Background: A previous report of lung biopsies from a patient with coronavirus disease
2019 (COVID-19) and acute respiratory distress syndrome (ARDS) identified mononuclear
cell infiltration but not the type of mononuclear cells (1).
Objective: To describe the type of immune cells identified by imaging mass cytometry
in lung tissue from 2 patients with COVID-19 and fatal ARDS.
Case Reports: The first patient was a 94-year-old woman who had close contact with
a confirmed case of COVID-19 eleven days earlier. She presented with an 8-day history
of lethargy and fever (maximum temperature, 39 °C), and her history included 10 years
of coronary heart disease. Physical examination found wheezing in both lungs and peripheral
edema, and chest radiography identified right pulmonary infiltration and consolidation
with right pleural effusion. She had a C-reactive protein level of 115.1 mg/L (normal
level, <3 mg/L) and leukocyte count of 10.2 × 109 cells/L with 9% lymphocytes. Tests
processed by the Beijing Centers for Disease Control confirmed that she had COVID-19.
At the direction of her relatives, we provided noninvasive therapy that included supplemental
oxygen and symptomatic treatment. She gradually developed dyspnea and hypoxemia and
had a fatal cardiac arrest on day 10 of the illness.
The second patient was a 65-year-old man whose wife had COVID-19. He presented with
a 4-day history of dry cough, anorexia, and fever (maximum temperature, 38.6 °C).
On admission, computed tomography of the chest showed bilateral pneumonia. He had
a C-reactive protein level of 13.5 mg/L and a leukocyte count of 3.0 × 109 cells/L
with 27.4% lymphocytes. Tests processed by the Beijing Centers for Disease Control
confirmed that he had COVID-19. We initiated supportive therapy and administered moxifloxacin
to prevent secondary infection. On day 15 of the illness, he required invasive ventilatory
support. We changed his antibiotics to vancomycin and imipenem for suspected sepsis
and administered intravenous methylprednisolone and immune globulin to attenuate systemic
inflammation. On day 16, he had a C-reactive protein level of 244.4 mg/L, leukocyte
count of 10.1 × 109 cells/L with 1.6% lymphocytes, and blood lactic acid level of
3.13
mmol/L. He developed septic shock and died on day 21 of the illness.
Lung tissue was obtained by autopsy in both patients and by percutaneous biopsy in
a control patient, a 54-year-old man with a pulmonary nodule. When we examined tissues
stained with hematoxylin–eosin, the first patient had the typical histologic features
of ARDS, with diffuse alveolar damage, alveolar septum edema, epithelial cell proliferation,
hyaline membrane formation, infiltration of lymphocytes and monocytes into interstitial
and alveolar spaces, and a small number of neutrophils. The second patient had the
typical histologic features of bacterial pneumonia, with alveolar damage; alveolar
septum edema; epithelial cell proliferation; and desquamation of pneumocytes, cellular
fibromyxoid exudates, many phagocytes, neutrophil debris, and pus cells in alveolar
cavities. The control patient had infiltration of interstitial inflammatory cells.
Imaging mass cytometry on lung tissue from the first patient (Figure) found diffuse
infiltration of CD4 T lymphocytes, macrophages (CD68), and a focal infiltration of
natural killer cells (CD16 and CD107A). Tissue from the second patient had a cluster
infiltration of neutrophils (CD11b and CD16) and activated macrophages (Arg1), a diffuse
infiltration of mature T cells (CD45RA and CD4), and a scattered infiltration of natural
killer cells and dendritic cells (CD276 and CD14), which distributed differently from
macrophages and were accompanied by local overexpression of type 1 collagen. The distributions
of infiltration by macrophages, mature T cells, and natural killer cells in the 2
patients were different, as was the expression of type 1 collagen, and the second
patient had more dendritic cells. Both patients had relatively independent distributions
of cell subsets by t-distributed stochastic neighbor embedding and PhenoGraph
analysis. The first patient had more infiltration by immune cells. This patient had
a cluster distribution of mature T cells (CD45RA) and macrophages. The second patient
had a cluster distribution of mature T cells (CD45RA and CD45RO) and macrophages,
which correlated with bacterial infection. More CD45RA+ T cells were recruited in
the first patient, whereas mostly CD45RO+ T cells were recruited in the second patient.
Additional data and figures are available from the authors on request.
Figure.
Imaging mass cytometry with markers of interest.
Representative mass cytometry images for each panel are different colors, and expression
levels are in parentheses. Iridium-DNA staining is shown in blue. All images are from
the same tissue sections. We designed a metal isotope–labeled antibody panel to detect
multiple markers simultaneously in 1 slide by using the imaging mass cytometry system.
For detailed methods, please refer to the Imaging Mass Cytometry Staining Protocol
for FFPE Sections on the Fluidigm website (http://cn.fluidigm.com/search?query=IMC+Staining+Protocol&resourceTypes=protocol).
All raw data were acquired using a Hyperion Imaging System (Fluidigm). The resulting
files were in MCD format and were exported as TIFF files using the MCD Viewer. FFPE
= formalin-fixed paraffin-embedded.
Discussion: Two previous studies of lung tissue from patients with severe acute respiratory
syndrome found mononuclear cell infiltration and hyaline membrane formation (2, 3),
whereas 1 previous study of a patient with COVID-19 and ARDS found interstitial infiltration
by mononuclear cells (1). Our study suggests that these infiltrated mononuclear cells
are CD4 T cells, CD8 T cells, natural killer cells, and macrophages; that recruitment
of aberrant CD45RA+ T cells is the immunologic feature of COVID-19; and that once
bacterial pneumonia occurs, some phagocytes recruited by CD4 T cells begin to play
a major role in lung injury. We conclude that additional studies are needed to understand
the immune response in patients with COVID-19.