3.1 Demographic and clinical characteristics
As of February 15, 2020, 74 patients who were confirmed to have COVID-19 on admission to the First Hospital of Changsha, were included in our study (Table 1). Among these, 17 patients (23.0%) were clinically diagnosed with severe infections, with the remaining 57 patients being categorized as non-severe. The average patient age was 49.95 ± 19.28 years, and 35 patients (47.3%) were men. A total of 67 patients (90.54%) had a history of exposure to potential transmission sources (having a history of travel or residence in Wuhan and its surrounding areas, or contact with infected individuals). Of the 74 patients, 25 (33.78%) patients had underlying diseases, including hypertension, diabetes, liver cirrhosis, and cardiovascular diseases. A higher percentage of comorbidities was found in the severe patients (41.18%, n = 17) than that in the non-severe patients (31.58%, n = 57). Compared with non-severe patients, the severe patients were significantly older (65.29 ± 12.33 years vs. 45.37 ± 18.66 years; P < 0.001). There was no significant difference in sex between the severe and non-severe patients (P = 0.595). The most common symptoms revealed by our study were fever (54.05%), cough (51.35%), fatigue (32.43%), pharyngalgia (13.51%), shortness of breath (12.16%). Moreover, severe patients were significantly more likely to suffer from fever (76.47% vs. 47.37%) and shortness of breath (35.29% vs. 5.26%), compared to non-severe patients.
3.2 Laboratory findings
The laboratory findings in patients with different degrees of disease severity are shown and compared in Table 2. Among the 74 patients who underwent laboratory examinations on admission, most tended to have lower lymphocyte counts, elevated enzyme marker (i.e. creatine kinase, lactate dehydrogenase, and aspartate aminotransferase) and infection-related biomarker (i.e. erythrocyte sedimentation rate and C-reactive protein) levels, compared to laboratory reference ranges. There were also numerous differences in blood cell counts, infection related biomarkers, enzymes, and other biochemical markers between the severe and non-severe patients. Severe patients tended to have a higher percentage of neutrophils (78.04% vs. 61.19%; P < 0.001), much lower lymphocytes counts (0.69 ± 0.36 × 10⁹ vs. 1.46 ± 0.75 × 10⁹; P < 0.001), higher neutrophil-to-lymphocyte ratios (NLRs) (3.76 (3.15–5.51) vs. 2.07 (1.48–2.93); P < 0.001), and lower eosinophil counts (0.01 ± 0.01 vs. 0.05 ± 0.07; P < 0.001). Compared to non-severe patients, severe patients presented higher C-reactive protein levels, erythrocyte sedimentation rates, lactate dehydrogenase levels, aspartate aminotransferase levels, D-dimer levels, creatine kinase levels, and lower albumin levels (P < 0.05, all).
3.3 Lymphocyte subset analysis
Limited by the detection ability of our hospital, we were only able to test several common subtypes of lymphocytes in all of the 74 patients (Table 3). The total number of T cells, B cells, and natural killer (NK) cells were significantly decreased in patients with COVID-19, and this was more evident in the severe group (675.0 vs. 1379.0/mL; P < 0.001) than in the non-severe group. In patients with COVID-19, NK cells were below normal levels, and T and B cells were both within the lower levels of the normal range. The levels of the three main subsets of lymphocytes were shown to be more suppressed in severe cases, as their counts were nearly half of those in non-severe patients (500 vs. 1014/mL, P < 0.001; 95 vs. 210/mL, P < 0.001; 52 vs. 122/mL, P < 0.001).
Different subsets of T cells were further analyzed, including helper T cells (CD3+CD4+), suppressor T cells (CD3+CD8+), and regulatory T cells (CD3+CD4+CD25+CD127low+). The levels of both helper T cell (CD3+CD4+) and suppressor T cells (CD3+CD8+) were decreased in patients with COVID-19, and this was more pronounced in severe patients compared to that in non-severe patients (335.47 vs. 666.46/mL, P < 0.001; 158 vs 334 mL, P < 0.001). However, there was no significant difference in the percentage of regulatory T cells between severe and non-severe cases (P = 0.617). The helper T cell/suppressor T cell ratio (Th/Ts) remained in the normal range, and there was no difference between the two subgroups.
