Unbiased single cell spatial analysis localises inflammatory clusters of immature neutrophils-CD8 T cells to alveolar progenitor cells in fatal COVID-19 lungs

Single cell spatial interrogation of the immune-structural interactions in COVID -19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we developed a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method revealed a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and cytotoxic CD8 T cells, was found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings provide new insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the MRC Human Immunology Unit (grant (MC\_UU\_00008/1), Oxford University Medical Sciences Division COVID Funds, Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M- 2-002). LD and CV are supported by the NIHR Oxford Biomedical Research Centre. LPH, TD, GO and JK are COI Principal Investigators (Chinese Academy of Medical Science (CAMS) Oxford Institute (COI), University of Oxford, Oxford. LPH is supported in part by the NIHR Oxford Biomedical Research Centre. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of the University of Navarra, Spain gave ethical approval for the study on COVID-19 lungs (Approval 2020.192) Oxford A South-Central NHS REC (ref 19/SC/0173)gave ethical approval for the study in healthy lung. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript, all data are online via links provided in the manuscript [1]: pending:yes