Clinical and Immunological Features of Severe and Moderate Coronavirus Disease 2019
Clinical and Immunological Features of Severe and Moderate Coronavirus Disease 2019
Clinical and Immunological Features of Severe and Moderate Coronavirus Disease 2019
Since December 2019, an outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, and is now becoming a global threat. We aimed to delineate
and compare the immunological features of severe and moderate COVID-19.
In this retrospective study, the clinical and immunological characteristics of 21 patients (17 male and 4 female) with
COVID-19 were analyzed. These patients were classified as severe (11 cases) and moderate (10 cases) according to the
guidelines released by the National Health Commission of China.
The median age of severe and moderate cases was 61.0 and 52.0 years, respectively. Common clinical manifestations
included fever, cough, and fatigue. Compared with moderate cases, severe cases more frequently had dyspnea,
lymphopenia, and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase, C-reactive
protein, ferritin, and D-dimer as well as markedly higher levels of IL-2R, IL-6, IL-10, and TNF-α. Absolute numbers of T
lymphocytes, CD4+ T cells, and CD8+ T cells decreased in nearly all the patients, and were markedly lower in severe
cases (294.0, 177.5, and 89.0 × 10 6/L, respectively) than moderate cases (640.5, 381.5, and 254.0 × 106/L, […]
Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
BACKGROUND. Since December 2019, an outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, and is now becoming a global threat. We aimed to delineate and
compare the immunological features of severe and moderate COVID-19.
METHODS. In this retrospective study, the clinical and immunological characteristics of 21 patients (17 male and 4 female)
with COVID-19 were analyzed. These patients were classified as severe (11 cases) and moderate (10 cases) according to the
guidelines released by the National Health Commission of China.
RESULTS. The median age of severe and moderate cases was 61.0 and 52.0 years, respectively. Common clinical
manifestations included fever, cough, and fatigue. Compared with moderate cases, severe cases more frequently had
dyspnea, lymphopenia, and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase,
C-reactive protein, ferritin, and D-dimer as well as markedly higher levels of IL-2R, IL-6, IL-10, and TNF-α. Absolute numbers
of T lymphocytes, CD4+ T cells, and CD8+ T cells decreased in nearly all the patients, and were markedly lower in severe
cases (294.0, 177.5, and 89.0 × 106/L, respectively) than moderate cases (640.5, 381.5, and 254.0 × 106/L, respectively). The
expression of IFN-γ by CD4+ T cells tended to be lower in severe cases (14.1%) than in moderate cases (22.8%).
CONCLUSION. The SARS-CoV-2 infection may affect primarily T lymphocytes, particularly CD4+ and CD8+ T cells, resulting
in a decrease in numbers as well as IFN-γ production by CD4+ T cells. These potential immunological markers may be of
importance because of their correlation with disease severity in COVID-19.
TRIAL REGISTRATION. This is a retrospective observational study without a trial registration number.
FUNDING. This work is funded by grants from Tongji Hospital for the Pilot Scheme Project, and partly supported by the
Chinese National Thirteenth Five Years Project in Science and Technology for Infectious Disease (2017ZX10202201).
All patients (n = 21) Severe cases (n = 11) Moderate cases (n = 10) P value
Characteristics
Males, n (%) 17 (81.0%) 10 (90.9%) 7 (70.0%) 0.31
Age, yrs 56.0 (50.0–65.0) 61.0 (56.5–66.0) 52.0 (42.8–56.0) 0.043
>50 15 (71.4%) 10 (90.9%) 5 (50.0%) 0.043
Huanan Seafood Market exposure, n (%) 4 (19.0%) 1 (9.1%) 3 (30.0%) 0.31
Any comorbidity, n (%) 7 (33.3%) 5 (45.5%) 2 (20.0%) 0.36
Hypertension, n (%) 5 (23.8%) 4 (36.4%) 1 (10.0%) 0.31
Diabetes, n (%) 3 (14.3%) 2 (18.2%) 1 (10.0%) 1.00
Signs and symptoms
