key: cord-1027440-zbaoarla authors: Shahbazi, Mehdi; Moulana, Zahra; Sepidarkish, Mahdi; Bagherzadeh, Mojgan; Rezanejad, Maryam; Mirzakhani, Mohammad; Jafari, Mohammad; Mohammadnia-Afrouzi, Mousa title: Pronounce expression of Tim-3 and CD39 but not PD1 defines CD8 T cells in critical Covid-19 patients date: 2021-02-04 journal: Microb Pathog DOI: 10.1016/j.micpath.2021.104779 sha: 29a498f4a683efbd20993b292bed26414a64a4af doc_id: 1027440 cord_uid: zbaoarla BACKGROUND: During viral infection, inhibitory receptors play a key role in regulating CD8 T-cell activity. The objective of this research was to investigate programmed cell death protein 1 (PD-1), mucin domain-containing protein-3 (TIM-3), and CD39 exhaustion markers in CD8 T cells of new coronavirus disease-2019 (COVID-19) patients. METHODS: A total of 44 patients with COVID-19 (17 subjects in a critical group and 27 patients in a non-critical group) and 14 healthy controls, who were admitted to Hospitals in Babol, were recruited to the study. In subjects' peripheral blood mononuclear cells (PBMCs), we compared the phenotype of CD8 T lymphocytes, expressing PD-1, TIM-3, or CD39, both alone and in various combinations. RESULTS: The findings showed that the percentage of CD8(+) cell counts was significantly lower in non-critical patients. Critical and non-critical patients were more likely than healthy controls to have an escalated frequency of CD8(+) TIM-3(+), CD8(+) CD39(+), and CD8(+) TIM-3(+) CD39(+) cells. No significant differences were observed between all groups in the CD8(+) PD-1(+) cell counts. There was also no difference between three groups regarding the counts of CD8+TIM-3+PD-1 (+), CD8(+) PD-1(+) CD39(+), and CD8(+) TIM-3(+) PD-1(+) CD39(+) cells. The counts of non-exhausted cells were significantly lower in critical and non-critical individuals compared to the healthy individuals’ value. CONCLUSION: Patients, infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), altered exhausted CD8 T lymphocytes with CD39 and TIM-3 exhaustion markers, which may account the dysregulated immune response found in COVID-19. The findings showed that the percentage of CD8 + cell counts was significantly lower in non-critical patients. Critical and non-critical patients were more likely than healthy controls to have an escalated frequency of CD8 + TIM-3 + , CD8 + CD39 + , and CD8 + TIM-3 + CD39 + cells. No significant differences were observed between all groups in the CD8 + PD-1 + cell counts. There was also no difference between three groups regarding the counts of CD8 + TIM-3 + PD-1 + , CD8 + PD-1 + CD39 + , and CD8 + TIM-3 + PD-1 + CD39 + cells. The counts of non-exhausted cells were significantly lower in critical and non-critical individuals compared to the healthy Today, an outbreak of new coronavirus disease-2019 (COVID-19) due to RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is threatening the public health (1). The World Health Organization (WHO) has listed the outbreak of COVID-19 as a Public Health Emergency of International Concern (PHEIC) (2, 3) . Most of the infected patients have mild disease, diagnosed with shared signs and symptoms such as dry cough, fever, fatigue, dyspnea, and myalgia (3) . Some of the patients especially older ones with comorbidities, such as diabetes, hypertension, chronic obstructive pulmonary disease (COPD), cardiomyopathy, chronic renal failure, and weak immunity, seemed to be at a higher risk of developing a critical illness (4) . Tools for the early prediction of critical illness are very important for prognosis and treatment of COVID-19 patients. The development of these tools depends on the knowledge of the disease pathogenesis and helps to discover new treatment and develops vaccine strategies. Several studies showed that the adaptive immune system responds to coronavirus, as well as to SARS-CoV-2, and is necessary for effective clearance of the virus (5, 6) . The reduced numbers of CD4 and CD8 T cells were seen in patients infected with SARS-CoV-2, suggesting impaired cellular immunity, especially in critical patients (7, 8) . It has been found that, during chronic viral infections, virus-specific CD8 T cells, which gradually lose effector functions, can eventually become exhausted (9) . Several studies have identified the presence or high-level expression of a number of inhibitory receptors such as programmed death protein 1 (PD-1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), CD39 (ENTPD1), Lymphocyte-activation gene 3 (LAG3), and 2B4 (CD244) on exhausted T cells (9) (10) (11) (12) . However, there is poor information regarding the exhausted CD8 T cells in COVID-19. Therefore, we aimed to study exhaustion markers of PD-1, CD39, and TIM-3 on CD8 T cells of patients infected with SARS-CoV-2. J o u r n a l P r e -p r o o f Forty-four patients with newly diagnosed COVID-19 (17 subjects in a critical group and 27 patients in a non-critical group) and 14 healthy control subjects were included in the study. COVID-19 patients were diagnosed with typical clinical symptoms (i.e., dry cough, fever, and shortness of breath) and radiological characteristics (i.e., chest X-ray or chest computed The medical records of all cases were collected for demographic characteristics and clinical features. All variables were recorded by the healthcare specialists caring for the patients in the hospitals. One of the authors abstracted the medical records of all patients by using a hospital data collection instrument. In this research, 5 mL of heparinized venous blood was collected from each person, and the Ficoll-Hypaque density gradient centrifugation procedure was used for isolation of the peripheral blood mononuclear cells (PBMCs). In brief, the whole blood samples were centrifuged for 5 min at 350 g, and the plasma was discarded. The phosphate-buffered saline (PBS) was added at a 1:1 ratio to the packed cells. Then, the tubes were centrifuged for 20 min at 400 g in RT, and a buffy coat ring was collected. Human TruStain FcX (Biolegend, USA) was used for preventing background dye signals, caused by FCγRs of lymphocyte and monocytes. This treatment allows the antibodies specifically attach to the given markers. Continuous variables were described by mean ± standard deviation (SD). Categorical variables are shown as numbers and percentages. We compared the distributions of cytotoxic T cells across the groups initially by using appropriate univariate methods (χ2 test, one-way analysis of variance (ANOVA), and Student's t-test). Subsequently, linear mixedeffects models were employed to estimate the adjusted mean difference (MD adj ) between groups. The models were adjusted for the following potential confounders: age, sex, existing medical problem, smoking, white blood cell (WBC), and lymphocyte. Statistical analyses were performed on Stata 16.0 (Stata Corp, College Station, TX, USA). All statistical tests were two-tailed at the significance level of p < 0.05. Seventeen patients in critical and 27 patients in non-critical groups consecutively enrolled in this case-control study. The count of CD8 + cells in controls and two case groups is shown in Figures 1 and 3 No significant differences in the CD8 + PD-1 + cell counts were observed between all groups (Figures 1 and 3) . The adjusted MD did not change substantially and remained non- Table 2 ). The percentage of CD8 + CD39 + cell counts in healthy controls was 5.13 ± (2.17), which was lower than that of the critical patients (8.47 ± 1.55; p < 0.001) and non-critical patients (7.28 ± 3.52; p = 0.044) (Figures 1 and 3 The percentage of CD8 + TIM-3 + CD39 + cell counts was observed significantly more frequently in critical cases than in controls Table 2 ). In the crude and multivariate analysis, there was no difference between the three groups regarding the counts of CD8 + TIM-3 + PD-1 + , CD8 + PD-1 + CD39 + , and CD8 + TIM-3 + PD-1 + CD39 + ( Table 2 ). J o u r n a l P r e -p r o o f In the bivariate analysis, the counts of CD8 + TIM-3 -PD- T cells, especially CD8 lymphocytes, are pivotal for the host's cellular immune response against viral infection, as they can kill virus-infected cells directly (13, 14) . Therefore, CD8 T lymphocytes are crucial to generate an appropriate antiviral response in patients infected with SARS-CoV-2 (15) and also for viral clearance subsequent acute viral infections (16). The acute phase of SARS-CoV2 infection in humans was correlated with a severely decreased numbers of T cells in the blood, including a considerable loss of CD8 T cells (4, 17) . In the present study, the frequency of CD8 T lymphocytes was considerably reduced in patients with SARS-CoV-2 infection in both the critical and non-critical groups, which is in accordance with previous reports and demonstrates that the frequency of lymphocytes could be used as a diagnostic indicator of COVID-19 in the clinic (4, 18) . A single marker does not properly identify exhausted cells. Therefore, we evaluated the level of three exhaustion molecules, including