medRxiv preprint doi: https://doi.org/10.1101/2021.01.27.21250591; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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. 1 Title: High-dimensional profiling reveals phenotypic heterogeneity and disease-specific 2 alterations of granulocytes in COVID-19 3 4 Authors: Magda Lourda1,2, Majda Dzidic1, Laura Hertwig1, Helena Bergsten1, Laura M. Palma 5 Medina1, Egle Kvedaraite1,2, Puran Chen1, Jagadeeswara R. Muvva1, Jean-Baptiste Gorin1, 6 Martin Cornillet1, Johanna Emgård1, Kirsten Moll1, Marina García1, Kimia T. Maleki1, Jonas 7 Klingström1, Jakob Michaëlsson1, Malin Flodström-Tullberg1, Susanna Brighenti1, Marcus 8 Buggert1, Jenny Mjösberg1, Karl-Johan Malmberg1, Johan K. Sandberg1, Jan-Inge Henter2,3, 9 Elin Folkesson4,5, Sara Gredmark-Russ1,4, Anders Sönnerborg4,6, Lars I. Eriksson7,8, Olav 10 Rooyackers8,9, Soo Aleman4,6, Kristoffer Strålin4,6, Hans-Gustaf Ljunggren1, Niklas K. 11 Björkström1, Mattias Svensson1, Andrea Ponzetta1, Anna Norrby-Teglund1, Benedict J. 12 Chambers1; and the Karolinska KI/K COVID-19 Study Group 13 14 Authorship note: 15 ML, MD and LH share first authorship. 16 AP, ANT and BJC share last authorship. 17 Correspondence: Magda Lourda ([email protected]) 18 19 Affiliations: 20 1Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, 21 Karolinska University Hospital, Stockholm, Sweden 22 2Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska 23 Institutet, Stockholm, Sweden 24 3Theme of Children’s Health, Karolinska University Hospital, Stockholm, Sweden 25 4Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.01.27.21250591; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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. 26 5Division of Infectious Diseases, Department of Medicine Solna, Karolinska Institutet, 27 Stockholm, Sweden 28 6Division of Infectious Diseases and Dermatology, Department of Medicine Huddinge, 29 Karolinska Institutet, Stockholm, Sweden 30 7Department of Physiology and Pharmacology, Section for Anesthesiology and Intensive Care, 31 Karolinska Institutet, Stockholm, Sweden 32 8Function Perioperative Medicine and Intensive Care, Karolinska University Hospital, 33 Stockholm, Sweden 34 9Department of Clinical Interventions and Technology CLINTEC, Division for Anesthesiology 35 and Intensive Care, Karolinska Institutet, Stockholm, Sweden. 36 37 Conflict of interest statement: 38 K-JM is a Scientific Advisor and has a research grant from Fate Therapeutics and is a member 39 of the Scientific Advisory Board of Vycellix Inc. H-GL is a member of the board of XNK 40 Therapeutics AB and Vycellix Inc. J-IH serves as consultant for Sobi AB. The remaining 41 authors have declared that no conflict of interest exists. 42 2 medRxiv preprint doi: https://doi.org/10.1101/2021.01.27.21250591; this version posted January 31, 2021. The copyright holder for this preprint (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. 43 Abstract 44 Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate 45 immune responses play an important role in the disease development. A dysregulated 46 inflammatory state has been proposed as key driver of clinical complications in COVID-19, 47 with a potential detrimental role of granulocytes. However, a comprehensive phenotypic 48 description of circulating granulocytes in SARS-CoV-2-infected patients is lacking. In this 49 study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in 50 peripheral blood collected from COVID-19 patients during acute and convalescent phases. 51 Severe COVID-19 was associated with increased levels of both mature and immature 52 neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were 53 evident in COVID-19 patients, with altered expression of several receptors involved in 54 activation, adhesion and migration of granulocytes (e.g. CD62L, CD11a/b, CD69, CD63, 55 CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic 56 signature in the convalescent phase. The identified granulocyte immunotypes correlated with 57 distinct sets of soluble inflammatory markers supporting pathophysiologic relevance. 