High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in SARS-CoV-2-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion and migration of granulocytes (e.g. CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers supporting pathophysiologic relevance. Furthermore, clinical features, including multi-organ dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19. Significance Accumulating evidence shows that granulocytes are key modulators of the immune response to SARS-CoV-2 infection and their dysregulation could significantly impact COVID-19 severity and patient recovery after virus clearance. In the present study, we identify selected immune traits in neutrophil, eosinophil and basophil subsets associated to severity of COVID-19 and to peripheral protein profiles. Moreover, computational modeling indicates that the combined use of phenotypic data and laboratory measurements can effectively predict key clinical outcomes in COVID-19 patients. Finally, patient-matched longitudinal analysis shows phenotypic normalization of granulocyte subsets 4 months after hospitalization. Overall, in this work we extend the current understanding of the distinct contribution of granulocyte subsets to COVID-19 pathogenesis. ### Competing Interest Statement K-JM is a Scientific Advisor and has a research grant from Fate Therapeutics and is a member of the Scientific Advisory Board of Vycellix Inc. H-GL is a member of the board of XNK Therapeutics AB and Vycellix Inc. J-IH serves as consultant for Sobi AB. The remaining authors have declared that no conflict of interest exists. ### Funding Statement The study was supported by grants from Nordstjernan AB and Knut and Alice Wallenberg Foundation to H-GL, the Swedish Governmental Agency for Innovation Systems (VINNOVA) under the frame of NordForsk (Project no. 90456, PerAID) and the Swedish Research Council under the frame of ERA PerMed (Project 2018-151, PerMIT) to ANT, the Swedish Research Council to BJC and AP, the Swedish Cancer Foundation to BJC, Clas Groschinsky Foundation to AP, Centrum for Innovative Medicine to AP and ANT, the Swedish Children's Cancer Foundation (TJ2018-0128 and PR2019-0100) to ML, Ake Olsson Foundation to ML and AP and KI Research Foundation to ML, LH and AP. ### 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 in Stockholm, Sweden All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. 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 Curated flow cytometry data will be made available for exploration via the KI/K COVID-19 Immune Atlas, http://covid19cellatlas.com/. All other data needed to evaluate the conclusions of the paper are presented in the paper or in the Supplemental Material.