Fever, n/N (%) 20/20 (100%) 10/10 (100%) 10/10 (100%) NA
Highest temperature, °C 38.7 (38.5–39.1) 38.6 (38.4–39.3) 38.8 (38.6–39.0) 0.87
38.1°C–39.0°C, n/N (%) 12/19 (63.2%) 5/9 (55.6%) 7/10 (70.0%) 0.52
>39.0°C, n/N (%) 7/19 (36.8%) 4/9 (44.4%) 3/10 (30.0%)
Cough, n/N (%) 16/20 (80.0%) 7/10 (70.0%) 9/10 (90.0%) 0.58
Fatigue, n/N (%) 17/20 (85.0%) 10/10 (100.0%) 7/10 (70.0%) 0.21
Myalgia, n/N (%) 8/20 (40.0%) 5/10 (50.0%) 3/10 (30.0%) 0.65
Sputum production, n/N (%) 5/20 (25%) 2/10 (20.0%) 3/10 (30.0%) 1.00
Headache, n/N (%) 2/20 (10.0%) 1/10 (10.0%) 1/10 (10.0%) 1.00
Diarrhea, n/N (%) 4/20 (20.0%) 1/10 (10.0%) 3/10 (30.0%) 0.58
Chest tightness, n/N (%) 11/20 (55.0%) 8/10 (80.0%) 3/10 (30.0%) 0.07
Coma, n (%) 1 (4.8%) 1 (9.1%) 0 (0.0%) 1.00
Dyspnea, n (%) 11 (52.4%) 11 (100.0%) 0 (0.0%) 0.000
Days from illness onset to dyspnea 8.0 (7.0–10.0) 8.0 (7.0–10.0) NA NA
Systolic pressure, mmHg 122.0 (109.0–135.0) 124.0 (118.5–145.5) 120.0 (107.5–134.0) 0.17
>140 mmHg, n (%) 4 (19.0%) 4 (36.4%) 0 (0.0%) 0.09
Heart rate, bpm 89.0 (78.0–106.0) 95.0 (77.0–108.0) 89.0 (85.5–96.0) 0.90
Respiratory rate, per min 21.0 (20.0–25.0) 25.0 (22.5–31.0) 20.0 (20.0–20.8) 0.005
≥30, n (%) 5 (23.8%) 5 (45.5%) 0 (0.0%) 0.035
SpO2 ≤ 93% on room air, n (%) 11 (52.4%) 11 (100.0%) 0 (0.0%) 0.000
PaO2/FiO2 172.0 (102.1–350.0) 104.8 (94.6–119.0) 371.7 (350.0–422.7) 0.001
>300, n/N (%) 3/10 (30.0%) 0/6 (0.0%) 4/4 (100.0%) 0.007
200–300, n/N (%) 2/10 (20.0%) 1/6 (16.7%) 0/4 (0.0%)
100–200, n/N (%) 2/10 (20.0%) 2/6 (33.3%) 0/4 (0.0%)
≤100, n/N (%) 3/10 (30.0%) 3/6 (50.0%) 0/4 (0.0%)
Data are the median (IQR), n (%), or n/N (%), where N is the total number of patients with available data. P values comparing severe cases and moderate
cases are from χ2 test, Fisher’s exact test, or unpaired 2-sided Student’s t test. COVID-19, coronavirus disease 2019; FiO2, inspiratory oxygen fraction; IQR,
interquartile range; PaO2, arterial oxygen tension; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SpO2, percutaneous oxygen saturation.
of breath, and acute respiratory distress syndrome. Additionally, hospital patients with pneumonia were identified as laborato-
a study of the first 41 laboratory-confirmed cases with COVID-19 ry-confirmed SARS-CoV-2 infection at Tongji Hospital. Of these
showed that 63% of patients had lymphopenia, and cytokine patients, only 4, including a familial cluster of 3 confirmed cases,
storm could be associated with disease severity. However, infor- had direct exposure to the Huanan Seafood Market. According to
mation on immunological features between severe and moderate the guidelines for diagnosis and management of COVID-19 (6th
COVID-19 is scarce (8). edition, in Chinese) issued by the National Health Commission
In this study, we performed a comprehensive evaluation of of China (9), 11 (52.4%) patients with percutaneous oxygen sat-
characteristics of 21 patients with COVID-19 admitted to Tongji uration (SpO2) of 93% or lower or respiratory rates of 30/min or
Hospital, Wuhan. We aimed to compare the clinical and immuno- greater on room air who required high-flow nasal cannula or non-
logical features between severe cases and moderate cases. These invasive mechanical ventilation using the bilevel positive airway
findings may help us extend our understanding of the risk factors pressure (BiPAP) mode to correct hypoxemia, were classified as
associated with disease severity in the SARS-CoV-2 infection. having severe COVID-19, whereas 10 (47.6%) patients not reach-
ing the criteria for severe COVID-19 were considered moderate.
Results There were more male patients in both severe cases and moderate
Patient demographics and baseline characteristics of severe and mod- cases. The median age of the severe cases (61.0 years) was signifi-
erate COVID-19. As of January 27, 2020, a total of 21 admitted cantly higher than moderate cases (52.0 years) (Table 1). More
Normal range All patients (n = 21) Severe cases (n = 11) Moderate cases (n = 10) P value
