key: cord-0836926-y9lxxar7
authors: Jiang, Mei; Guo, Yang; Luo, Qing; Huang, ZiKun; Zhao, Rui; Liu, ShuYuan; Le, AiPing; Li, JunMing; Wan, LaGen
title: T cell subset counts in peripheral blood can be used as discriminatory biomarkers for diagnosis and severity prediction of COVID-19
date: 2020-05-07
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa252
sha: ce25390a4d5ae93dbb73bb6deb5e73a45abaaa9a
doc_id: 836926
cord_uid: y9lxxar7

This study evaluated the significance of lymphocyte subsets detection in peripheral blood in the diagnosis and prognosis of coronavirus disease 2019 (COVID-19). Our results revealed that CD3+T, CD4+T, CD8+T cells and NK cells were significantly decreased in COVID-19 patients. COVID-19 patients had a relatively slight decrease in CD4+T cells but a severe decrease of CD8+T cells. The significantly elevated CD4/CD8 ratio was observed in COVID-19 patients. T cell subset counts were related to the severity and prognosis of COVID-19. The counts of CD8+T and CD4+T cells can be used as diagnostic markers of COVID-19 and predictors of disease severity.

Coronavirus disease 2019 (COVID-19) is viral pneumonia that affects humans. It is caused by a novel coronavirus that the International Committee on Taxonomy of Viruses identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). The infection induces groups of severe respiratory illnesses that are similar to severe acute respiratory syndrome coronavirus and are associated with intensive care unit (ICU) admission [1] . Recent studies have reported that T lymphocyte, as well as the counts of inflammatory cytokines in the peripheral blood, are correlated with the severity of COVID-19 [2, 3] . Yet, the significance of lymphocyte subsets in peripheral blood in the diagnosis and prognosis of COVID-19 still needs to be elucidated. In this study, we investigated the counts of lymphocyte subsets in COVID-19 patients and evaluated the significance of detection of lymphocyte subsets in peripheral blood in the diagnosis, disease assessment, and prognosis of COVID-19.

A total of 103 COVID-19 patients (58 males and 45 females) with a median age of 46 years (17~88) treated at the First Affiliated Hospital of Nanchang University between January 30, 2020 and February 16, 2020, were enrolled in the study. Among them, 86 (47 males and 39 females) were mild-to-moderate patients with a median age of 44 years (17~83) and 17 (11 males and 6 females) were severe patients treated in ICU with a median age of 62 years (41~88). All patients were confirmed to have SARS-CoV2 infection by virus nucleic acid test.

Thirteen healthy controls (HCs) who did not have any infectious disease and were unrelated M a n u s c r i p t 4 to the COVID-19 patients were enrolled in the study as the control group.

The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University and was performed in compliance with the Declaration of Helsinki. Informed consent forms were obtained from all participants.

The EDTA-anticoagulated peripheral blood samples were collected from all subjects.

Lymphocyte subsets were detected and counted by Cytomics 

Significant decreases were observed in the counts of CD3+T, CD4+T, CD8+T, NK cells, as well as increases in the ratio of CD4/CD8 in COVID-19 patients compared to HCs (all P<0.05). There was no statistical difference detected in B cells between the two groups (P>0.05)( Table S1 ).

Severe COVID-19 patients showed significant decreases in lymphocyte subsets counts compared to mild-to-moderate patients, especially in CD3+T, CD4+T, and CD8+T cells (all P<0.05). There was no significant difference in the percentage of Treg cells between mild-to-moderate and severe COVID-19 patients (P>0.05)( Table S2) .

