key: cord-0770462-szp9jp3d
authors: Jiang, Yujie; Wei, Xin; Guan, Jingjing; Qin, Shuang; Wang, Zhongyong; Lu, Hong; Qian, Jingjing; Wu, Lianfeng; Chen, Yingxiao; Chen, Yanfan; Lin, Xiangyang
title: COVID-19 pneumonia: CD8(+) T and NK cells are decreased in number but compensatory increased in cytotoxic potential
date: 2020-06-20
journal: Clin Immunol
DOI: 10.1016/j.clim.2020.108516
sha: d850b04b136a9bca718828e4ecfe3c19f64dcb95
doc_id: 770462
cord_uid: szp9jp3d

BACKGROUND: Coronavirus disease 2019 (COVID-19) is posing a huge threat to human health worldwide. We aim to investigate the immune status of CD8(+) T and NK cells in COVID-19 patients. METHODS: The count and immune status of lymphocytes were detected by flow cytometry in 32 COVID-19 patients and 18 healthy individuals. RESULTS: As the disease progression in COVID-19 patients, CD8(+) T and NK cells were significantly decreased in absolute number but highly activated. After patients' condition improved, the count and immune status of CD8(+) T and NK cells restored to some extent. GrA(+)CD8(+) T and perforin(+) NK cells had good sensitivity and specificity for assisting diagnosis of COVID-19. CONCLUSIONS: As the disease progression, the declined lymphocytes in COVID-19 patients might lead to compensatory activation of CD8(+) T and NK cells. GrA(+)CD8(+) T and perforin(+) NK cells might be used as meaningful indicators for assisting diagnosis of COVID-19.

Coronavirus disease 2019 (COVID-19) caused by a new coronavirus SARS-CoV-2 has spread widely and rapidly around the world since it was first reported in Wuhan, China, in December 2019. Until May 16, 2020, locations with confirmed COVID-19 cases included 216 countries and 4347935 cases were confirmed. A total of 297241 patients died from this viral infection until May 16 [1] . WHO officially announced COVID-19 as a "pandemic". A model-based analysis combining about 70,000 COVID-19 cases at home and abroad reported that the fatality rate of COVID-19 increased with age, and that the mortality in patients over 80 years of age even reached 10 times of the overall mortality in China [2] . However, the phenomenon of low incidence and mild symptoms in children with COVID-19 was common in many countries [3] . Guan WJ et al. confirmed the following points by analysis of inpatients in 575 domestic hospitals: COVID-19 patients with comorbidity would be poorer in clinical outcomes than those without, and a large number of comorbidities were closely correlated with worse clinical outcomes [4] . Besides, the diagnosis and treatment protocol for COVID- 19 in China has been updated to the trial version 7, and the clinical classification of the disease remained mild, moderate, severe and critical cases. A confusing phenomenon is that why some people suffer from severe or even critical illness while some others only show slight clinical symptoms, and the prognosis is so various. Yufang Shi et al. proposed that traditional immunology viewpoints couldn't explain the broad spectrum of the disease, and suggested that the J o u r n a l P r e -p r o o f Journal Pre-proof course of COVID-19 should be divided into immune defense-based protective phase and inflammation-driven damaging phase [5] .

The morbidity and mortality of infectious diseases can be caused by direct damage to the host by the pathogen or collateral damage to the host tissue by the excessive immune response to the pathogen [6, 7] . After infected with COVID-19, whether for clinical or basic research, the patients' immune status both need to be clarified urgently. The guideline of new edition for COVID-19 as mentioned above suggests that the progressive decline of peripheral lymphocytes is one of the clinical early warning indicators for severe and critical cases in adults, and many studies have also reported lymphopenia, mainly for the reduction of CD8 + T cells, in COVID-19 patients [8] [9] [10] [11] [12] [13] . In an autopsy report about one died case with COVID-19, Fu-Sheng Wang and his colleague found that the count of peripheral CD8 + T cells was substantially reduced, while cell immune status was hyperactivated; meanwhile, CD8 + T cells were found to harbour high concentrations of cytotoxic granules including perforin and granzyme [14] . It is well known that as primary cytotoxic lymphocytes, CD8 + T and NK cells play a vital role in the control of pathogen infection by mediating cellular immunity and cytotoxic function [15] [16] [17] . However, the immune some experience in the diagnosis and treatment of COVID-19 patients. In this study, we focus on the dynamic change in the number of lymphocyte subsets and monitor the immune status of CD8 + T and NK cells in COVID-19 patients with different severity. It may help provide new ideas for understanding the immune response mechanism of COVID-19 patients with different severity.

