Clinical characteristics and predictive value of low CD4+T cell count in patients with moderate and severe COVID-19: A multicenter retrospective study


 BackgroundIn December 2019, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, Hubei, China. And, it has become a global pandemic. Describe the patient's clinical symptoms in detail, finding markers that predict the prognosis of patients with COVID-19 are of great value.MethodsIn this multicenter, retrospective study, 476 patients with COVID-19 were recruited from a consecutive series. After screening, a total of 395 patients were included in this study. All-cause death was the primary endpoint. All patients were followed up from admission till discharge or death.ResultsThe dominant symptoms observed in the study included fever on admission, cough, fatigue, and shortness of breath. The most frequent comorbidities were hypertension and diabetes. Compared with patients with higher CD4+T cell levels, patients with lower CD4+T cell levels were older and were more frequently male. Reduction of CD8+T cell is an indicator of the severity of COVID-19. Both decreased CD4+T cell [HR:13.659; 95%CI: 3.235-57.671] and CD8+T cell [HR: 10.883; 95%CI: 3.277-36.145] were associated with in-hospital death in COVID-19 patients, but only decreased CD4+T cell was an independent predictor of in-hospital death in COVID-19 patients.ConclusionsReductions in lymphocytes and lymphocyte subsets were common in COVID-19 patients, especially in severe cases. It was the CD8+T cell, not the CD4+T cell, that reflected the severity of the patient’s disease. Only CD4+T cell reduction was independently associated with increased in-hospital death in COVID-19 patients.Trial registration: Prognostic Factors of Patients With COVID-19, NCT04292964. Registered 03 March 2020. https://clinicaltrials.gov/ct2/show/NCT04292964.


Background
In December 2019, an outbreak of coronavirus disease 2019 (COVID- 19), an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in mainland China. Although the overall case fatality rate of patients with COVID-19 is relatively low [1], the number of deaths related to COVID-19 has already exceeded the sum of SARS and MERS, which has brought great harm to human beings. Moreover, the fatality rate of patients with severe COVID-19 is higher and the harm is bound to be greater [2]. Describe the patient's clinical symptoms in detail, nding markers that predict the prognosis of patients with COVID-19 are of great value.
The decline of T-lymphocytes in peripheral blood is a typical laboratory characteristic of patients with COVID-19, especially in severe COVID-19 patients [3,4]. A recent study recruited 21 patients with COVID-19 including 11 severe COVID-19 patients and 10 moderate COVID-19 patients. The study showed an absolute number of T-lymphocytes, CD4 + T and CD8 + T cells decreased in almost all the patients with COVID-19, and signi cantly lower in severe COVID-19 patients (294.0, 177.5 and 89.0×10 6 /L) than moderate COVID-19 patients (640.5, 381.5 and 254.0×10 6 /L). Meanwhile, most patients did not show a decrease in B-lymphocytes count but showed a tendency to an increased B-lymphocytes count. This phenomenon suggested that SARS-CoV-2 infection may primarily affect T-lymphocytes particularly CD4 + T and CD8 + T cell [4]. T-lymphocytes play a critical role in antiviral immunity. CD4 + T lymphocyte subsets secrete a high level of effector cytokines, especially interferon-γ (IFN-γ), which are essential for virus clearance [5,6]. A previous study also showed that the drastic reduction in total lymphocytes indicated the consumed immune cells and the destructed cellular immune function by coronavirus [7].
However, there are not enough studies on whether CD4 + T cell predicts the prognosis of COVID-19 patients.

Subjects
Medical records from 476 patients with con rmed COVID-19 were collected in Hubei General Hospital and Chongqing Three Gorges Central Hospital. Missing CD4 + T cell count or CD + 8T cell count data (n=58), malignant tumor (n=8), younger than 18 years (n=11), eGFR≤30ml/min (n=3), and pregnant (n=1) were excluded, patients with immune system diseases or HIV, which may affect lymphocyte and subsets, were also excluded. Finally, 395 patients with COVID-19 were analyzed in this study ( Figure 1). The positive infected cases were con rmed by testing new coronavirus nucleic acid by real-time uorescent Polymerase Chain Reaction (RT-PCR). Patients with severe COVID-19 were de ned according to the New Coronavirus Pneumonia Prevention and Control Program issued by the National health commission of the People's Republic of China (5th edition). Patients with respiratory distress (respiratory rates ≥30 per/min or resting oxygen saturation ≤93% or partial pressure of arterial oxygen (PaO2)/inspired oxygen fraction (FiO2) ≤300mmHg or respiratory failure requiring mechanical ventilation, were de ned as severe COVID-19, and the remaining patients were de ned as moderate COVID-19 patients. CD4 + T cell count, CD8 + T cell count, and lymphocytes count were divided into lower group and higher group according to the low value of laboratory reference values. The study was a multicenter, retrospective, observational registry with clinicaltrials.gov identi er NCT04292964. All study procedures were approved by the local ethics committee (approval NO. 20200701). All data were collected by experienced researchers using blinded methods.