White blood cell count, ×109/L 3.5–9.5 5.7 (4.6–8.3) 8.3 (6.2–10.4) 4.5 (3.9–5.5) 0.003
<4, n (%) 3 (14.3%) 0 (0.0%) 3 (30.0%) 0.017
4–10, n (%) 15 (71.4%) 8 (72.7%) 7 (70.0%)
≥10, n (%) 3 (14.3%) 3 (27.3%) 0 (0.0%)
Neutrophil count, ×109/L 1.8–6.3 4.8 (2.8–6.9) 6.9 (4.9–9.1) 2.7 (2.1–3.7) 0.002
Lymphocyte count, ×109/L 1.1–3.2 0.9 (0.7–1.1) 0.7 (0.5–0.9) 1.1 (1.0–1.2) 0.049
<0.8, n (%) 9 (42.9%) 8 (72.7%) 1 (10.0%) 0.008
Hemoglobin, g/L 130–175 137.0 (127.0–147.0) 136.0 (125.5–144.5) 139.5 (132.8–146.0) 0.78
Platelet count, ×109/L 125–350 160.0 (137.0–189.0) 157.0 (134.0–184.5) 175.6 (148.3–194.0) 0.88
<100, n (%) 1 (4.8%) 0 (0.0%) 1 (10.0%) 0.48
Alanine aminotransferase, U/L ≤41 26.0 (16.0–42.0) 42.0 (32.5–50.0) 16.0 (13.3–21.8) 0.000
Aspartate aminotransferase, U/L ≤40 27.0 (21.0–47.0) 47.0 (28.0–74.5) 24.0 (21.5–26.5) 0.014
>40, n (%) 6 (28.6%) 5 (45.5%) 0 (0.0%) 0.035
Albumin, g/L 35.0–52.0 33.7 (29.6–37.4) 29.6 (28.6–33.0) 37.2 (35.8–38.8) 0.013
<32 g/L, n (%) 8 (38.1%) 7 (63.6%) 1 (10.0%) 0.024
Total bilirubin, mmol/L ≤26 8.8 (6.8–10.3) 8.8 (7.9–10.5) 7.8 (6.4–9.5) 0.24
Blood urea nitrogen, mmol/L 3.1–8.0 5.1 (4.1–6.4) 6.1 (5.2–9.1) 4.0 (3.4–4.8) 0.015
Creatinine, μmol/L 59–104 81.0 (67.0–85.0) 82.0 (67.5–91.5) 76.5 (63.3–81.0) 0.21
Creatine kinase, U/L ≤190 73.0 (63.0–287.0) 214.0 (90.0–329.0) 64.0 (57.5–83.5) 0.16
Lactate dehydrogenase, U/L 135–225 336.0 (221.0–537.0) 537.0 (433.5–707.5) 224.0 (200.3–251.8) 0.001
>300 U/L, n (%) 11 (52.4%) 10 (90.9%) 1 (10.0%) 0.000
Prothrombin time, seconds 11.5–14.5 13.7 (13.0–14.5) 14.3 (13.6–14.6) 13.4 (12.8–13.7) 0.15
Activated partial thromboplastin time, seconds 29.0–42.0 39.4 (33.6–44.5) 33.7 (32.1–38.4) 44.0 (42.6–47.6) 0.002
D-dimer, μg/mL <0.5 0.5 (0.4–1.8) 2.6 (0.6–18.7) 0.3 (0.3–0.4) 0.029
Procalcitonin, ng/mL 0.02–0.05 0.11 (0.05–0.24) 0.18 (0.13–0.81) 0.05 (0.04–0.06) 0.059
<0.1, n/N (%) 7/18 (38.9%) 0/10 (0.0%) 7/8 (87.5%) 0.002
0.1–0.25, n/N (%) 6/18 (33.3%) 6/10 (60.0%) 0/8 (0.0%)
0.25–0.5, n/N (%) 2/18 (11.1%) 1/10 (10.0%) 1/8 (12.5%)
≥0.5, n/N (%) 3/18 (16.7%) 3/10 (30.0%) 0/8 (0.0%)
High-sensitivity C-reactive protein, mg/L <1 108.4 (28.0–139.5) 139.4 (86.9–165.1) 22.0 (14.7–119.4) 0.003
>60, n/N (%) 14/20 (70%) 11/11 (100.0) 3/9 (33.3%) 0.002
Ferritin, μg/L 30–400 1424.6 (337.4–1780.3) 1598.2 (1424.6–2036.0) 337.4 (286.2–1275.4) 0.049
>800, n/N (%) 12/19 (63.2%) 9/9 (100.0%) 3/10 (30.0%) 0.003
Bilateral involvement of chest CT scan 17/21 (81.0%) 10/11 (90.9%) 7/10 (70.0%) 0.31
on admission, n/N (%)
Data are the median (IQR), n (%), or n/N (%), where N is the total number of patients with available data. P values comparing severe cases and moderate
cases are from χ2, Fisher’s exact test, or unpaired 2-sided Student’s t test. COVID-19, coronavirus disease 2019; IQR, interquartile range; SARS-CoV-2,
severe acute respiratory syndrome coronavirus 2.
severe cases had comorbidity. The median time from onset of mon in severe cases. In addition, tachypnea and dyspnea were only
symptoms to first hospital admission was 8.0 days in severe cases developed in severe cases. All the severe cases developed dyspnea,
and 7.0 days in moderate cases. and 9 of them with SpO2 of 93% or lower showed no improved
Four of 11 severe cases died an average of 20 days after the SpO2 even with high-flow nasal cannula; these 9 cases were then
onset of the illness. Of these 4 deceased patients, all of them were ventilated using the BiPAP mode to treat hypoxemia. An arterial
male and aged 50 years and older, with 2 cases having hyperten- blood gas (ABG) test was performed in 10 patients on admission
sion. The median age of deceased cases was 64.0 years. Three (6 severe and 4 moderate cases). Among them, the PaO2/FiO2 ratio
of the deceased cases had an arterial oxygen tension/inspiratory was significantly lower in severe cases (104.8) than moderate cases
oxygen fraction ratio (PaO2/FiO2) of 100 or less on admission. (371.7), with 3 out of 6 severe patients below 100.
