key: cord-0853626-bsgqsdbx
authors: Chen, Guang; Wu, Di; Guo, Wei; Cao, Yong; Huang, Da; Wang, Hongwu; Wang, Tao; Zhang, Xiaoyun; Chen, Huilong; Yu, Haijing; Zhang, Xiaoping; Zhang, Minxia; Wu, Shiji; Song, Jianxin; Chen, Tao; Han, Meifang; Li, Shusheng; Luo, Xiaoping; Zhao, Jianping; Ning, Qin
title: Clinical and immunologic features in severe and moderate forms of Coronavirus Disease 2019
date: 2020-02-19
journal: nan
DOI: 10.1101/2020.02.16.20023903
sha: d4cfb1fc4fc53abbc1d4b0ca60276c6af6632c3c
doc_id: 853626
cord_uid: bsgqsdbx

Background Since late December, 2019, an outbreak of pneumonia cases caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, and continued to spread throughout China and across the globe. To date, few data on immunologic features of Coronavirus Disease 2019 (COVID-19) have been reported. Methods In this single-centre retrospective study, a total of 21 patients with pneumonia who were laboratory-confirmed to be infected with SARS-CoV-2 in Wuhan Tongji hospital were included from Dec 19, 2019 to Jan 27, 2020. The immunologic characteristics as well as their clinical, laboratory, radiological features were compared between 11 severe cases and 10 moderate cases. Results Of the 21 patients with COVID-19, only 4 (19%) had a history of exposure to the Huanan seafood market. 7 (33.3%) patients had underlying conditions. The average age of severe and moderate cases was 63.9 and 51.4 years, 10 (90.9%) severe cases and 7 (70.0%) moderate cases were male. Common clinical manifestations including fever (100%, 100%), cough (70%, 90%), fatigue (100%, 70%) and myalgia (50%, 30%) in severe cases and moderate cases. PaO2/FiO2 ratio was significantly lower in severe cases (122.9) than moderate cases (366.2). Lymphocyte counts were significantly lower in severe cases (7000 million/L) than moderate cases (11000 million/L). Alanine aminotransferase, lactate dehydrogenase levels, high-sensitivity C-reactive protein and ferritin were significantly higher in severe cases (41.4 U/L, 567.2 U/L, 135.2 mg/L and 1734.4 ug/L) than moderate cases (17.6 U/L, 234.4 U/L, 51.4 mg/L and 880.2 ug /L). IL-2R, TNF-α and IL-10 concentrations on admission were significantly higher in severe cases (1202.4 pg/mL, 10.9 pg/mL and 10.9 pg/mL) than moderate cases (441.7 pg/mL, 7.5 pg/mL and 6.6 pg/mL). Absolute number of total T lymphocytes, CD4+T cells and CD8+T cells decreased in nearly all the patients, and were significantly lower in severe cases (332.5, 185.6 and 124.3 million/L) than moderate cases (676.5, 359.2 and 272.0 million/L). The expressions of IFN-γ by CD4+T cells tended to be lower in severe cases (14.6%) than moderate cases (23.6%). Conclusion The SARS-CoV-2 infection may affect primarily T lymphocytes, particularly CD4+T cells, resulting in significant decrease in number as well as IFN-γ production, which may be associated with disease severity. Together with clinical characteristics, early immunologic indicators including diminished T lymphocytes and elevated cytokines may serve as potential markers for prognosis in COVID-19.

Coronaviruses (CoV) are a large family of respiratory viruses that can cause diseases ranging 8 ≤ 100 on admission.

Excluding one comatose patient without a clear history (classified as severe case), the most 2 3 9

common clinical manifestations at onset of illness include fever (100% of total cases), cough 2 4 0 (70% of severe cases, 90% of moderate cases), fatigue (100% of severe cases, 70% of 2 4 1 moderate cases) and myalgia (50% of severe cases, 30% of moderate cases). Less common 2 4 2 symptoms include sputum production (20% of severe cases, 30% of moderate cases), diarrhea 2 4 3 (10% of severe cases, 30% of moderate cases), headache (10% of severe cases, 10% of 2 4 4 moderate cases) and hemoptysis (10% of severe cases). All the severe cases developed 2 4 5 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi. org/10.1101 org/10. /2020 1 1 dyspnea, and nine of them had SpO2<93% even with high-flow nasal cannula, who were then 2 4 6 ventilated using the BiPAP mode to treat hypoxemia. The mean duration from illness onset to 2 4 7 dyspnea was 8.6 days. Arterial blood gas (ABG) test was performed in 10 patients on 2 4 8 admission (six severe and four moderate cases). Of them, PaO2/FiO2 ratio was significantly 2 4 9 lower in severe cases (122.9 mmHg) than moderate cases (366.2 mmHg). Three severe cases The routine blood tests on admission of three (30%) moderate cases showed mild leucopenia 2 5 4

(white blood cell count (WBC) < 4 × 10 /L). WBC counts were normal or slightly increased 2 5 5 in all the severe cases. Both WBC and neutrophil counts were significantly higher in severe 2 5 6 cases (9.2 × 10 /L and 8.0 × 10 /L) than moderate cases (4.7 × 10 /L and 3.1 × 10 /L).