PD-1, TIM-3, and CD39 in CD8 T cells by flow cytometry. CD8 T cells of COVID-19 patients (notably critical ones) showed increased exhaustion markers, i.e., CD39 and TIM-3, which suggests that impaired antiviral immunity may play a key role in the severity and pathogenesis of SARS-CoV-2 infection. In our study, the frequency of CD8 + TIM-3 -PD-1 -CD39cells was significantly lower in the COVID-19 cases (especially in critical ones), which shows that SARS-CoV-2 triggers an early over-activation and high cytotoxicity of lymphocytes in the onset of the disease, continued by exhaustion of them. Thus, it is suggested that the excessive exhaustion of CD8 T lymphocytes weakens cellular immunity to SARS-CoV-2 in critical patients. Several studies have demonstrated that exhausted CD8 T cells with CD39 and TIM-3 exhaustion markers increased in the peripheral blood of patients with a variety of viral infections caused by RNA viruses, such as HBV, HCV, HIV, and LCMV (10, (19) (20) (21) . In addition, the interaction of TIM-3 on T cells with its ligand galectin-9 results in apoptosis of CD8 T lymphocytes in viral infections (22, 23) . In this study, the TIM-3 exhaustion marker was upregulated in CD8 T lymphocytes of COVID-19 patients, which describes the observed lymphopenia. Additionally, CD39 is extensively expressed by virus-specific CD8 T lymphocytes, and its expression correlates with viral load in patients infected with HIV and HCV (11) . The elevated expression level of CD39 in COVID-19 patients, notably in the critical group, suggests that high viral antigen load may lead to exhaustion in the virus-specific pool of CD8 T lymphocytes by increasing CD39 expression. However, there are some limitations in this study. First, the sample size was relatively small in our study. Second, we only studied peripheral blood CD8 lymphocytes in the context of exhaustion. A further study of CD8 cells from alveolar lavage fluid is demanded. Third, an additional analysis of lymphocytes with other exhaustion markers, such as CTLA-4, 2B4, and LAG-3, helps to better understand the disease immunopathology. Fourth, the study of exhaustion in SARS-CoV-2 specific CD8 T cells gives a precise concept. In summary, our study suggested the appearance of TIM-3 and CD39 as exhaustion markers in CD8 lymphocytes of SARS-CoV-2 patients. Critical patients have more frequency of CD8+ TIM-3 + and CD8 + TIM-3 + CD39 + lymphocytes than non-critical patients, implying that immunological dysfunction (T cell exhaustion) may exacerbate the patients' status. Thus, preventing T cell exhaustion may contribute to better function of the immune system against COVID-19 patients. J o u r n a l P r e -p r o o f A novel coronavirus from patients with pneumonia in China Statement on the second meeting of the International Health Regulations (2005) Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV) Clinical features of patients infected with 2019 novel coronavirus in Wuhan Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study The trinity of COVID-19: immunity, inflammation and intervention T cell-mediated immune response to respiratory coronaviruses Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 Reduction and Functional Exhaustion of T Cells in Patients With Coronavirus Disease 2019 (COVID-19) T cell exhaustion CD8 T-cell exhaustion during chronic viral infection CD39 Expression Identifies Terminally Exhausted CD8+ T Cells TIM-3 as a marker of exhaustion in CD8+ T cells of active chronic hepatitis B patients T cell responses to viral infections -opportunities for Peptide vaccination The CD8 T Cell Response to Respiratory Virus Infections Contribution of Resident Memory CD8+ T Cells to Protective Immunity against Respiratory Syncytial Virus and Their Impact on Vaccine Design Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection TIM-3 as a marker of exhaustion in CD8+ T cells of active chronic hepatitis B patients. Microbial pathogenesis A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection Galectin-9/TIM-3 interaction regulates virus-specific primary and memory CD8 T cell response We thank the staff members of Shahid Yahya Nezhad, Ayatollah Rouhani, and ShahidBeheshti Hospitals for the collection of human samples. We also thank the staff members of the Immunology Department for cooperation in experimental procedures.J o u r n a l P r e -p r o o f The authors declare no competing financial interests. The authors alone are responsible for the content and writing of the paper.