Excluding 1 patient without a clear history due to a disorder Laboratory findings and CT scans of severe and moderate
of consciousness (coma) (classified as a severe case), the most COVID-19. Compared with the normal range, the whole blood
common clinical manifestations at onset of illness included fever, count on admission of 3 (30%) moderate cases showed mild leu-
cough, fatigue, and myalgia. Less common symptoms included copenia, while white blood cell (WBC) counts were normal or
sputum production, diarrhea, headache, and hemoptysis. Com- slightly increased above the upper limit of normal (ULN) in all
pared with moderate cases, chest tightness tended to be more com- the severe cases (Table 2). Both WBC and neutrophil counts were
Discussion
This is the first preliminary study to our knowledge
descriptively evaluating the immunological char-
acteristics of patients with laboratory-confirmed
SARS-CoV-2 infection. Both clinical and epidemi-
ological features of patients with COVID-19 have
recently been reported (5, 6, 8, 10). However, there is
insufficient knowledge of pathophysiological param-
Figure 2. Plasma cytokine levels in patients with COVID-19. Series of comparisons of plasma eters, particularly immunological indicators, to
cytokine levels between severe cases (n = 9) and moderate cases (n = 7). All data presented understand the mechanism involved in COVID-19.
as the mean ± SEM. Differences were tested using unpaired 2-sided Student’s t test. Consistent with previous reports (8), the present
study showed that a male predominance in the inci-
dence of COVID-19 has been noted, similarly to
and natural killer (NK) cell counts below 77 × 106/L. Of these 6 that of SARS-CoV, indicating males are more susceptible to SARS-
patients, 3 (50%) eventually died. CoV-2 infection than females. Older males (>50 years old), partic-
The frequencies of regulatory T cells (Tregs) (CD4+CD25+ ularly those with underlying comorbidities, may be more likely to
CD127lo) and CD45RA+ Tregs were reduced (below the LLN) develop severe COVID-19. The most common clinical manifesta-
in nearly all the severe and moderate cases, and the CD45RA+ tions at onset of illness included fever, cough, fatigue, and myalgia.
Treg proportion was markedly lower in severe cases (0.5%) than Severe cases more frequently had dyspnea and developed acute
in moderate cases (1.1%). The reduced expression of interferon respiratory distress syndrome. In terms of laboratory findings,
γ (IFN-γ) by CD4+ T, CD8+ T, and NK cells below the LLN was leukocytosis (≥10 × 109/L) and lymphopenia (<0.8 × 109/L) were
observed in some patients with severe (50%, 16.7%, and 16.7%, more common in severe cases than in moderate cases. ALT, LDH,
respectively) or moderate COVID-19 (14.3%, 0%, and 14.3%, D-dimer, and inflammatory markers including hsCRP and ferritin
respectively). The expression of IFN-γ by CD4+ T cells tended to be were significantly higher in severe cases than in moderate cases.
lower in severe cases (14.1%) than moderate cases (22.8%) (Table Serum concentrations of both proinflammatory cytokines and anti-
3 and Figure 2C). However, there was no significant difference in inflammatory cytokines, including IL-2R, IL-6, TNF-α, and IL-10
terms of mean fluorescence intensity of IFN-γ production by CD4+ increased in the majority of severe cases and were markedly higher
T, CD8+ T, or NK cells (data not shown). Overall, we found a signif- than those in moderate cases, suggesting cytokine storms might be
icant reduction in CD4+ T cell count and a borderline reduction in associated with disease severity. Similarly, SARS was also charac-
IFN-γ expression in severe cases. terized by exuberant inflammatory responses and lung damage. A
Complications and clinical outcomes of COVID-19. With regard previous study using a mouse model of SARS demonstrated that
to complications as shown in Supplemental Table 2, common com- rapid kinetics of SARS-CoV replication and delay in IFN-I signal-
plications observed in severe cases included acute respiratory dis- ing promoted inflammatory monocyte-macrophage accumulation,
tress syndrome (100.0% of patients with available ABG data) and resulting in elevated lung cytokine/chemokine levels, vascular
respiratory failure (83.3%). Less common complications among the leakage, and suboptimal T cell responses (11). The underlying cel-
Figure 3. Number of immune cell subsets and proportion of IFN-γ expression in patients with COVID-19. (A) Flow cytometric analysis of NK cells, CD4+ T
cells, CD8+ T cells, and Tregs as well as production of IFN-γ by CD4+ T cells, CD8+ T cells, and NK cells from a representative patient. (B) A series of compari-
sons of absolute number of total T and B lymphocytes, CD4+ T cells, CD8+ T cells, and NK cells between severe cases (n = 8) and moderate cases (n = 6).
