key: cord-0867609-qng05jry
authors: Wan, S.; Yi, Q.; Fan, S.; Lv, J.; Zhang, X.; Guo, L.; Lang, C.; Xiao, Q.; Xiao, K.; Yi, Z.; Qiang, M.; Xiang, J.; Zhang, B.; Chen, Y.
title: Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP)
date: 2020-02-12
journal: nan
DOI: 10.1101/2020.02.10.20021832
sha: b2b59e4f0d8ebbd2921608653a990bdd2b7a6ab4
doc_id: 867609
cord_uid: qng05jry

Background: To explore the cellular immunity and cytokines status of NCP patients and to predict the correlation between the cellular immunity levels, cytokines and the severity of patients. Methods: 123 NCP patients were divided into mild and severe groups. Peripheral blood was collected, lymphocyte subsets and cytokines were detected. Correlation analysis was performed on the lymphocyte subsets and cytokines, and the differences between the indexes of the two groups were analyzed. Results: 102 mild and 21 severe patients were included. Lymphocyte subsets were reduced in two groups. The proportion of CD8 + T reduction in the mild and severe group was 28.43% and 61.9%, respectively; The proportion of B cell reduction was 25.49% and 28.57%; The proportion of NK cell reduction was 34.31% and 47.62%; The detection value of IL-6 was 0 in 55.88% of the mild group, mild group has a significantly lower proportion of patients with IL-6 higher than normal than severe group; There was no significant linear correlation between the lymphocyte subsets and cytokines, while significant differences were noticed between the two groups in CD4 + T, CD8 + T, IL-6 and IL-10. Conclusions: Low levels of CD4+T and CD8+T are common in severe NCP. IL-6 and IL-10 levels were higher in severe patients. T cell subsets and cytokines can be used as one of the basis for predicting the transition from mild to severe. Large number of samples are still needed to confirm the "warning value" of CD4 + T, CD8 + T IL-6 and IL-10.

CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were diagnosed according to the World Health Organization interim guidance for NCP and divided 73 into mild and severe (including severe and critical) groups 74

Blood samples of the patients were collected by the nurse according to the doctor's order, and 76 all patients were not treated before the blood sampling or did not receive the standardized treatment 77 according to the diagnosis and treatment scheme of NCP. 78

Two test tubes were taken for each patient, and were numbered A and B respectively. 5 μL of 80 CD3/ CD8/ CD45/ CD4 antibody (Beijing Tongshengshidai Biotechnology Co., Ltd) was added to 81 the tube A and 5 μL of CD16 + 56/ CD45/ CDl9 antibody was added to tube B. After adding 50 μL 82 of EDTA anticoagulant whole blood to each tube, the tubes were vortexed and kept at room 83 temperature for 15 minutes in the dark. Then the samples were detected by four-color fluorescence 84 labeled flow cytometry (Mindray BriCyte E6; Mindray, Shenzhen, China). 85 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.10.20021832 doi: medRxiv preprint

The detection reagent of cytokine was provided by Qingdao Raisecare Biotechnology Co., Ltd 87 (calibrator lot number: 20190801). Six cytokines including IL-4, IL-6, IL-10, IL-17, TNF and IFN  88 were detected by multiple microsphere flow immunofluorescence according to the manufacturer's 89 instructions. After the blood sample and the corresponding flow tube be numbered 101, 102, 103, 90 104, and 105, EDTA-K2 anticoagulant whole blood was centrifuged at 2365 r / min for 30 min. 91

Then 25 μL of experimental buffer, 25 μL of centrifuged plasma, 25 μL of capture microsphere 92 antibody, and 25 μL of detection antibody were added to the corresponding flow tube. After 93 incubating at room temperature for 2 hours in the dark with gentle shaking, 25 μL SA-PE was added 94 into the flow tube respectively, and then incubation was continued for 30 minutes. Subsequently, 95 the diluted wash buffer (1:10) was added to the flow tube. After a few seconds of vortex shaking, 96 the flow tube was centrifuged at 1500r / min for 5 minutes, the liquid was slowly poured out, and 97 the flow tube was inverted on the absorbent paper. Then 100 μL of diluted washing buffer (1:10) 98 was added to the flow tube according to the requirements of the flow cytometer, and the test was 99 performed after shaking for 10 seconds. 100

All statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). 102

Descriptive analyses were performed for categorical variables such as gender. Continuous variables 103 such as inspection results were expressed as x ± s and compared using the independent samples t-104

test. Correlation analysis results were expressed by Pearson correlation coefficient and a larger r 2 105 indicates better linear correlation. P<0.05 was considered as statistically significant. 106

This study was approved by the ethical committee of Chongqing Three Gorges Central 108

Hospital and informed consent was obtained from each patient. 109

The study population included 138 hospitalized patients with confirmed NCP. 102 NCP 112 patients (55 males and 47 females) with a mean age of 43.05 ± 13.12 (15~82) years in the mild 113 group and 21 NCP patients (11 males and 10 females) with a mean age of 61.29±15.55 (34~79) 114 years in the severe group were enrolled. 115 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.10.20021832 doi: medRxiv preprint 6

According to the results of each index, CD4 + T, CD8 + T, B cell, NK cell, CD4 + T / CD8 + 117 T were divided into below normal value, within normal value and above normal value. The 118 corresponding quantities and proportions were respectively calculated, and the results were shown 119

in Table 1 In severe patients, CD4 + T was lower than normal in 20 patients (95.24%), within normal in 128 1 patient (4.76%); CD8 + T was lower than normal in 13 patients (61.90%), within normal in 8 129 patients (38.10%); B cell was lower than normal in 6 patients (28.57%), within normal in 15 patients 130 (71.43%); NK cell was lower than normal in 10 patients (47.62%), and within normal in 11 patients 131 (52.10%) 38%, 18 (85.71%) patients had CD4 + / CD8 + ratio within the normal value, 2 (9.53%) 132 patients were lower than the normal value, 1 (4.76%) was higher than the normal value. 133

