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      Functional exhaustion of antiviral lymphocytes in COVID-19 patients

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          Abstract

          In December 2019, a novel coronavirus was first reported in Wuhan, China. 1 It was named by the World Health Organization as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is responsible for coronavirus disease 2019 (COVID-19). Up to 28 February 2020, 79,394 cases have been confirmed according to China’s National Health Commission. Outside China, the virus has spread rapidly to over 36 countries and territories. Cytotoxic lymphocytes such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are necessary for the control of viral infection, and the functional exhaustion of cytotoxic lymphocytes is correlated with disease progression. 2 However, whether the cytotoxic lymphocytes in patients infected with SARS-CoV-2 become functionally exhausted has not been reported. We showed that the total number of NK and CD8+ T cells was decreased markedly in patients with SARS-CoV-2 infection. The function of NK and CD8+ T cells was exhausted with the increased expression of NKG2A in COVID-19 patients. Importantly, in patients convalescing after therapy, the number of NK and CD8+ T cells was restored with reduced expression of NKG2A. These results suggest that the functional exhaustion of cytotoxic lymphocytes is associated with SRAS-CoV-2 infection. Hence, SARS-CoV-2 infection may break down antiviral immunity at an early stage. SARS-CoV-2 has been identified as a genus β-coronavirus, and it shares 79.5% sequence homology with SARS-CoV. 3 In our cohort of 68 COVID-19 patients admitted to The First Affiliated Hospital (Hefei) and Fuyang Hospital (Fuyang), both of which are part of Anhui Medical University in China, there were 55 cases of mild disease (MD) and 13 cases of severe disease (SD). Patients were aged 11–84 years, and the median age of patients was 47.13 years. The percentage of male patients was 52.94%. Consistent with previous studies, many patients had fever (80.88%), cough (73.53%), and sputum (32.36%) upon admission. The prevalence of other symptoms (e.g., headache, diarrhea) was relatively low (Supplementary Table 1). The clinical features of patients infected with SARS-CoV-2 was consistent with those reported by Chen and colleagues. 4 Upon admission, the neutrophil count was remarkably higher in SD patients than in MD cases, whereas the lymphocyte count was significantly lower in SD cases than in MD cases. The concentration of total bilirubin, D-dimer, and lactate dehydrogenase in blood was higher in SD patients than that in MD patients. Levels of alanine aminotransferase and aspartate aminotransferase were slightly higher in SD cases than those in MD cases. Levels of albumin and hemoglobin were lower in SD patients than those in MD patients (Supplementary Table 2). Specifically, T cell and CD8+ T cell counts were decreased significantly in MD and SD patients compared with those in healthy controls (HCs). The number of T cells and CD8+ T cells was significantly lower in SD patients than that in MD cases. The counts of NK cells were reduced remarkably in SD patients compared with those in MD cases and HCs (Fig. 1a). Fig. 1 NKG2A+ cytotoxic lymphocytes are functionally exhausted in COVID-19 patients. a Absolute number of T cells, CD8+ T cells, and NK cells in the peripheral blood of healthy controls (n = 25) and patients with mild (n = 55) and severe (n = 13) infection with SARS-CoV-2. b Percentages of NKG2A+ NK cells and NKG2A+CD8+ T cells in the peripheral blood of healthy controls (n = 25) and patients infected with SARS-CoV-2 (n = 68). c Expression of intracellular CD107a, IFN-γ, IL-2, and granzyme-B in gated NK cells and CD8+ T cells and percentage of TNF-α+ NK cells in the peripheral blood of patients infected with SARS-CoV-2 and healthy controls. d Total number of T cells, CTLs, and NK cells in the peripheral blood of COVID-19 patients and convalescing patients. e Percentages of NKG2A+ NK cells and NKG2A+ CTL in the peripheral blood of COVID-19 patients and convalescing patients. Data are mean ± SEM. Unpaired/paired two-tailed Student’s t tests were conducted. p < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; N.S., not significant As an inhibitory receptor, NKG2A has been demonstrated to induce NK cell exhaustion in chronic viral infections. 