Clinical Observation of the Number of Lymphocytes and T Cell Subsets in Patients with COVID-19 at Different Stages

To explore the changes of lymphocytes and T cell subsets at different stages in patients with COVID-19. 86 patients with COVID-19 were enrolled, and the dynamic changes of peripheral blood lymphocytes and T cell subsets of CD3+, CD4+, and CD8+ were measured on admission, after treatment for1 week, 2 weeks, and before discharge. There were no signicant differences in the number of white blood cells and lymphocytes between admission and 2 weeks after treatment or before discharge in severe patients. The counts of CD3+, CD4+, and CD8+ T cells decreased signicantly on admission. After 2 weeks of treatment, the CD3+ counts were signicantly higher than that on admission. The CD4+ and CD8+ counts increased signicantly after 1 week of treatment, and went up remarkably before discharge compared with that on admission. There was no signicant difference in the number of CD3+ cells between the mild group and the control group on admission, but it was signicantly lower in the severe group than that in the control group and the mild group. The CD4+ and CD8+ counts decreased signicantly in both mild and severe patients on admission, and increased signicantly before discharge. At the time of discharge, the CD4+ counts in the severe and mild groups were still signicantly lower than in the control group, but there was no signicant difference in CD8+ counts among the three groups. The counts of CD3+,CD4+,and CD8+ T cells in the patients with COVID-19 is signicantly correlated with the short-term prognosis, and is more sensitive than lymphocytes. In the earliest stage, the numbers of CD4+ and CD8+ cells are more sensitive to early reduction and faster to late recovery.


Introduction
At present, more than three million people worldwide are infected with the Corona Virus Disease 2019(COVID- 19), and the number of deaths in some countries has increased 1,2,3. The epidemic of COVID-19 in China has entered the middle and late stages, and now we mainly treat the hospitalized severe ill cases and screen the imported cases. We have accumulated some experience during the treatment process. It is important to identify those individuals with COVID-19 who will become critically ill and to treat them with the right methods. We observed that in severe COVID-19, although patients had lymphocytopenia, the lymphocytes were activated. Howerer, the dynamic changes in the number and function of lymphocyte subsets, and what to do in the early stage of the severe illness have not yet been known. In the present study, some patients with severe and mild COVID-19 were selected to observe the dynamic changes of lymphocytes and T cell subsets during the treatment, and to observe the prognosis of the patients, in order to nd new biomarkers to guide the therapy at the earliest stages of hospitalization4,5.

Materials And Methods
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All patients signed informed consent. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University(WDRY2020-K031).

Study design and participants
This is a two-center prospective cohort study, from February 1 to March 1, 2020. We included consecutive patients with laboratory con rmed COVID-19 who were admitted to Renmin Hospital of Wuhan University, Shouyi district and Optics Valley district, Wuhan city, Hubei province.
Procedures 5ml of venous blood was taken from the enrolled patients on admission, 1 week, 2 weeks and discharge. White blood cells (WBC), lymphocytes, and T cell subsets of CD3+, CD4+, and CD8+ were measured to observe the dynamic changes in different periods. Samples for T lymphocyte subsets were analyzed using FACSCanto-II cytometers (Becton Dickinson, San Jose, CA). The following human T lymphocyte subsets were determined (expressed as cells/μL): CD3+, CD4+, and CD8+ cells. Lymphocyte counts were performed as controls on 8 healthy individuals.
Critically ill patients were de ned as those admitted to the intensive care unit (ICU) who required mechanical ventilation or had a fraction of inspired oxygen (FiO2) of at least 60% or more6,7,8. Laboratory con rmation of SARS-CoV-2 infection was performed by the clinical lab of our hospital authorized by Wuhan Center for disease control and prevention. The criteria for discharge were absence of fever for at least 3 days, substantial improvement in both lungs in chest CT, clinical remission of respiratory symptoms, and two throat-swab samples negative for SARS-CoV-2 RNA obtained at least 24 h apart9. All analyses were performed at a single laboratory. General clinical data of all patients were recorded, including a patient's history and physical examination and results of haematological, biochemical, radiological, and microbiological investigations, treatment (oxygen therapy, vasoconstrictive agents, antiviral agents, antibacterial agents, corticosteroids, and immunoglobulin). We got the counts of T lymphocyte subsets of the patients at different stages during hospitalization.

Data analysis
Statistical analysis was performed using IBM SPSS Version 20.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5. The data were reported as N (%) or as median (

Results
A total of 86 new COVID-19 patients were enrolled, including 44 severe patients, 42 mild patients, and 8 healthy volunteers as controls. Mild patients were treated in the common isolation ward, while severe patients were treated in the ICU isolation ward. Among the 44 patients with severe diseases, 4 died rapidly after admission to ICU, 4 patients had incomplete data, and 4 patients requested to withdraw from the study voluntarily. A total of 32 patients with severe diseases completed the research in the end. Two critically ill patients died between the rst week and the second week, and two died between the second week and discharge. Among the mild patients, 3 patients had incomplete data, 5 patients withdrew voluntarily, 3 patients became severe and were transferred to ICU, and a total of 31 patients completed the study. The data of enrolled patients are shown in table 1 and table 2.  There were no signi cant differences in the number of WBC and lymphocytes on admission of severe patients compared with that after 2 weeks of treatment and before discharge. No signi cant differences was found in the CD3+ counts between 1 week and admission, but there was a signi cant increase (P<0.01) in the counts between 2 weeks and admission, and it continued to increase before discharge.CD4+ and CD8+ counts were increased signi cantly at 1 week compared with that on admission (P<0.05). See Figure 1. Figure 2 showed that there was no difference in WBC counts among the three groups on admission, and WBC counts decreased signi cantly in severe patients before discharge(P<0.05), while there was no difference in the rest. However, there was a signi cant decrease in the number of lymphocytes in the severe group compared with other groups (all P<0.01).Before discharge, there was no difference in lymphocytes counts among the three groups. There was no signi cant difference in the CD3+ counts between the mild group and the control group on admission, but the CD3+ counts in the severe group was signi cantly lower than that in the control group and the mild group (P <0.001). There was no signi cant difference in the CD3+ counts between the mild group and the control group before discharge, but the CD3+ counts in the severe group was signi cantly lower than that in the other two groups (P <0.001).On admission, the CD4+ counts in the mild and severe groups decreased signi cantly compared with the control group, and gradually increased after treatment. There was no signi cant difference in the CD4+ counts between the severe group and the mild group before discharge, but it still decreased signi cantly compared with the control group (P <0.001).The CD8+ counts decreased signi cantly in both mild and severe patients on admission, but there was no signi cant difference between the severe and the mild group. Before discharged, the CD8+ counts recovered well and there were no signi cant differences among the three groups.