23 newly diagnosed COVID-19 patients were followed up within 2 weeks. After the follow-up, the counts of CD3+T, CD4+T and CD8+T cells dramatically recovered in most patients whose virus nucleic acid test turned negative (Figure1a-c), but showed no significant difference in patients with a persistent positive nucleic acid test (Figure S1a Previous studies have reported that a significant decrease in both CD4+T and CD8+T cells was observed in patients with SARS compared to HCs. CD4+T cells are more severely damaged by the SARS virus than CD8+T cells [4, 5] . Our results revealed significant decreases in the counts of CD3+T, CD4+T, CD8+T, NK cells, especially CD3+T, CD8+T, and NK cell, as well as increases in the CD4/CD8 ratio in COVID-19 patients compared to those in the HCs. This implied that T lymphopenia, and in a particular decrease of CD8+T, was more common among COVID-19 patients than CD4+T, which differs from SARS-CoV infection. It has also been reported that the counts of T lymphocytes are related to the severity of SARS patients [4] . We found that severe patients admitted in ICU showed significant decreases in a count of lymphocyte subset compared to mild-to-moderate patients, especially CD3+T, CD4+T, and CD8+T cells. This indicated that T lymphocytes were more suppressed in severe patients compared to B cells and NK cells, which is consistent with recent studies [2, 3] . Researchers reported on two causes of T lymphopenia in SARS: sequestration into the lung of β-chemokine-recruited lymphocytes and IFN--induced apoptosis [6] . Nevertheless, the underlying mechanism of the decrease of T lymphocytes in COVID-19 patients remains unclear and needs to be elucidated by further studies.

A c c e p t e d M a n u s c r i p t 8 tissue damage [7] . However, they can also be abnormally induced by some viruses maintaining viral infection, such as hepatitis C virus, hepatitis B virus, and Epstein-Barr virus [8] [9] [10] .Our study revealed that there was no significant difference in the percentage of Treg cells between mild-to-moderate patients and severe patients. We speculated that Treg cells might not have a critical role in SARS-CoV2 infection, and multicenter researches are needed to confirm our hypothesis.

According to the diagnosis and treatment of pneumonia caused by a new coronavirus infection (trial version 7) [11], two negative new coronavirus nucleic acid tests were one of the conditions for releasing patients from isolation, which to some extent indicated that the patient was recovering from an infection. We found that the counts of CD3+T, CD4+T and CD8+T cells dramatically recovered in most follow-up patients whose virus nucleic acid test turned negative, but showed no significant difference for patients with the persistent positive nucleic acid test. It has been confirmed that T-cell immune response has an important role in recovery from SARS-CoV2 infection. Liu et al [3] also found that the decrease of T cells in the severe patient group reached its peak within the first week during the disease course, after which T cell numbers gradually increased from the second week and recovered to a count that was comparable count to the mild patient group in the third week; all the severe patients survived the disease. These results implied that the restoration of T lymphocytes was a favorable outcome. neutrophil-to-lymphocyte ratio (NLR) and neutrophil-to-CD8+T cell ratio (N8R), which had even better performance than NLR in the ROC curve analysis [3, 12] . Their kinetic analysis revealed that CD8+ T cells are the major lymphocyte subset, which decreases in cell numbers during COVID-19 [3] , which is consistent with our findings.

In summary, our results demonstrated that CD3+T cells, CD4+T cells, CD8+T cells, and NK cells were significantly decreased in COVID-19 patients and related to the severity and prognosis of COVID-19. Consequently, the counts of CD8+T and CD4+T cells can be used as diagnostic markers of COVID-19 and predictors of disease severity. To the best of our A c c e p t e d M a n u s c r i p t 10 knowledge, this is the first work that described Treg cells in COVID-19 patients; still, we found no significant difference in Treg cells percentage between mild-to-moderate patients and severe patients. This suggests that Treg cells may not have a critical role in SARS-CoV2 infection. Of note, the sample size in our study was relatively small, and multicenter researches are needed to confirm our hypothesis. To conclude, we found the risk value of 

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP)

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients

Significant Changes of Peripheral T Lymphocyte Subsets in Patients with Severe Acute Respiratory Syndrome

Expression of Lymphocytes and Lymphocyte Subsets in Patients with Severe Acute Respiratory Syndrome

An interferon-Υ-related cytokine storm in SARS patients

Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it?

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Regulatory T cells secreting IL-10 dominate the immune response to EBV

A c c e p t e d M a n u s c r i p t 11 We declare no conflicts of interest.