(C) A series of comparisons of production of IFN-γ by CD4+T cells, CD8+ T cells, and NK cells between severe cases (n = 6) and moderate cases (n = 7). All
data presented as the mean ± SEM. Differences were tested using unpaired 2-sided Student’s t test.
lular origin and mechanism involved in cytokine accumulation in severe cases than moderate cases. In contrast, both the proportion
COVID-19 warrants further exploration. and number of B cells were not reduced in most patients, with
Additionally, we noted that SARS-CoV-2 infection can cause 75.0% of severe cases showing an increased proportion of B cells.
a significant reduction in circulating lymphocytes and T cell sub- This could be partly due to the more significant decrease in T lym-
sets. Although the proportions of T cell subsets in peripheral blood phocytes in these patients. It is notable that 6 out of 8 severe cas-
remained within the normal range in most patients, decreased es and none of the moderate cases with available immunological
CD4+ and CD8+ T cell counts below the LLN were considerably data exhibited a broad, significant decline in all the lymphocyte
frequent in both severe and moderate cases. More important- subsets, excluding B cells. Of these 6 patients, 3 eventually died.
ly, the number of CD4+ and CD8+ T cells was markedly lower in Moreover, the production of IFN-γ by CD4+ T cells but not CD8+
All patients (n = 21) Severe cases (n = 11) Moderate cases (n = 10) P value Normal range
Total T lymphocytes, % 60.5 (54.4–70.3) 55.1 (52.2–60.5) 68.8 (64.7–75.2) 0.020 50–84
Total T lymphocyte count, ×106/L 486.5 (267.0–664.8) 294.0 (169.3–415.3) 640.5 (588.3–789.5) 0.011 955–2860
Decreased, n/N (%) 13/14 (92.9%) 8/8 (100.0%) 5/6 (83.3%) 0.43
<400, n/N (%) 6/14 (42.9%) 6/8 (75.0%) 0/6 (0.0%) 0.010
Total B lymphocytes (%) 16.9 (10.8–22.4) 20.2 (17.6–39.5) 10.8 (10.3–12.4) 0.025 5–18
Increased, n/N (%) 7/14 (50.0%) 6/8 (75.0%) 1/6 (16.7%) 0.10
Total B lymphocyte count, ×106/L 115.5 (57.8–249.3) 184.0 (42.8–273.3) 115.5 (102.8–133.5) 0.35 90–560
Decreased, n/N (%) 4/14 (28.6%) 3/8 (37.5%) 1/6 (16.7%) 0.58
CD4+ T cells, % 36.7 (32.0–40.0) 36.7 (30.7–37.3) 36.4 (32.0–40.6) 0.56 27–51
CD4+ T cell count, ×106/L 241.5 (135.0–363.8) 177.5 (104.0–249.8) 381.5 (255.0–451.0) 0.018 550–1440
Decreased, n/N (%) 14/14 (100.0%) 8/8 (100.0%) 6/6 (100.0%) NA
CD8+ T cells, % 22.2 (15.7–26.9) 17.4 (14.7–23.4) 25.2 (22.8–34.2) 0.093 15–44
CD8+ T cell count, ×106/L 169.5 (86.0–281.5) 89.0 (61.5–130.3) 254.0 (183.3–312.8) 0.035 320–1250
Decreased, n/N (%) 12/14 (85.7%) 7/8 (87.5%) 5/6 (83.3%) 1.00
<150, n/N (%) 6/14 (42.9%) 6/8 (75.0%) 0/6 (0.0%) 0.010
NK cells, % 14.8 (10.3–21.9) 14.7 (7.5–21.0) 15.1 (11.6–22.8) 0.62 7–40
NK cell count, ×106/L 89.0 (58.8–207.0) 60.5 (27.5–109.0) 180.5 (115.0–228.0) 0.27 150–1100
Decreased, n/N (%) 8/14 (57.1%) 6/8 (75.0%) 2/6 (33.3%) 0.28
<77, n/N (%) 6/14 (42.9%) 6/8 (75.0%) 0/6 (0.0%) 0.010
CD28+CD4+ T cells/CD4+ T, % 98.3 (96.8–98.8) 97.5 (96.8–98.7) 98.6 (97.2–99.0) 1.00 84.11–100.00
CD28+CD8+ T cells/CD8+ T, % 64.8 (44.6–75.9) 44.6 (37.5–73.1) 70.3 (63.3–76.9) 0.20 48.04–77.14
HLA-DR+CD8+ T cells/CD8+ T, % 42.3 (30.9–48.2) 46.2 (42.3–48.2) 28.6 (25.4–37.9) 0.19 20.73–60.23
CD45RA+CD4+ T cells/CD4+ T, % 32.8 (31.7–40.3) 32.8 (31.8–36.4) 36.0 (29.3–40.5) 0.54 29.41–55.41
CD45RO+CD4+ T cells/CD4+ T, % 67.2 (59.7–68.3) 67.2 (63.6–68.2) 64.0 (59.5–70.7) 0.54 44.44–68.94
Treg, % 4.1 (3.5–4.9) 4.7 (2.6–5.4) 3.9 (3.6–4.3) 0.92 5.36–6.30
CD45RA+ Treg, % 0.8 (0.5–1.1) 0.5 (0.3–0.7) 1.1 (1.0–1.3) 0.020 2.07–4.55
CD45RO+ Treg, % 3.3 (2.4–3.8) 3.8 (1.9–4.9) 2.9 (2.5–3.4) 0.59 1.44–2.76
IFN-γ–expressing CD4+ T cells, % 19.1 (13.0–22.8) 14.1 (9.4–18.8) 22.8 (18.8–25.4) 0.063 14.54–36.96
IFN-γ–expressing CD8+ T cells, % 50.1 (44.2–53.6) 47.2 (39.2–52.7) 51.2 (47.3–54.1) 0.49 34.93–87.95
IFN-γ–expressing NK cells, % 73.3 (65.7–79.7) 71.2 (63.8–72.9) 79.7 (71.9–81.5) 0.25 61.2–92.65
Data are the median (IQR) or n/N (%), where N is the total number of patients with available data. P values comparing severe cases and moderate cases
are from χ2, Fisher’s exact test, or unpaired 2-sided Student’s t test. COVID-19, coronavirus disease 2019; IQR, interquartile range; SARS-CoV-2, severe
acute respiratory syndrome coronavirus 2.
