA, USA). A number of 10,000 leukocytes were counted for each sample and the results were analyzed using Flowjo software (Treestar, Ashland, OR, USA), Also, the MET of CD4 and CDS of T-lymphocytes were caleulated. 29, Statisical analyse Statistical analyses were performed in $SS version 16.0 (SPSS, Inc (Chicago, I, USA) and presented as mean = standard deviation (SD). test was used to assess significant differences between the groups. P- value of < 0.05 was considered statistically significant. 23. Results 1, Bod cls analyzing [As fig. 1 shows, WBC (ig. 1A), lymphocyte (Fig. 1B), and Pl (ig. 10) numbers were reduced significantly (P< 0.05) in COVID-19 infected patients compared with healthy individuals. 3.2, CD4CDS ratio and MEI analyzing ‘After calculating CD4:CDS ratio and MPL analyzing (Fig. 2A-€), ‘here i significant increase of CDS MEF in COVID-19 infected patients than healthy individuals @P = 0.002) (Fg. 3B); however, there was no significant difference in CD4:CDS count ratio (Fig. 3A), CD4 MEL (ig. 8C), CDS T-cell frequency (Fig. 3D), and CD4 T-cell frequency (ig. SE) between studied groups (P > 0.05), 4. Discussion Response to viral infeetions is accompanied by activation of the fnnate and acquired immune system, The most effective response against a variety of vial infections fs the activation of the cellular Jmmune response especially T cell activation (7]. CDS + cytotoxic T calls (CTLs) by secreting a number of molecules, including perforin, granzyme, and interferons (IFNs) ean eliminate viruses from the host body 171. CD# helper T cells (Th) cells also help to eliminate viral Infection by helping eytotoxieT cells and B cells (181. ‘Asa feature ofthe immune system, the CD4:CD8 ratio in the normal state is about 2:1 1197, But in some viral infections, as expected and Inased on research into the immune response in the human body, this ratio is disrupted and inverted CD4:CDS ratio, = 1:1 [20]. Based on the study by Saing etal. finding this ratio can act as a potential diagnostic smarker (9). In the present study, flow cytometry studies were performed to measure the number of CD‘ and CD8 T eels in peripheral blood sam ples of COVID-19 patients to measure the CD4:CDS ratio in patients ‘with COVID.19, Als, the ME was calculated ofthese cellular markersA Gat a to determine the expression ofthese markers. The results ofthe present study indicated that the number of Iym- Dhocytes in the peripheral blood of COVID-19-infected individuals was significantly reduced, which is consistent with the results of previous studies [11-15]. Also, peripheral blood platelet counts in COVID-19 patients were significantly decreased. It isin harmony with Lippi etal. stidy that platelet count reduetion isan indicator af worsening disease conditions [21] In the patients with COVID-19, CD4:CD8 ratio was the same as the normal vale of 21 that indicates no significant difference compared to the control group. Consistent with this finding, the results of Hou ct a. study showed that in COVID-19 patients the CD&CDS ratio is in the ‘normal range [221 Also, the present study showed a significant inerease in MFT of CDS marker in the patient group compared to the healthy individuals, whereas MEI of CD4 marker did not show a slgalfican diference be: ‘ween the patient and control groups. Unchanged CD4:CD8 ratio and a significant increase in CD8 MFI may indicate that the immune response of infeted persons to COVID-19 virus Is maintained by maincaining ‘normal CDA:CDE ratio and by increasing CD cell marker expression level. Since, CD8 molecule Is important for T-cell activity, T lympho: cytes try to Increase their cytotoxic activity by increasing CDS protein [23], However, some viral diseases, such as AIDS, have associated with 1 decrease in €DB molecule, which the difference eould be due to the short duration of COVID-19 infection compared with the long duration of chronic infection of AIDS (23]. The findings ofthis study shed light ‘on changes in immunologic markers in COVID-19 patients. Conducting such studies in larger populations and on other immunologic markers may open the way for diagnosing and treating this disease. In conclusion, in COVID-19 infected patients, the alterations of (CD4CDS ratio and CD4 ME were not significant compared to the control group. However, the expression of CDS on the CTLs in the pa- tient group raised significantly. Therefore it can be inferred that the ‘immune response to the COVID-19 infection is through overexpression of CDS and hyperactivation of CTL antiviral responses and aot by changes in CD4:CD8 ratio and CD4 MF. Funding This study was supported by Arak University of Medical Sciences. (CRedit authorship contribution statement Ali GanjiConceptualization, Methodology, Software Iman FarahaniData curation, Writing original draft Behzad Khansarinejad:Visualization, Investigation Ali GhazaviSofware, Validation.Ghasem Mosayebi‘Supervision, Writing - review & editing Declaration of competing interest ‘The authors report no conflicts of interest. Bledel, Mle nd Dees 882020) 12487 Acknowledgments We woul like to thank: Arak University of Medical Sciences fr their support. References (1) 5. 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