The diagnosis and clinical classification of COVID- 19 

Laboratory confirmation of the SARS-CoV-2 was performed by The First Affiliated Hospital of Wenzhou Medical University according to the Chinese CDC protocol. Throat-swab specimens were collected from all patients and all samples were maintained in viral-transport medium for laboratory testing. Infection with other respiratory viruses including influenza A virus, influenza B virus, Coxsackie virus, respiratory syncytial virus, parainfluenza virus, and enterovirus were excluded by RT-PCR. Specimens, including sputum or alveolar lavatory fluid, blood, urine, and feces, were cultured to identify pathogenic bacteria or fungi that might be associated with the SARS-CoV-2 infection. All tests were performed according to the product manual.

To detect the phenotypic and functional characteristics of cytotoxic lymphocytes (CD8 + T cells and NK cells), 2 mL of peripheral blood was used for cell surface markers staining, and then fixed and permeabilized for intracellular cytokine staining. 

Data were expressed as the mean (Standard deviation) and median (Interquartile range) for continuous variables with and without normal distribution, and were analyzed by GraphPad Prism version 8.0 (GraphPad Software, Inc., San Diego, CA, confidence interval was utilized to calculate the sensitivity, specificity and consistency, and the cut-off value was selected when the Jordan index was at its maximum. For all tests, a two-sided P value < 0.05 was considered to be significant. Statistical significance is indicated as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

We detected the absolute number of circulating total lymphocytes and found that lymphocytes in COVID-19 patients decreased significantly as compared with HCs ( Figure 1A ). In COVID-19 patients, the sustained decrease of lymphocytes in the Critical group was observed as compared with the other three groups ( Figure 1B) . To determine the changes of different lymphocyte subsets in COVID-19 patients, we performed flow cytometry to determine total T cells, CD4 + T and CD8 + T cell subsets, B cells, and NK cells. Similar to the findings of total lymphocytes, sustained decrease in total T, CD8 + T and NK cell count was observed in COVID-19 patients compared to HCs ( Figure 1A ). In COVID-19 patients, the sustained decrease of total T and NK cells in the Critical group was observed compared to the other three groups ( Figure   1B ), and CD8 + T cell count in the Critical group was significantly decreased as compared with HCs and the Mild group ( Figure 1B) . However, no significant difference in CD4 + T and B cell count was observed between COVID-19 patients and HCs ( Figure 1A ).

J o u r n a l P r e -p r o o f

Expression of CD38 and HLA-DR was detected to analyze the activation status of CD8 + T cells and found that circulating CD8 + T cells from COVID-19 patients had higher expression of those two markers compared to HCs (Figure 2A) , while the expression of CD38 and HLA-DR in CD8 + T cells was not significantly different among the severity of the disease ( Figure 2B ). We then detected the expression of GrA, GrB and perforin on CD8 + T and NK cells to understand their cytotoxic potential. We found that CD8 + T cells in COVID-19 patients had higher expression of cytotoxic granules (GrA, GrB and perforin) compared to HCs ( Figure 3A ). Moreover, in COVID-19 patients, we found that the expression of GrB and perforin on CD8 + T cells in the Critical group was significantly increased as compared with the Severe group and Mild group ( Figure 3B ), respectively; while GrA expression on CD8 + T cells was not significantly different among different severity of disease ( Figure 3B ).

In addition, we found that the expression of GrA and perforin on NK cells in COVID-19 patients was significantly increased compared to HCs ( Figure 3A ). In comparing diseases of different severity, we found the expression of GrA on NK cells in the Mild and Severe groups was significantly increased as compared with HCs and the Critical group ( Figure 3B ). The expression of perforin on NK cells in the Severe group was significantly increased as compared with the Mild group ( Figure 3B ).

However, the expression of GrB on NK cells in COVID-19 patients was significantly decreased compared to HCs ( Figure 3A ), but it had no significant relationship with the severity of the disease ( Figure 3B ).