Discussion
The critically ill COVID-19 have some special characteristics in common: autopsy results show that the spleen and multiple lymph nodes atrophy accompanied by a signi cant decrease in the number of lymphocytes, the injured lung tissue has a large number of monocytes and macrophages in ltration, but the number of lymphocytes is less. In terms of clinical symptoms, severe patients usually experience sudden deterioration 7-14 days after infection, which may be related to the cytokine storm. The result of peripheral venous blood test showed that lymphocytes were mostly decreased. With the cytokine storm, the patients showed unstable symptoms, accompanied by bacterial infection and coagulation disorders, and some patients had poor clinical outcomes10,11,12,13,14.
Now it seems that there are widespread infections in many countries, and the virus is likely to coexist with us for a long time.
The key to the treatment of COVID-19 is to identify the patients who are likely to convert to severe infection at an earliest stage, and to give effective treatment to prevent the patients from progressing to acute respiratory distress syndrome (ARDS). At present, there is no simple and feasible biomarker or imaging test that can identify high-risk patients in early stage, thus saving ICU resources and improving the prognosis of patients. The conventional WBC and lymphocyte counts are affected by multiple factors, so they are not accurate and sensitive enough. Lung CT examination can see the in ammatory in ltration of the lung tissues of the patients, but the symptoms of the patients are more serious when there is obvious in ltration shadow in the double lungs. It has been widely observed that the decline of lymphocytes is signi cantly correlated with the poor prognosis of patients, but the dynamic changes of lymphocytes and their subsets at different stages and the changes of lymphocyte function in different subsets have not been observed15, 16,17,18,19. Our study dynamically observed the changes of peripheral blood lymphocytes and T cell subsets in mild and severe patients.
We found that the CD4+ counts of mild and severe patients decreased signi cantly compared with the control group (P<0.001) on admission. Before discharge, the CD4+ counts in these patients increased signi cantly compared with that on admission. However, the CD4+ counts in the severe group were still signi cantly decrease compared with the control group. The CD8+ counts also decreased signi cantly in mild patients (P<0.01) on admission, and the decline was more pronounced in severe patients, while they all recovered to normal before discharge. After infection, T lymphocyte subsets immediately reduced signi cantly, which are more sensitive than WBC and lymphocytes, and are unaffected by other factors. The lower lymphocyte subsets are directly related to the poor prognosis of patients with COVID-1920.
The total number of leukocytes and lymphocytes were affected by many factors. In the early stage, some severe patients were infected with secondary leukocytes elevation. There was no signi cant difference in leukocytes counts between the mild group and the control group on admission, which could not be used as an earliest screening index. Therefore, the measurement of the number of lymphocytes and their subsets may be a more effective and sensitive indicator for early identi cation. While we are concerned about the number of lymphocytes and their subsets, we should also pay attention to the changes of the function of lymphocyte subsets. In some cases, the counts of peripheral CD4+ and CD8+ T cells were substantially reduced, while their status was hyperactivated. The excessive hyperfunction and lower number of the lymphocytes may be the source of cytokine storm. Our results imply that overactivation of lymphocyte subsets accounts for the severe immune injury in these patients.
Drugs targeting lymphocyte proliferation or apoptosis (IL-7, PD1/PD-L1 inhibitors) could help to prevent lymphopenia or improve lymphocyte counts in severe patients. It is the key point to give the immunoregulatory treatment in the earliest stage of the patients with COVID-19.We need to improve the counts of lymphocytes and the subsets to prevent cytokine storm.
Infusion of immunoglobulin, low dose hormone therapy, and traditional Chinese medicine conditioning are the choices of the clinical treatment 21. On February 14, our guidelines explicitly recommended the use of immunoglobulin therapy in critically ill patients. We recommend traditional Chinese medicine to the mild patients, such as lianhua qingwen capsule, and pneumonia prescription 1. These methods have achieved good outcomes. One of the reasons why traditional Chinese medicine is effective is related to the improvement of patients' immune function. Anticytokine therapy alone may be unpromising, anti-IL-6 is not effective, and blood replacement therapy (arti cial liver therapy) is not effective, because immune activation may be involved in multiple-factors22.
This study suggests that the detection of the number of peripheral blood lymphocytes and subsets in patients with COVID-19 is a sensitive indicator to understand the progress and direction of the disease. We should pay attention to increase the number of lymphocytes and subsets and improve the cellular function in earliest stage.

Data availability
Data produced and processed in this study are included in the published article and the supplementary les. The datasets can be acquired from the corresponding author upon appropriate purposes.

Figure 1
Counts of White blood cells, Lymphocytes and their subsets in critically ill patients with COVID-19 at different stages.