Two distinct immunopathological profiles in lungs of lethal COVID-19

Immune responses in lungs of Coronavirus Disease 2019 (COVID-19) are poorly characterized. We conducted transcriptomic, histologic and cellular profiling of post mortem COVID-19 and normal lung tissues. Two distinct immunopathological reaction patterns were identified. One pattern showed high expression of interferon stimulated genes (ISGs) and cytokines, high viral loads and limited pulmonary damage, the other pattern showed severely damaged lungs, low ISGs, low viral loads and abundant immune infiltrates. Distinct patterns of pulmonary COVID-19 immune responses correlated to hospitalization time and may guide treatment and vaccination approaches.

ISG low samples, and observed a higher frequency of CD8+PD1+ T-cells in the ISG low subgroup, potentially indicative of advanced disease (Extended Data Figure 3a,b). Histological analysis of COVID-19 lung tissues revealed striking pulmonary damage exclusively in ISG low samples, with distinct peri-alveolar foci of infiltrating CD68+ macrophages and CD8+ T cells (Figure 1e).
Expression of ISGs was tightly correlated with pulmonary viral load (Figure 2a), and immunohistochemical staining showed SARS-CoV-2 nucleocapsid protein mostly in pneumocytes of ISG high lungs (Figure 2b). Since a cytokine storm has been proposed to cause adverse outcome of COVID-19 7 , and cytokines were highly expressed in bronchoalveolar lavages (BALs) of COVID-19 patients 8 , we investigated expression of a pro-inflammatory cytokine signature (TNF, IL6, IL1b, CCL2, IFNA17, IFNB1, CXCL9, CXCL10, CXCL11) in lung samples from lethal COVID-19. The proinflammatory gene signature was significantly enriched in the ISG high subset (Figure 2c), but was not associated with alveolar hemorrhage (Figure 2d). Within this cytokine signature, co-regulated subgroups (IL1B/IL6/TNF, IFNB1/IFNA17, CCL2/CXCL9/CXCL19/CXCL11) were identified ( Figure   2e). Importantly, only the CXCL9/10/11 sub-signature was positively associated with alveolar hemorrhage (Figure 2f, Extended Data Figure 4). This is in line with observations that these chemokines compromise endothelial integrity via CXCR3 9 , and that CXCL10 is a key determinant of severe COVID-19 10 . Interestingly, basal levels of CXCL9 or CXCL10 are elevated in elderly, hypertensive and obese individuals, which were strongly represented in our autopsy cohort and are predisposed to severe COVID-19 11,12 . Of note, our study could not take extrapulmonary cytokine sources or effects into account.
Since none of the above pulmonary cytokine sub-signatures was positively associated with diffuse alveolar damage (DAD, Extended Data Figure 5), we investigated which other local immune signature showed this association. We found a strong association of DAD with low expression of ISGs (Figure 2g), and an activated CD8+ T cell signature (CD38, GZMA, GZMB, CCR5, Figure 2h), yet not with pulmonary CD8+ T-cell infiltration (Figure 2i). In addition, the activated CD8+ signature was inversely correlated to viral counts, particularly in ISG low cases (Figure 2j). Therefore we speculate that activated CD8+ T cells are essential for virus elimination, similar to murine models of coronavirus infection 13 , yet it is possible that they contribute to pulmonary damage as well. Of note, ISG low samples also expressed elevated p53 and Ki67 (Figure 2k), i.e. reactive markers of DAD which indicate lung remodeling 14 . Since the ISG low pattern showed lower viral counts, higher accumulation and activation of CD8+ T cells in tissues and accrual of pulmonary damage and remodeling, the ISG low phase may follow an earlier ISG high phase during the course of infection. This was supported by significantly 4 instead show low viral loads yet strong complement activation in lungs (Figure 2m) and thus may potentially benefit from complement inhibition 16 . In addition, the ISG low pattern suggests that CD8+ T cells are involved in antiviral protection and should be considered for vaccination efforts.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  Table 1a. Detailed autopsy findings for each patient were recently published, and the identifiers (with the prefix "C") for each COVID-19 patient are consistent with the description of this Swiss COVID-19 autopsy cohort 3 . In this study, we analysed formalin fixed and paraffin embedded (FFPE) lung tissue of distinct areas of the lungs of 16 of these COVID-19 patients. All 16 COVID-19 patients had positive nasopharyngeal swabs collected while alive. In all COVID-19 patients, diagnosis was confirmed by detection of SARS-CoV-2 in postmortal lung tissues. 5/16 patients were additionally tested by postmortal nasopharyngeal swabs which were positive for SARS-CoV-2 in all 5 cases.
As a control cohort, we selected 6 autopsies performed between January 2019 and March 2020 at the Institute of Pathology Liestal ("normal" patients N1 -N6). These control patients died of other, non-infectious causes and had a similar age, gender and cardiovascular risk profile. Patients with infections were excluded from this control cohort. Another control cohort consisted of 4 autopsies of patients suffering from various infections mainly with bacteria affecting the lung (patients with lung pathology, P1 -P4). Details for both control cohorts are listed in Extended Data (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Quantification of SARS-CoV-2 in FFPE tissue samples The OIRRA is a targeted gene expression assay designed for the Ion™ next-generation sequencing (NGS) platform. This gene expression assay was originally designed to interrogate the tumor microenvironment to enable mechanistic studies and identification of predictive biomarkers for immunotherapy in cancer. The assay is optimized to measure the expression of genes involved in immune cell interactions and signaling, including genes expressed at low levels and involved in inflammatory signaling. The 398 genes covered by this assay are listed in Extended Data Table 2.
The NGS libraries were prepared as recommended by the supplier. In brief, 30ng of total RNA were (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Antibodies, staining protocols and conditions are detailed in Extended Data Table 4.
Qualitative and semiquantitative assessment of histopathological lung damage and neutrophilic infiltration Hematoxylin and eosin (H&E) and Elastica van Gieson (EvG) stained sections of all lung tissues used in this study were independently evaluated by two experienced and board certified pathologists (VZ and KDM) (Extended Data Table 5). Both pathologists evaluated the presence of diffuse alveolar damage (DAD), and if present, its stage, intra-alveolar edema and hemorrhage. In addition, both All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20133637 doi: medRxiv preprint pathologists evaluated the severity of histopathological changes in COVID-19 lungs (1 = mild / discrete alterations, 2 = moderate, 3 = severe changes) based on resemblance between normal and pathologically altered lung tissues. Parameters that were taken into account included reduction of alveolar air-filled spaces, typical histologic features of DAD with hyaline membrane formation, infiltration of lymphocytes, monocytes and neutrophils into interstitial and alveolar spaces, type 2 pneumocyte hyperplasia, desquamation of pneumocytes, histologic features of organizing pneumonia including intra-alveolar fibrin deposition and fibrosis (acute fibrinous and organizing pneumonia, AFOP) 17, 18  For cell-level analysis, color deconvolution for DAB, AP and hematoxylin channels was performed and nuclear segmentation was optimized using cell-morphometric parameters. Marker-positive cells in stromal and epithelial regions were quantified. For CD3, CD4, CD8, CD20, CD68, CD123, CD163 and PD1, staining detection was optimized for the cytoplasmic / membranous compartment and marker expression was measured on a continuous scale at single cell resolution. For assessment of CD8/PD1 double stains, color deconvolution was optimized for separation of DAB (PD1) and AP (CD8) staining products. Internal controls (non-immune cells) and external controls (tonsil) were used to calibrate the detection limits and cross-validated by visual review. For each tissue sample, the total area of lung tissue in mm 2 , the absolute number of marker-positive cells, cell morphometric parameters and staining intensity were recorded.
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The copyright holder for this preprint this version posted June 19, 2020. Expression of gene signatures was calculated as median of log2(cpm + 1) of selected genes.