5 Notably, NKG2A expression on NK and CD8+ T cells results in functional exhaustion of NK and CD8+ T cells. 6 In patients infected with SARS-CoV-2, NKG2A expression was increased significantly on NK and CD8+ T cells compared with that in HCs (Fig. 1b). Next, to identify the role of NKG2A on the function of NK and CD8+ T cells, levels of CD107a, interferon (IFN)-γ, interleukin (IL)-2, granzyme B, and tumor necrosis factor (TNF)-α were measured through staining of intracellular cytokines. We found lower percentages of CD107a+ NK, IFN-γ+ NK, IL-2+ NK, and TNF-α+ NK cells and mean fluorescence intensity (MFI) of granzyme B+ NK cells in COVID-19 patients than those in HCs. Consistent with these findings, COVID-19 patients also showed decreased percentages of CD107a+ CD8+, IFN-γ+CD8+, and IL-2+CD8+ T cells and MFI of granzyme B+CD8+ T cells, compared with those in HCs (Fig. 1c). Taken together, these results suggest the functional exhaustion of cytotoxic lymphocytes in COVID-19 patients. Hence, SARS-CoV-2 may break down antiviral immunity at an early stage. In our setting, ~94.12% of patients were administered antiviral therapy (Kaletra®). Chloroquine phosphate was used in 7.35% of patients, and the proportion of patients treated with IFN was 64.71%. In addition, 48.53% patients received antibiotic treatment (Supplementary Table 3). Comparison of the total number of cytotoxic lymphocytes (including CTLs and NK cells) after therapy was carried out. The total number of T cells and NK cells recovered in the convalescent period in four of the five patients, and the total count of CTLs was restored in the convalescent period in three of the five patients (Fig. 1d). Hence, efficacious therapy was accompanied by an increased number of T cells, CTLs, and NK cells. Importantly, the percentage of NKG2A+ NK cells was decreased in the convalescent period compared with that before treatment among five patients. Similarly, five patients showed a decreased percentage of NKG2A+ CTLs in the convalescent period (Fig. 1e). These findings suggest that downregulation of NKG2A expression may correlate with disease control in COVID-19 patients. We showed that NKG2A expression was upregulated on NK cells and CTLs in COVID-19 patients with a reduced ability to produce CD107a, IFN-γ, IL-2, granzyme B, and TNF-α. Also, the percentage of NKG2A+ cytotoxic lymphocytes was decreased in recovered patients infected with SARS-CoV-2, which strongly suggests that NKG2A expression may be correlated with functional exhaustion of cytotoxic lymphocytes and disease progression in the early stage of COVID-19. Although exhaustion of T and NK cells occurs in human chronic infection and tumorigenesis, T cell apoptosis (which is regarded as the host mechanism involved in chronic infection and cancer) also occurs in SARS-CoV infection. 7 Thus exhausted NKG2A+ cytotoxic lymphocytes may be present in COVID-19 patients. With regard to our finding that the percentage of NKG2A+ cytotoxic lymphocytes was decreased after antiviral therapy in COVID-19 patients, efficacious control of SARS-CoV-2 infection is related to reduce expression of NKG2A on cytotoxic lymphocytes. Therefore, in COVID-19 patients with severe pulmonary inflammation, SARS-CoV-2-induced NKG2A expression may be correlated with functional exhaustion of cytotoxic lymphocytes at the early stage, which may result in disease progression. Moreover, immune inhibitory “checkpoint” receptors that result in exhaustion of NK and T cells have been demonstrated in chronic infection and cancer. Importantly, checkpoint inhibitors such as anti-PD-1 and anti-TIGIT help to reinvigorate exhausted responses from T or NK cells in the context of chronic infection and cancer. 8,9 NKG2A is thought to be a novel inhibitory molecule on immune-checkpoint blockade. 10 Taken together, these data highlight the importance of improving the immune response of NK cells and CTLs and avoiding exhaustion of cytotoxic lymphocytes at the early stage of SARS-CoV-2 infection. Therefore, targeting NKG2A may prevent the functional exhaustion of cytotoxic lymphocytes and consequently contribute to virus elimination in the early stage of SRAS-CoV-2 infection. Supplementary information Supplementary Materials