Whereas lymphocyte counts were significantly lower in severe cases (0.7 × 10 /L) than 2 5 8 moderate cases (1.1 × 10 /L). Lymphopenia (lymphocyte count <0.8 × 10 /L) was found in 2 5 9 8 (72.7%) severe cases and only 1 (10.0%) moderate cases (table 2) .

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were higher in severe cases (567.2 U/L) than moderate cases (234.4 U/L).

Concentrations of serum high-sensitivity C-reactive protein (hsCRP) and ferritin on tended to be higher in severe cases (0.5 ng/mL) than moderate cases (0.1 ng/mL). Activated 2 6 9 partial thromboplastin time (APTT) on admission was significantly shorter in severe cases 2 7 0 (36.2s) than moderate cases (44.6s). D-dimer levels on admission were markedly greater in 2 7 1 severe cases (8.2 ug/mL) than moderate cases (0.4 ug/mL).

Interstitial lung abnormalities were observed in chest CT scans of all patients on admission.

Of the 21 patients, 10 (90.1%) severe cases and 7 (70%) moderate cases had bilateral 2 7 4 All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity. showed bilateral ground glass opacity ( figure 1C ). Later chest CT images revealed bilateral 2 8 0 ground-glass opacity had been resolved (figure 1D). and IL-10 concentrations on admission were significantly higher in severe cases (1202.4 2 9 0 pg/mL, 10.9 pg/mL and 10.9 pg/mL) than moderate cases (441.7 pg/mL, 7.5 pg/mL and 6.6 2 9 1 pg/mL).

Preliminary analysis of circulating immune cells subsets showed that absolute number of total 2 9 3

T lymphocytes, CD4 + T cells and CD8 + T cells decreased in the majority of patients with either 2 9 4 severe (100%, 100% and 87.5%) or moderate COVID-19 (83.3%, 100% and 83.3%), and 2 9 5 total T lymphocytes, CD4 + T cells and CD8 + T cells counts were reduced more profoundly in author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 1 3 significant decrease in all the lymphocytes subsets excluding B cells, with total T 3 0 4 lymphocytes counts below 400 × 10 6 /L, CD8 + T cells counts below 150 × 10 6 /L, and NK cells 3 0 5 counts below 77 × 10 6 /L. Of these six patients, three (50%) eventually died.

Moreover, the frequencies of Treg (CD4 + CD25 + CD127 low+ ) and CD45RA + Treg decreased in 3 0 7

nearly all the severe (60% and 100%) and moderate cases (100% and 100%), with 3 0 8

CD45RA + Treg decreased more profoundly in severe cases (0.5%) than moderate cases (1.1%).

The expressions of IFN-γ by CD4 + T, CD8 + T and NK cells were decreased in some patients bilateral ground-glass opacity and subsegmental areas of consolidation.

Of 21 author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10. 1101 /2020 1 4 laboratory-confirmed SARS-CoV-2 infection.

All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.02.16.20023903 doi: medRxiv preprint 1 5

This is the first preliminary study evaluating descriptively the immunologic characteristics of including hsCRP and ferritin were significantly higher in severe cases than moderate cases.

Plasma concentrations of both pro-inflammatory cytokines and anti-inflammatory cytokines,

including IL-2R, TNF-α and IL-10 increased in the majority of severe cases and was 3 5 2 significantly higher than did those in moderate cases, suggesting cytokine storms might be 3 5 3 associated with disease severity.