T cells or NK cells tended to be lower in severe cases than mod- eases (12). Whether the altered Treg proportions observed in the
erate cases. These data suggest that SARS-CoV-2 infection induc- current study account for the severity of COVID-19, or correlate to
es lymphopenia, particularly in CD4+ and CD8+ T cells, as well as the viremia, warrants further investigation.
suppressed IFN-γ production by CD4+ T cells, which correlates CD4+ T cells play a pivotal role in regulating immune respons-
with disease severity of COVID-19. es, orchestrating the deletion and amplification of immune cells,
Although the total Treg proportion was comparable between especially CD8+ T cells. CD4+ T cells facilitate virus-specific anti-
severe and moderate cases, severe cases showed a significantly body production via the T-dependent activation of B cells (13).
lower proportion of CD45RA+ naive Tregs (nTregs) and a slight- However, CD8+ T cells exert their effects mainly through 2 mech-
ly higher proportion of their memory counterparts, CD45RO+ anisms, cytolytic activities against target cells or secretion of cyto-
memory Tregs (mTregs). nTregs might be activated in the periph- kines, including IFN-γ, TNF-α, and IL-2, as well as many chemo-
ery by antigen and subsequently converted to mTregs, and thus kines (14). The production of IFN-γ is essential for the resistance
are thought to represent precursor cells of antigen-experienced against infection of various pathogens such as viruses, bacteria,
mTregs and possess an equivalently strong suppressive capac- and parasites (15). As a major source of IFN-γ, the ability of T cells
ity as compared with mTregs (12). It is reported that peripheral to respond to infection is part of the adaptive immune response
homeostatic mechanisms are crucial in the control of Treg diver- that takes days to develop a prominent IFN-γ response.
sity and concomitantly in the maintenance of immune tolerance In this study, although decreased numbers of CD8+ T cells
in healthy individuals. Disturbances within these mechanisms were detected in severe cases, the proportion of CD8+HLA-DR+ T
may have detrimental consequences and could contribute to the cells was slightly greater than that in moderate cases, which is in
development of certain diseases, particularly autoimmune dis- agreement with a recent case report (16). Circulating CD8+ T cells
were found to harbor high concentrations of cytotoxic granule known about the mechanism underlying the lymphopenia caused
components, including perforin and granulysin (16). Furthermore, by SARS-CoV-2 infection. In this study, we could not exclude the
a cytokine storm was exhibited in nearly all these populations; the possibility that some of the lymphopenia may be worse due to the
only currently available histological examination of a severe case use of steroids during hospitalization. Further research is required
who died of SARS-CoV-2 demonstrated lung interstitial mononu- to determine the effects of corticosteroids on lymphocytes in the
clear inflammatory infiltrates, dominated by lymphocytes, and context of COVID-19.
multinucleated syncytial cells with atypical enlarged pneumocytes Our study has some limitations. First, we mainly evaluated
in the intra-alveolar spaces (16). These findings suggested that the number of T cell subsets and NK cells as well as their IFN-γ
overactivation of cytotoxic CD8+ T cells, along with overproduc- production; the function of these cells and the role of activated
tion of proinflammatory cytokines, might account for, at least in macrophages and lymphocytes infiltrating the pulmonary intersti-
part, the immunogenicity of COVID-19. Nevertheless, the cellular tium remain to be elucidated. Second, this study only included a
source (T cells, dendritic cells, or macrophages) of these cytokines small number of patients; thus, the results should be interpreted
remains to be determined. with caution, and statistical nonsignificance may not rule out dif-
The roles of T cell responses in the context of SARS-CoV and ferences between severe and moderate cases. Third, because data
MERS-CoV infection have been previously studied. Patients who regarding the viremic profile of SARS-CoV-2 are not available, fur-
survived SARS-CoV and MERS-CoV infections usually had better ther studies are needed to investigate the correlation between the
immune responses than those who did not (17). The immune sys- viral load kinetics and the dynamics of cellular immune responses.
tem plays an important role in both diseases, but it is differentially Clarification of these questions will allow further dissection of the
affected by the 2 viruses (18). A study in a mouse SARS-CoV model complex SARS-CoV-2 pathogenesis, with potential implications
has shown that depletion of CD8+ T cells at the time of infection for the development of therapeutics and vaccines.
does not affect viral replication or clearance. However, depletion In conclusion, the SARS-CoV-2 infection induced cytokine
of CD4+ T cells leads to an enhanced immune-mediated intersti- storm and lymphopenia, particularly a decrease in CD4+ and
tial pneumonitis and delayed clearance of SARS-CoV from the CD8+ T cell counts, as well as suppressed IFN-γ production by
lungs, demonstrating the vital role of CD4+ T cells but not CD8+ CD4+ T cells, which might be correlated with disease severity of
T cells in primary SARS-CoV infection (19). A Chinese study in COVID-19. Gaining a deeper understanding of the factors that
SARS-CoV–infected patients has demonstrated that the majority affect lymphocytes, particularly T lymphocyte counts and their
of infiltrative inflammatory cells in the pulmonary interstitium association with the disease severity in patients with SARS-CoV-2
are CD8+ T cells that play an important role in virus clearance as infection, is of importance for clinical management of COVID-19.