Baseline data and follow-up
Demographic and clinical characteristics were collected from the electronic medical record system. Data collection of laboratory results were de ned by the results of the rst test after admission. The absolute number of lymphocytes was measured by an automatic blood cell analyzer. Peripheral blood lymphocyte subsets were detected by ow cytometry. Data from both clinical centers were standardized, and standardized forms were used to collect clinical data from COVID-19 patients. All COVID-19 patients in the study were followed up from admission till death or discharge. The outcome was de ned as the inhospital death rate.

Statistical analysis
Continuous data were expressed as mean ± standard deviation (SD) or median (interquartile range) according to the distribution. Categorical variables were presented as frequency rates with percentages. Continuous variables with normal distribution were compared using independent group T-test; otherwise, the Mann-Whitney U test. Categorical data were tested using the Chi-square test and Fisher's exact Chisquare test. Cox proportional-hazards models were used to perform univariate analyses and multivariate analyses to identify the association between CD4 + T cell count and in-hospital death. Kaplan-Meier survival analysis with a log-rank test was performed to estimate the cumulative survival rate of groups with higher or lower CD4 + T cell count. Statistical analyses were performed by the IBM SPSS Statistics 26.0 software. P (two-sided) value less than 0.05 was considered statistical signi cance.
According to the low value of laboratory reference values of CD4 + T cell count, the 395 COVID-19 patients were divided into two groups: lower CD4 + T cell group and higher CD4 + T cell group. Patients in the lower group were older (55.0±16. 5   There was no signi cant change in the proportion of CD4 + T cell lower than the lower limit of normal in moderate and severe COVID-19 patients, but the proportion of CD8 + T cells lower than the lower limit of normal in moderate and severe COVID-19 patients accounted for 36.0% (71/197) and 51.5% (102/198), respectively. (Figure 2A). The analysis also found that it is the CD8 + T cell count that re ects the severity of the patient's condition, not the CD4 + T cell count. ( Figure 2B).
In terms of computed tomography ndings, in moderate COVID-19 patients, compared with patients in the higher group, patients in the lower group more often represented as local patchy shadowing (45 [47.4%] vs 33 [32.4%], P=0.031). Ground-glass opacity and local patchy shadowing did not differ between the two groups in the entire patient population. (Table1).

Treatment and Clinical outcome
In all cases, the proportion of use of oxygen inhalation, and mechanical ventilation were 84.3% (328/389), and 7.7% (30/388), respectively. The most common therapy is treatment with antiviral treatment (388/395, 98.2%), followed by antibiotic treatment (  The low CD4 + T cell group had a higher in-hospital death rate than the high CD4 + T cell group during the follow-up period (log rank<0.001). The same trend was also found in severe COVID-19 patients (log rank<0.001). Kaplan-Meier survival analysis was not performed on moderate COVID-19 patients because no patients died during follow-up.

Results of Cox proportional hazards analyses of in-hospital death
Cox proportional hazard regression analysis was performed to test the associations between the lower factors, we thus concluded that reduced CD4 + T cell was a better predictor of in-hospital death. After adjusting for age, a history of hypertension, shortness of breath, white blood cell count platelet count, Ddimer, and CD4/CD8 (Mode 6), the HR of the lower CD4 + T cell count group for in-hospital death was 7.656 (95%CI: 1.610-36.396, P=0.010). Multivariate analysis demonstrated that presenting with lower CD4 + T cell count was an independent risk factor for in-hospital death. Variables like age, white blood cell count, and shortness of breath also showed signi cance for independently predicting in-hospital death in this study (Figure4). Similarly, Cox proportional hazards analyses were also performed on severe COVID-19 patients, and the results also suggested that lower CD4 + T cell count was an independent risk factor for in-hospital death (Supplementary table4, Supplementary table5, Supplementary gure1).

