Single-Cell Analysis Reveals Macrophage-Driven T Cell Dysfunction in Severe COVID-19 Patients

The vastly spreading COVID-19 pneumonia is caused by SARS-CoV-2. Lymphopenia and cytokine levels are tightly associated with disease severity. However, virus-induced immune dysregulation at cellular and molecular levels remains largely undefined. Here, the leukocytes in the pleural effusion, sputum, and peripheral blood biopsies from severe and mild patients were analyzed at single-cell resolution. Drastic T cell hyperactivation accompanying elevated T cell exhaustion was observed, predominantly in pleural effusion. The mechanistic investigation identified a group of CD14+ monocytes and macrophages highly expressing CD163 and MRC1 in the biopsies from severe patients, suggesting M2 macrophage polarization. These M2-like cells exhibited up-regulated IL10, CCL18, APOE, CSF1 (M-CSF), and CCL2 signaling pathways. Further, SARS-CoV-2-specific T cells were observed in pleural effusion earlier than in peripheral blood. Together, our results suggest that severe SARS-CoV-2 infection causes immune dysregulation by inducing M2 polarization and subsequent T cell exhaustion. This study improves our understanding of COVID-19 pathogenesis.


INTRODUCTION
respiratory dendritic cell migration from the lung to the draining lymph nodes (Zhao et

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. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) by frequency ( Figures 1C,1D, S1D and S1E). The percentage of FOXP3 + CD25 + Treg 154 and CD25 expression were higher in PFMC, and a group of IL10 + T cells was 155 exclusive in the patient's PFMC ( Figures 1C, 1D and S1F and S1G). Conventional 156 cell lineage markers and flow cytometry analysis further validated the clustering and 157 annotation results ( Figures 1D, 1E and S1B). These data reflected the changes in 158 immune cell composition in pulmonary and peripheral in response to viral infection.

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Of note, the expression of SARS-CoV-2 entry receptor angiotensin-converting 160 enzyme II (ACE2) was also examined in scRNA-seq (Zhou et al., 2020). Minimal 161 ACE2 expression was found in all immune cell types ( Figure S1H). CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.23.20100024 doi: medRxiv preprint would be preferentially recruited to the infected site. We also observed mild and

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To elucidate the mechanism causing changes in T cell function and exhaustion post-203 SARS-CoV-2 infection in the lung, macrophages in PFMC were scrutinized. CCL2 204 was highly induced in the PFMC macrophages ( Figure 2B) and reported to shape 205 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 26, 2020.

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Interestingly, the majority of CD163 + cells express PD-1 while CD206 + cells express 227 TIM-3, and Poly I:C induced IL-10 expression was primarily found in CD206 + TIM-3 high 228 cells ( Figure S3D). Moreover, CCL-2 and IL-10 concentrations were higher in pleural 229 effusion than those in patient plasma, agreeing with the M2 macrophage-driven 230 microenvironment ( Figure 3E). Collectively, these results supported the notion that 231 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 26, 2020.

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Our study provides a framework to systematically investigate immune profile   the lung, leading to exudate pleural effusion Light, 2006, 2008). Thus,

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PFMC was reasoned to share closer immune properties in the SARS-CoV-2-infected 303 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted May 26, 2020. LGALS 3 LG AL S3  Selected genes are indicated on the right.
(E) Gene ontology (GO) analysis for the differential expressed genes from panel D.
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PBMC PFMC
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(A) Flow plots showing CD38 and HLA-DR expression by IFN-g + TNF + and IFN-g + TNFvirus-specific CD4 + and CD8 + T cells from the patient's pleural fluid and peripheral blood.
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The following anti-human monoclonal antibodies were used: . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We thank the patient who took part in this study.

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