well as in immune-mediated injury (20). After comparing T cell–
deficient mice and B cell–deficient mice, it was found that T cells Methods
are able to survive and kill virus-infected cells in the MERS-CoV–
infected lung (21). These data highlight the importance of T lym- Study participants
phocytes, CD4+ T cells in particular, but not B cells in controlling From late December 2019 to January 27, 2020, a total of 21 cases who
and fine-tuning the pathogenesis and outcomes of SARS-CoV initially presented with fever or respiratory symptoms, with pulmo-
and MERS-CoV infection. However, a cohort study investigating nary infiltrates on chest CT scans in the isolation ward of the Depart-
adaptive immune responses to SARS-CoV infection revealed that ment of Infectious Disease, Tongji Hospital, were later confirmed to
despite no significant correlation between the total T cell respons- be infected with SARS-CoV-2 by the local health authority. Four cases
es and disease progression, the disease severity correlates strongly had a history of exposure to the Huanan Seafood Market.
with high-level CD4+ T cell responses but not the CD8+ memory We retrospectively evaluated and analyzed the medical history,
T cell response (22). It is noteworthy that the immune responses physical examination, and hematological, biochemical, radiological,
evaluated in this study were in patients who recovered fully; thus, microbiological, and immunological evaluation results obtained from
whether these responses contribute to recovery or disease pro- these 21 patients with COVID-19. Epidemiological, clinical, laborato-
gression remains unclear (22). ry, and radiological characteristics and treatment as well as outcome
Chu et al. demonstrated that MERS-CoV, but not SARS-CoV, data were obtained from electronic medical records. The data collec-
can efficiently infect T cells from the peripheral blood and from tion forms were reviewed independently by 2 researchers.
human lymphoid organs and induce apoptosis in T cells, which
involves the activation of both the extrinsic and intrinsic apoptosis Clinical classifications and complication definitions
pathways (23). This may partly explain the lymphopenia observed According to the guidelines for diagnosis and management of COVID-19
in MERS-CoV–infected patients (23). SARS-CoV can also signifi- (6th edition, in Chinese) released by National Health Commission of
cantly decrease peripheral CD4+ and CD8+ T lymphocyte subsets China (9), the clinical classifications of COVID-19 are as follows:
and this was related to the onset of illness (24). Several poten- Mild cases: The clinical symptoms are mild and no pneumonia
tial mechanisms may be involved, including the development of manifestation can be found in imaging.
autoimmune antibodies or immune complexes triggered by viral Moderate cases: Patients have symptoms like fever and respira-
infection and directly infecting and promoting the growth inhi- tory tract symptoms, etc., and pneumonia manifestation can be seen
bition and apoptosis of hematopoietic stem and progenitor cells. in imaging.
The use of glucocorticoids may also account for the decrease Severe cases: Meeting any of the following — respiratory distress,
in lymphocytes in some SARS patients (25). At present, little is respiratory rate ≥ 30 breaths/min; SpO2 ≤ 93% at rest; and PaO2/FIO2 ≤
300. Patients with greater than 50% lesion progression within 24 to 48 Evaluation of peripheral blood immunological indicators
hours in pulmonary imaging should be treated as severe cases. The proportions and numbers of NK, CD4+ T, CD8+ T, Treg, and
Critically ill cases: Meeting any of the following — respiratory failure B cells, and the expression of cell surface markers as well as IFN-γ
occurs and mechanical ventilation is required, shock, and complications expression by CD4+ T, CD8+ T, and NK cells were studied in these
from other organ failure that require monitoring and treatment in the ICU. patients with laboratory-confirmed SARS-CoV-2 infection. Flow
Acute respiratory distress syndrome and shock were defined cytometry antibodies against human surface and intracellular
according to the interim guidance of WHO for SARS-CoV-2 (26). molecules are commercially available. The following antibodies
Hypoxemia was defined as a PaO2/FiO2 ratio of less than 300. were used: anti-CD28 (CD28.2, PE, 555729), anti-CD8 (RPA-T8,
Acute kidney injury was identified and classified based on the PE-Cy7, 557746), anti-CD45 (2D1, PerCP, 347464), anti–HLA-
highest serum creatinine level or urine output criteria according to the DR (G46-6, APC, 560744), anti-CD3 (SK7, APC-Cy7, 557832),
Kidney Disease Improving Global Outcomes (KDIGO) classification. anti-CD4 (RPA-T4, V450, 560345), anti-CD45RA (HI100, FITC,
Acute liver injury was defined as jaundice with a total bilirubin 555488), anti-CD45RO (UCHL1, PE, 5618898), anti-CD127 (HIL-
level of 3 mg/dL or higher and an acute increase in ALT of at least 5 7R-M21, PE-Cy7, 560822), anti-CD45 (2D1, PerCP, 347464),
times the upper limit of the normal range and/or an increase in alka- anti-CD25 (M-A251, APC, 561399), anti-CD3 (SK7, APC-Cy7,
line phosphatase of at least twice the upper limit of the normal range. 557832), anti-CD4 (RPA-T4, V450, 560345), anti-CD3 (UCHT1,
Cardiac injury was diagnosed if serum levels of cardiac biomark- FITC, 561806), anti-CD8 (RPA-T8, PE, 555367), anti-CD56 (B159,
ers (e.g., troponin I) were greater than the 99th percentile upper refer- PE-Cy7, 557747), anti–IFN-γ (4S.B3, APC, 551385), and anti-CD4
ence limit, or new abnormalities were shown in electrocardiography (RPA-T4, APC-Cy7, 557871). All reagents were purchased from
and echocardiography. Becton, Dickinson, and Company (BD). All samples were detected
Secondary infection including bacterial and fungal was diagnosed by a BD FACSCanto II flow cytometry system and analyzed with the
if the patients had clinical symptoms or signs of nosocomial pneumo- BD FACSDiva software.