Additionally, we also noted that SARS-CoV-2 infection can cause a significant reduction in in peripheral blood remained within the normal range in most patients, the prevalence of 3 5 7

decreasing CD4 + T cell counts and CD8 + T cell counts was considerably high in both severe 3 5 8

and moderate cases. It is notable that both the proportion and number of B cells were not 3 5 9

frequently decreased in these patients. More importantly, the number of CD4 + T cells and

CD8 + T cells was markedly lower in severe cases than moderate cases. Albeit diminished 3 6 1 CD8 + T cells, the proportion of HLA-DR + expression on CD8 + T cells over 35% was more 3 6 2 common in severe cases than those of moderate cases. The expression of HLA-DR on human 3 6 3 All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the total T lymphocyte counts below 400 × 10 6 /L, CD8 + T cells below 150 × 10 6 /L, and NK cells 3 6 9

below<77 × 10 6 /L, of these six patients, three (50%) eventually died, indicating that COVID-19 and extremely high mortality. Moreover, the production of IFN-γ by CD4 + T cells 3 7 2

but not CD8 + T cells or NK cells tended to be lower in severe cases than moderate cases.

These data suggest that SARS-CoV-2 infection may affect primarily T lymphocytes,

particularly CD4 + T cells, resulting in diminished number as well as their IFN-γ production,

which might be correlated with disease severity of COVID-19.

CD4 + T cells play a pivotal role in regulating immune responses, orchestrating the deletion 3 7 7

and amplification of immune cells, especially CD8 + T cells. CD4 + T cells facilitate 3 7 8

virus-specific antibody production via the T-dependent activation of B cells 11 . However, CD8 + 3 7 9

T cells exert their effects mainly through two mechanisms, cytolytic activities against target 3 8 0 cells or cytokines secretion, including IFN-γ, TNF-α, and IL-2 as well as many chemokines 12 .

The production of IFN-γ is essential for the resistance against infection of various pathogens 3 8 2 such as virus, bacteria, and parasite 13 . As a major source of IFN-γ, the ability of T cells to leads to an enhanced immune-mediated interstitial pneumonitis and delayed clearance of 3 9 2 All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity. demonstrated that the majority of infiltrative inflammatory cells in the pulmonary interstitium 3 9 5

are CD8 + T cells that play an important role in virus clearance as well as in immune-mediated 3 9 6

injury 17 . After comparing T cell-deficient mice and B cell-deficient mice, it is found that T 3 9 7

cells are able to survive and kill virus-infected cells in the MERS-CoV infected lung 18 . These 

Although nothing is known about mechanism underlying the lymphopenia caused by 4 1 7

SARS-CoV-2 infection, in this present study, some patients were administered 4 1 8 methylprednisolone, the average dosage was a bit higher in severe cases (40-80mg once a day) 4 1 9

than moderate cases (20-40mg once a day). Since corticosteroids have a profound effect on 4 2 0 circulating T lymphocytes, which may involve their movement out of the intra-vascular 4 2 1 All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity. to determine the effects of corticosteroid on lymphocytes in the patient with COVID-19.

Our study has some limitations. First of all, we mainly evaluated the number of T cell subsets 4 2 6

and NK cells as well as their IFN-γ production, the function of these cells, and the role of implications for the development of therapeutics and vaccines.

In conclusion, the SARS-CoV-2 infection may affect primarily T lymphocytes, especially 4 3 6

CD4 + T cells, resulting in significant decrease in number as well as IFN-γ production, which author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the integrity of data and the accuracy of the data analysis.

GC and DW contributed to patient recruitment, data collection, data analysis, data 4 4 7

interpretation, literature search, and writing of the manuscript.

WG and YC had roles in patient recruitment, data collection, and clinical management.

DH, HW, TW and XZ had roles in the experiments, data collection, data analysis, and data 4 5 0

interpretation.

All authors contributed to data acquisition, data analysis, or data interpretation, and reviewed author/funder, who has granted medRxiv a license to display the preprint in perpetuity. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 2 3 bilateral ground-glass opacity and subsegmental areas of consolidation on day 6 after 5 2 7

symptom onset (A), and typical presentation of a "white lung" appearance with bilateral 5 2 8 multiple lobular and subsegmental areas of consolidation on day 8 after symptom onset (B).

Chest CT axial view lung window from a 32-year-old male with moderate COVID-19

showing bilateral ground-glass opacity on day 7 after symptom onset (C), and resolved 5 3 1

bilateral ground-glass opacity on day 11 after symptom onset (D). All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 2 4 All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 2 6 author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Mann-Whitney U test.

All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.02.16.20023903 doi: medRxiv preprint 2 8 5 5 5

All rights reserved. No reuse allowed without permission.

author/funder, who has granted medRxiv a license to display the preprint in perpetuity. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the Data are mean (S.D.) or n/N (%), where N is the total number of patients with available data.

p values comparing severe cases and moderate cases are from χ ², Fisher's exact test, or

Mann-Whitney U test. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10. 1101 /2020 

author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the

High-sensitivity