Functional exhaustion of antiviral lymphocytes in COVID-19 patients

In December 2019, a novel coronavirus was first reported in Wuhan, China. 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. 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. 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. 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). As an inhibitory receptor, NKG2A has been demonstrated to induce NK cell exhaustion in chronic viral infections. Notably, NKG2A expression on NK and CD8 T cells results in functional exhaustion of NK and CD8 T cells. In patients infected with SARSCoV-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-γCD8, and IL-2CD8 T cells and MFI of granzyme BCD8 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).