nia or bacteremia, and was combined with a positive culture of a new The steps of intracellular staining for IFN-γ in immune cells were
pathogen from a respiratory tract specimen or from blood samples tak- as follows: GolgiStop (BD Biosciences, 554724) was added to cell cul-
en 48 hours or more after admission. tures for 4 hours and then the cells were resuspended in FACS buffer
for flow cytometry antibody staining. Peripheral blood mononucle-
Laboratory measurements ar cells were stained for surface antibody at 4°C for 30 minutes and
Real-time reverse transcription PCR assay for SARS-CoV-2. Respiratory washed with FACS buffer followed by fixation/permeabilization (BD
specimens were collected by the local CDC and then shipped to des- Cytofix/Cytoperm, 554722) at 4°C for 20 minutes in the dark. Fixed/
ignated authoritative laboratories to detect SARS-CoV-2. The pres- permeabilized cells were washed twice with Perm/Wash buffer (BD
ence of SARS-CoV-2 in respiratory specimens was detected by real- Biosciences, 554723) and then thoroughly resuspended in 50 μL of
time reverse transcription (RT-PCR) methods. Primers and probe Perm/Wash buffer containing a predetermined optimal concentration
targeting the CoV envelope gene were used and the sequences were of a fluorochrome-conjugated anti–IFN-γ antibody or appropriate neg-
as follows: forward primer, 5′-TCAGAATGCCAATCTCCCCAAC-3′; ative control and incubated at 4°C for 30 minutes in the dark. Cells
reverse primer 5′-AAAGGTCCACCCGATACATTGA-3′; and the probe were washed twice with Perm/Wash buffer and resuspended in FACS
5′CY5-CTAGTTACACTAGCCATCCTTACTGC-3′BHQ1. Conditions buffer prior to flow cytometric analysis.
for the amplifications were 50°C for 15 minutes, 95°C for 3 minutes,
followed by 45 cycles of 95°C for 15 seconds and 60°C for 30 seconds. Statistics
Clinical laboratory measurements. Initial clinical laboratory inves- Continuous variables were expressed as median (IQR) and compared
tigation included a complete blood count, serum biochemical test with the unpaired 2-sided Student’s t test; categorical variables were
(including liver and renal function, creatine kinase, LDH, and elec- expressed as number (%) and compared by χ2 test or Fisher’s exact test
trolytes), coagulation profile, as well as immunological test (including between moderate and severe case groups. A 2-sided α of less than
serum cytokines, peripheral immune cell subsets, and the expression 0.05 was considered statistically significant. Statistical analyses were
of IFN-γ by immune cells). Respiratory specimens, including nasal and done using SPSS (version 19.0, IBM).
pharyngeal swabs or sputum, were tested to exclude evidence of other
viral infections, including influenza, respiratory syncytial virus, avian Study approval
influenza, parainfluenza, and adenovirus. Routine bacterial and fun- The study was performed in accordance with Good Clinical Practice
gal examinations were also performed. and the Declaration of Helsinki principles for ethical research. The
Cytokine measurement. To explore the influence of SARS-CoV-2 study protocol was approved by the Institutional Review Board of
infection on the secretion of cytokines, IL-1β, IL-2R, IL-6, IL-8 (also Tongji Hospital, Tongji Medical College, Huazhong University of Sci-
known as CXCL8), IL-10, and TNF-α were assessed in serum samples ence and Technology (Wuhan, China). Written informed consent was
drawn shortly after hospital admission by chemiluminescence immu- waived due to the rapid emergence of this infectious disease.
noassay (CLIA) performed on a fully automated analyzer (Siemens
Immulite 1000, DiaSorin Liaison, or Roche Diagnostics Cobas e602) Author contributions
for all patients according to the manufacturers’ instructions. CLIA kits QN and DW designed the study and had full access to all data in
for IL-1β (LKL11), IL-2R (LKIP1), IL-8 (LK8P1), IL-10 (LKXP1), and the study and take responsibility for the integrity of data and the
TNF-α (LKNF1) were purchased from DiaSorin. An IL-6 kit (05109442 accuracy of the data analysis. GC and DW contributed to patient
190) was purchased from Roche Diagnostics. recruitment, data collection, data analysis, data interpretation,
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