IL-4, IL-6, IL-10, IL-17, TNF, and IFN were divided into values of 0, within the normal value 135

(normal values other than 0), and above the normal value according to the results of various 136

indicators. The corresponding quantities and proportions were counted ( is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.10.20021832 doi: medRxiv preprint patients (95.24%), and 1 patient (4.76%) was higher than the normal values. 146

Considering that the value of IL-6 changed most in the mild and severe groups, the correlation 148 between the lymphocyte subsets and the cytokine IL-6 was analyzed. The data with IL-6 value of 0 149 in each group (57 cases in light group and 3 cases in severe group) were excluded, and 45 cases in 150 the mild group and 18 cases in severe group were included in the correlation analysis. As shown in 151 Table 3 , the correlation analysis between IL-6 and lymphocyte subsets showed that the Pearson 152 correlation coefficients of IL-6 and CD4 + T, CD8 + T, B cell, NK cell, CD4 + T / CD8 + T were 153 very low, and there was no significant linear correlation. 154

and severe NCP 156

The data (57 cases of IL-6 and 3 cases of IFN in the mild group) with the index value of 0 in 157 the two groups were excluded. Two independent-samples t test was performed on the lymphocyte 158 subsets and cytokines of the mild group and the severe group, with α= 0.05 as the inspection level. 159

Significant differences were observed in CD4 + T, CD8 + T, IL-6, IL-10 between the mild group 160 and the severe group (P < 0.05), while no significant difference was detected in B cell, NK cell, 161

CD4 + T / CD8 + T, IL-4, IL-17, TNF, IFN between the two groups (P > 0.05, Table 4 ). 162

Lymphocyte subsets play an important role in the body's cellular immune regulation, and each 164 cell restricts and regulates each other. This study found that among NCP patients, the reduction rate 165 of CD4 + T accounted for 52.90% in the mild group, and 95.24% in the severe group; the reduction 166 rate of CD8 + T accounted for 28.40% in the mild group, and 61.90% in the severe group, indicating 167 that T lymphocytes were more inhibited in severe patients when the body is resistant to 2019-nCoV 168

infection. This was consistent with the research conclusions of Huabiao Chen 2 in SARS coronavirus, 169 revealing that the body responds in the same way when coping with homologous coronavirus 170 infection. The reduction ratio of B cell was 25.49% and 28.57% in the mild group and the severe 171 group, respectively, with no significant difference. The reduction ratio of NK cells accounted for 172 34.31% in the mild group and 47.62% in the severe group, which suggested that 2019-nCoV 173 infection limited the activity of NK cells to a certain extent, and in view of the fact that immune 174 adjuvant IL-2 can improve the activity of NK cells, the above research results may provide new 175 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The results of this study suggested that there was no significant linear correlation between 193 lymphocyte subsets and cytokines. By analyzing the differences of lymphocyte subsets and 194 cytokines in peripheral blood between the mild and severe patients, we found that only CD4 + T, 195 CD8 + T, IL-6, IL-10 had statistical significance between the mild and severe groups, suggesting 196 that the immunosuppression of severe patients with 2019-nCoV infection was more obvious, which 197 was consistent with the opinions of many experts. 5, 6 For several results of this study, for example, 198 the proportion of patients with an IL-6 value of 0 in the mild group was as high as 55.88%, there 199 was no significant difference in terms of IL-17, TNF, IFN, and IL-4 between the two groups of is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.10.20021832 doi: medRxiv preprint initiation of a specific immune response; (4) Other reasons that have not yet been confirmed. 206

This study has several limitations. Firstly, as the largest diagnosis and treatment center for 207 patients with NCP in Chongqing area, our hospital has more than 123 patients so far. The sample 208

size was relatively small compared with Wuhan, where the disease originated, which may have some 209 impact on the statistical results. But on the whole, the number of patients in this area was in the 210 middle level for other parts of the country except Wuhan, and the research results were relatively 211 reliable. Secondly, the humoral immunity level of the included patients was not monitored, so there 212 was a certain deficiency in the evaluation of the immune system. Thirdly, due to the large-scale 213 outbreak of the epidemic restricting the flow of people, data on healthy patients are lacking as blank 214

In future studies, data will be collected from healthy patients as blank controls to further 216 explore the predictive value of peripheral blood lymphocyte subsets and cytokines for patients with 217

2019-nCoV infection. At the same time, we will cooperate with other designated treatment units to 218 carry out multicenter research to include more confirmed 2019-nCoV infected patients, expand the 219 sample size, and design more rigorous randomized controlled trials. In addition, we will strengthen 220 the follow-up of patients who are cured and discharged, and regularly detect the patient's peripheral 221 blood lymphocyte subsets and cytokines. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.10.20021832 doi: medRxiv preprint

The new england journal of medicine

Study on immune response of SARS coronavirus-specific cytotoxic T 252 lymphocytes

Effect of atypical influenza virus infection on NK cell activity

Clinical features of patients infected with 256 2019 novel coronavirus in Wuhan, China.The lancet

Diagnosis and treatment of pneumonia with a new coronavirus infection (trial version 5)

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.10.20021832 doi: medRxiv preprint