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          A Novel Coronavirus from Patients with Pneumonia in China, 2019

          Summary In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.)
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            Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

            Summary Background In December, 2019, a pneumonia associated with the 2019 novel coronavirus (2019-nCoV) emerged in Wuhan, China. We aimed to further clarify the epidemiological and clinical characteristics of 2019-nCoV pneumonia. Methods In this retrospective, single-centre study, we included all confirmed cases of 2019-nCoV in Wuhan Jinyintan Hospital from Jan 1 to Jan 20, 2020. Cases were confirmed by real-time RT-PCR and were analysed for epidemiological, demographic, clinical, and radiological features and laboratory data. Outcomes were followed up until Jan 25, 2020. Findings Of the 99 patients with 2019-nCoV pneumonia, 49 (49%) had a history of exposure to the Huanan seafood market. The average age of the patients was 55·5 years (SD 13·1), including 67 men and 32 women. 2019-nCoV was detected in all patients by real-time RT-PCR. 50 (51%) patients had chronic diseases. Patients had clinical manifestations of fever (82 [83%] patients), cough (81 [82%] patients), shortness of breath (31 [31%] patients), muscle ache (11 [11%] patients), confusion (nine [9%] patients), headache (eight [8%] patients), sore throat (five [5%] patients), rhinorrhoea (four [4%] patients), chest pain (two [2%] patients), diarrhoea (two [2%] patients), and nausea and vomiting (one [1%] patient). According to imaging examination, 74 (75%) patients showed bilateral pneumonia, 14 (14%) patients showed multiple mottling and ground-glass opacity, and one (1%) patient had pneumothorax. 17 (17%) patients developed acute respiratory distress syndrome and, among them, 11 (11%) patients worsened in a short period of time and died of multiple organ failure. Interpretation The 2019-nCoV infection was of clustering onset, is more likely to affect older males with comorbidities, and can result in severe and even fatal respiratory diseases such as acute respiratory distress syndrome. In general, characteristics of patients who died were in line with the MuLBSTA score, an early warning model for predicting mortality in viral pneumonia. Further investigation is needed to explore the applicability of the MuLBSTA score in predicting the risk of mortality in 2019-nCoV infection. Funding National Key R&D Program of China.
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              Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells

              Summary Checkpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.
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                Author and article information

                Contributors
                mjzheng@mail.ustc.edu.cn
                xyhong1964@163.com
                tzg@ustc.edu.cn
                Journal
                Cell Mol Immunol
                Cell. Mol. Immunol
                Cellular and Molecular Immunology
                Nature Publishing Group UK (London )
                1672-7681
                2042-0226
                19 March 2020
                : 1-3
                Affiliations
                [1 ]ISNI 0000 0004 1771 3402, GRID grid.412679.f, Department of Clinical Laboratory, , First Affiliated Hospital of Anhui Medical University, ; Hefei, Anhui China
                [2 ]ISNI 0000 0000 9490 772X, GRID grid.186775.a, Department of Clinical Laboratory, Fuyang Second People’s Hospital, Fuyang Infectious Disease Clinical College, , Anhui Medical University, ; Fuyang, Anhui China
                [3 ]ISNI 0000000121679639, GRID grid.59053.3a, Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, , University of Science and Technology of China, ; Hefei, Anhui China
                [4 ]ISNI 0000000121679639, GRID grid.59053.3a, Institute of Immunology, , University of Science and Technology of China, ; Hefei, Anhui China
                Article
                402
                10.1038/s41423-020-0402-2
                7091858
                6e2d05e5-4c77-4355-a75c-d0b9feafd344
                © CSI and USTC 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: the National Natural Science Foundation of China #81771685
                Funded by: the National Natural Science Foundation of China #81788101 and Chinese Academy of Sciences (XDB29030000)
                Categories
                Correspondence

                Immunology

                immunology, cell biology

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