Discussion
This study revealed the relationship between the lymphocyte subsets of COVID-19 patients and the severity of COVID-19 and in-hospital mortality. The dominant symptoms observed in the study included fever on admission, cough, fatigue, and shortness of breath. The most frequent comorbidities were hypertension and diabetes. Compared with patients with higher CD4 + T cell levels, patients with lower CD4 + T cell levels were older and were more frequently male. In terms of laboratory ndings, lymphocytes count, CD4 + T cell count, CD8 + T cell count were signi cantly lower in the lower group. Reduction of CD8 + T cell was an indicator of the severity of COVID-19. Both decreased CD4 + T cell and CD8 + T cell were associated with in-hospital death of COVID-19 patients but only decreased CD4 + T cell was an independent predictor of in-hospital death of COVID-19 patients.
We found CD8 + T cell reduction was associated with the severity of COVID-19. Previous studies suggested that CD4 + T cell and CD8 + T cell were reduced in the vast majority of patients with either severe or moderate COVID-19 patients [4,8]. Reductions in CD4 + T cell and CD8 + T cell were associated not only with the severity of COVID-19 but also with adverse outcomes [8,9]. In the present study, we found that decreased CD4 + T cell and CD8 + T cell were common in COVID-19 patients; there was no signi cant difference in the reduction of CD4 + T cell between moderate and severe COVID-19 patients, while CD8 + T cell was more likely to be reduced in severe COVID-19 patients, suggesting that the reduction of CD8 + T cell could re ect the severity of the disease. This result was similar to the previous report, which pointed out that the reduction of CD8 + T lymphocyte subsets was associated with the severity of COVID-19 [10].
The reason may be that CD8 + T cytotoxic cells can promote virus clearance by producing many bioactive molecules such as perforin, granzyme and interferon; thus decreased CD8 + T cell can re ect the severity of COVID-19 [11].
We found it was CD4 + T cell reduction, not CD8 + T cell reduction, which was the independent risk for inhospital death of COVID-19 patients. We conducted the univariate analysis, the same as previous reports.
The results con rmed that decreased CD4 + T cell and CD8 + T cell was associated with poor prognosis of COVID-19 patients. We also performed Cox's proportional hazard regression, which suggested that after adjusting for other confounding factors, only CD4 + T cell reduction was the independent risk for inhospital death of COVID-19 patients. Lymphocyte and subsets play an important role in maintaining immune system function. CD8 + T cell is crucial to directly attacking and killing virus-infected cells, CD4 + T cell can affect the differentiation and maturation of other cells by producing cytokines and chemokines, and the secretion of interferon-γ is a T-cytokine with both antiviral and immune activity [12,13]. Patients infected with SARS-CoV-2 show a Th1 cell response and use cellular immunity to control the infection [14]. Viral infection causes comprehensive changes in cellular immunity, manifested by lymphopenia, changes in T cell subpopulation distribution, and increased cytokine concentration [15]. But the mechanisms of SARS-CoV-2 infection leading to decreased lymphocyte and subsets remains unclear. It was reported that the elevated concentration of IL-10, Interleukin-6 (IL-6), and TNF-α were negatively correlated with the total T-cell count, CD4 + T cell count, and CD8 + T cell count, respectively. Compared with patients in the illness period, levels of IL-10, IL-6, and TNF-α in the patients in the decline stage decreased signi cantly, while the total T-cell counts, CD4 + T cell count, and CD8 + T cell count were recovered [16,17]. The phenomena suggested the decrease of T-cells in COVID-19 patients may be due to the negative effects of high concentrations of TNF-α, IL-6, IL-10 in serum on the survival or proliferation of T-cells [16].And, studies reported angiotensin-converting enzyme 2 (ACE2) is expressed in white blood cells, lymphopenia may be due to the direct lethal effect of SARS-COV-2 on lymphocytes through its binding to ACE2 receptors [18,19].
Increased age and increased white blood cell count in our study were associated with in-hospital death, which was similar to several reports. It was shown that the total case fatality rate increased with age in COVID-19 patients, possibly because they often had other chronic diseases, as well as a decrease in lymphocyte and subsets with age [20]. A previous study suggested that white blood cell count and neutrophil count of dead patients were higher than those of surviving patients, which may be related to cytokine storm caused by the invasion of SARS-Cov-2 [21]. It was reported patients with malignancy or immune system diseases may have an increased risk of severe COVID-19 and death [22]. To avoid these confounders, our study excluded all patients with malignancy or immune system diseases.
This study was limited by sample size and lack of dynamic detection of CD4 + T cell and CD8 + T cell. First, our study only analyzed 395 patients with COVID-19, the relatively small sample sizes may affect the statistical power. Secondly, the patients included in this study lacked dynamic measurements of CD4 + T cell and CD8 + T cell, which made the evaluation of the relationship between CD4 + T cell and disease changes in patients with COVID-19 incomplete.

Conclusions
In conclusion, the main ndings of the study were that it was the CD8 + T cell, not the CD4 + T cell, that re ected the severity of the patient's disease; And, the high prognostic value of decreased CD4 + T cell in patients with COVID-19. Both decreased CD4 + T cell and CD8 + T cell were associated with in-hospital death of COVID-19 patients, but only CD4 + T cell reduction was independently associated with increased in-hospital death of COVID-19 patients. Thus, in this acute-care setting, CD4 + T cells can provide early prognostic information in patients with COVID-19.

Declarations
Ethics approval and consent to participate: All study procedures were approved by the local ethics committee (approval NO. 20200701). Due to the urgency of the disease at the time, the patient's verbal consent was obtained during the data collection.
Consent for publication: Not Applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: All authors declare no con ict of interest. Authors' Contributions: W.X. participated in study design, analyzing data analysis, and manuscript writing. G.L., C.X., J.D., H.B., C.Y., L.P., and T.X. were involved in data collection. Q.S., C.G., and Z.D. were responsible for the study concept, design, and nal approval of the manuscript. W.X. is the rst author. All authors have read and approved the nal manuscript.