J o u r n a l P r e -p r o o f

In order to study the relationship between lymphocyte changes and patients' condition, we first detected the absolute number of lymphocyte subsets before and after remission in 9 severe patients and 4 critical patients. We found that the absolute number of total lymphocytes, total T cells, and CD8 + T cells in the remission group was significantly higher than those in the illness group. Although the absolute number of NK cells was not changed significantly after remission, it had a tendency to increase compared to before remission ( Figure 4A ). Then, we detected the expression of CD38, HLA-DR, GrA, GrB, and perforin on CD8 + T and NK cells before and after remission in 5 severe patients and 2 critical patients. The expressions of CD38, HLA-DR, GrA, GrB, especially of perforin, on CD8 + T cells all showed a downward trend compared to those before remission ( Figure 4B ). However, there was no significant statistical change in perforin + CD8 + T cell count before and after remission in COVID-19 patients. The tendency of GrA, GrB, and perforin on NK cells did not change significantly before and after remission ( Figure 4B ).

Through ROC curve analysis, we found that GrA + CD8 + T (AUC, 0.995) and

perforin + NK (AUC, 0.977) cells showed an excellent predictive value for COVID-19

with high sensitivity (96.88%, 84.38%) and specificity (100.00%, 100.00%); followed by GrB + CD8 + T (0.894) and perforin + CD8 + T (0.826) with sensitivity of 78.13% and 78.13%, and specificity of 88.89% and 77.78%, respectively ( Figure 5A ). However, other indicators had relatively weak predictive value for COVID-19 (Detailed data are listed in Table 1 ). We further used parameters with different expressions and Table 2 ).

COVID-19 has emerged as a severe infectious disease and is affecting the whole world, threatening human health and life. Lymphocytes are core components of the immune system to fight against virus intrusion, especially of CD8 + T and NK cells which play a vital role in the clearance of virus [15, 16] . However, it's still largely unclear about the characteristic of lymphocyte subsets, especially of CD8 + T and NK cells, in COVID-19 patients.

In this study, we detected the characteristic of lymphocyte subsets in 32 COVID-19

patients with different severity. We found a significant reduction of circulating total lymphocytes in COVID-19 patients compared to healthy controls, which was consistent with previous studies [8] [9] [10] [11] [12] . We also found that the decreased lymphocytes in COVID-19 patients might mainly due to the reduction of CD8 + T and NK cells.

And with the progression of COVID-19, CD8 + T and NK cells continued to decline, expression on T cells as COVID-19 patients progressed from prodromal to overtly symptomatic stages [18] , which indicated that T cells might be functionally exhausted, such results seem to be inconsistent with our research, probably because the functional status of cells is determined by the co-regulation of many activation, inhibition and apoptosis pathways. Liao et al. [19] found that compared with patients in the moderate group, the number of CD8 + T cells in the severe group was reduced, and these CD8 + T cells expressed high levels of GrA, GrK, which was very similar with our results; As for the different results of GrA and GrK expression between patients with moderate and severe in Liao et al. study [19] and our study, we speculate which may be caused by the difference in the number of statistics. And, of course, there is also a great possibility that in those with severe cases, the number of granzyme A + / perforin + CD8 + T cells was still reduced due to the dramatic reduction in total cell counts.

We then detected the cytotoxic potential of NK cells, and found an increase of GrA and perforin expression on NK cells but a decrease of GrB expression in COVID-19

patients. Besides, we found that GrA expression on NK cells was significantly increased in the Mild and Severe groups, however, as the disease progressed, GrA expression on NK cells was significantly decreased in the Critical group, which might due to a compensatory enhancement in the mild and severe stages, but when the disease reached a critical stage, the cell immune status was significantly suppressed and couldn't be compensated. To some extent, this could be confirmed by the fact that the absolute number of NK cells in the Critical group decreased significantly compared to the other three groups.

Perforin is a key effector molecule for T and NK cells to mediate cytolysis. It could lyse cells by coordinating the delivery of GrA and GrB to target cells. The perforin/ granzyme cytolytic pathway is a two-edged sword in viral defense, which could result in viral clearance and host cell damage [20] [21] [22] . Therefore, we speculate that as the COVID-19 progression, the over-activated immune status of cytotoxic CD8 + T and NK cells might mediate pathological damage and lead to continuous deterioration of patients.

Besides, we analyzed the changes of absolute number and immune status of lymphocytes in COVID-19 patients before and after remission. We found that 

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