3.4 The immune cells factors related to the severity of COVID-19.
The result of univariable analysis demonstrated that immune cells including lymphocytes, neutrophils, eosinophils, T cells, NK cells, Th cells and Ts cells were related to the severity of COVID-19. In multivariate logistic regression analysis, we observed neutrophils (OR (95%CI): 3.79 (1.07, 13.46), P=0.039) and B cells (OR (95%CI): 1.01 (1.00, 1.02), P=0.049) were independent risk factors for assessing the severity of COVID-19 and lymphocytes (OR (95%CI): 0.01 (0.00, 0.57), P=0.027) and Th cells (OR (95%CI): 0.99 (0.99, 1.00), P=0.019) were protect factors of the severity of COVID-19 (Table 4).
3.5 The kinetics of immune response and correlation with disease severity and outcome
We analyzed the kinetics of white blood cells associated with disease severity and outcomes in patients with COVID-19. Significant increases in the neutrophil counts of the severe group were observed at day 8 and 15 compared to the non-severe group (Fig. 1A). With improved patient conditions, the number of neutrophils in severe patients decreased significantly after day 15. We took a generalized linear mixed model to find that the severity of disease (F = 0.719, P = 0.459) and curing time (F = 3.132, P = 0.136) were not related to neutrophil counts. The lymphocytes in the severe group was significantly lower than non-severe group at day 1,8,15,20 and 25 (Fig. 1B). With improved patient conditions, the number of lymphocytes in severe patients gradually increased. We found the severity of disease (F = 11.244, P = 0.044) was related to lymphocytes counts, but curing time (F = 3.228, P = 0.115) was not related to lymphocytes counts in generalized linear mixed model. At day 1, eosinophils of severe group were significantly decreased compared to the non-severe group (Fig. 1C). At other time points, we found no significant differences in eosinophils counts between the two groups.
We further analyzed the kinetics of lymphocyte subsets associated with disease severity and outcomes in patients with COVID-19. The similar trend was observed in T cells (Fig.2A), Ts cells (Fig.2B) and Th cells (Fig.2C). In severe patients, the numbers of T cells, Th cells and Ts cells increased from day 1 to day 20, and decreased after day 20. Before day 15, the numbers of T cells, Th cells and Ts cells in severe group were significantly lower than non-severe group. We found that the severity of disease (F = 6.208, P = 0.047) and curing time (F = 4.730, P = 0.017) were related to T cells and the severity of disease (F = 16.747, P = 0.009) and curing time (F =10.727, P = 0.002) were also related to Th cells. No significant differences in NK cells were observed between the two groups during the whole observation period (Fig. 2D).
We analyzed the kinetic changes of inflammatory cytokine levels, including IL-2 (Fig. 3A), IL-4 (Fig. 3B), IL-6 (Fig. 3C), IL-10 (Fig. 3D), IL-17A (Fig. 3E), IFN-γ (Fig. 3F) and TNF–α (Fig. 3G). There were no significant differences in the levels of IL-2, IL-4, IL-17A, IFN-γ and TNF–α between non-severe group and severe group at day 1,8,15,20 and 25. And all of them showed a gradual downward trend. But in generalized linear mixed model, we found the severity of disease (F = 10.535, P = 0.048) and curing time (F =10.439, P = 0.023) were related to IFN-γ levels and curing time (F =39.345, P < 0.001) were related to TNF–α levels. IL-6 levels showed sustained increases in the severe group compared to the non-severe group until day 20. The IL-6 levels of the non-severe group remained basically unchanged. And at day 1, 8, 15 and 20, The IL-6 levels in severe patients were significantly higher than non-severe patients. We observed IL-10 levels showed sustained increases in the severe group from day 1 to day 8. Reductions in serum IL-10 levels in the severe group started at day 8. At day 1, 8 and 15, IL-10 levels in the severe group were significantly increased compared to the non-severe group.