Functional enrichment analysis
Biological processes enrichment was performed using the enrichGO function of the package clusterProfiler 20 setting all the genes included in the assay as universe.

Statistical analysis
All the analyses and graphical representations were performed using the R statistical environment software 21 and the following packages: ggplot2 22 , circlize 23 , ComplexHeatmap 24 , ggfortify 25 , reshape2 26 and factoextra 27 . Correlation between transcripts and viral counts was performed using Pearson's correlation. Association between continuous and categorical data were tested using Wilcoxon rank sum test.
Box-plots elements indicate the median (center line), upper and lower quartiles (box limits) and show all the data points. Whiskers extend to the most extreme value included in 1.5x interquartile range.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020.     (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Data availability
The datasets generated and analysed during this study can be accessed in GEO (GSE151764) and are available from the corresponding author upon request. Competing interests VHK has served as an invited speaker on behalf of Indica Labs. TH and TJ are employees of Novartis.
The other authors declare no competing interests.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Patients were suffering from other infections of the lung (bacterial or viral pneumonia). Detailed analysis of individual pathogens is shown in Extended Data Figure 1.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. At least two different tissue blocks from different areas of the lungs were evaluated for each case. 1 1 = slight to moderate changes; 2 = moderate changes; 3 = severe changes 2 1 = exudative; 2 = proliferating/organizing; 3 = fibrotic 3 1 = yes; 0 = no 4 1 = very few or few; 2 = moderate; 3 = numerous All rights reserved. No reuse allowed without permission.
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