58 Furthermore, clinical features, including multi-organ dysfunction and respiratory function, 59 could be predicted using combined laboratory measurements and immunophenotyping. This 60 study provides a comprehensive granulocyte characterization in COVID-19 and reveals 61 specific immunotypes with potential predictive value for key clinical features associated with 62 COVID-19. 3 medRxiv preprint doi: https://doi.org/10.1101/2021.01.27.21250591; this version posted January 31, 2021. The copyright holder for this preprint (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. 63 Significance 64 Accumulating evidence shows that granulocytes are key modulators of the immune response 65 to SARS-CoV-2 infection and their dysregulation could significantly impact COVID-19 66 severity and patient recovery after virus clearance. In the present study, we identify selected 67 immune traits in neutrophil, eosinophil and basophil subsets associated to severity of COVID- 68 19 and to peripheral protein profiles. Moreover, computational modeling indicates that the 69 combined use of phenotypic data and laboratory measurements can effectively predict key 70 clinical outcomes in COVID-19 patients. Finally, patient-matched longitudinal analysis shows 71 phenotypic normalization of granulocyte subsets 4 months after hospitalization. Overall, in this 72 work we extend the current understanding of the distinct contribution of granulocyte subsets 73 to COVID-19 pathogenesis. 74 4 medRxiv preprint doi: https://doi.org/10.1101/2021.01.27.21250591; this version posted January 31, 2021. The copyright holder for this preprint (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. 75 Introduction 76 Caused by the novel coronavirus SARS-CoV-2, the current COVID-19 pandemic poses an 77 unprecedented threat to the global health systems, with over 2 million confirmed deaths 78 reported worldwide in January 2021 (https://www.who.int/emergencies/diseases/novel- 79 coronavirus-2019). About 80% of COVID-19 patients present with a mild or moderate disease 80 course, while 15-20% develop severe complications (1, 2), including respiratory failure, 81 coagulation abnormalities and life-threatening acute respiratory distress syndrome (ARDS). 82 A dysregulated inflammatory state has been proposed as a key driver of clinical complications 83 of COVID-19 (3) and has been associated with increased mortality (4). High levels of several 84 pro-inflammatory mediators (e.g. Interleukin-1β (IL-1β), IL-6, Tumor necrosis factor ⍺ 85 (TNF⍺) and CXCL8) are detected early after viral infection (5), and the resolution of this 86 inflammatory response seems impaired in patients with severe disease progression (5, 6). Many 87 of these pro-inflammatory cytokines are associated with granulocyte activation and recruitment 88 (7), and it has been reported that an increased neutrophil-to-lymphocyte ratio (NLR) in 89 peripheral blood can be used as a prognostic marker of higher disease severity in COVID-19 90 patients, similar to SARS-CoV-1 and MERS (8-10). 91 The cellular components of the polymorphonuclear granulocyte family – neutrophils, 92 eosinophils and basophils – arise from a common myeloid progenitor in humans (11) and 93 represent the very first line of defense against invading microbes. The role of granulocytes in 94 viral infection has mostly been confined to neutrophils (12, 13), though eosinophils and 95 basophils have been implicated in the host response to viruses as well (14, 15). In COVID-19, 96 an increased neutrophil abundance in circulation is mirrored by a substantial enrichment of 97 granulocytes in the inflamed lung (16, 17). Several reports highlighted the presence of 98 immature neutrophils in blood of COVID-19 patients (18, 19), implying a marked emergency 99 granulopoiesis occurring in the bone marrow. The presence of immature neutrophil populations 5 medRxiv preprint doi: https://doi.org/10.1101/2021.01.27.21250591; this version posted January 31, 2021. The copyright holder for this preprint (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. 100 is phenotypically reminiscent of granulocytic myeloid-derived suppressor cells (G-MDSCs), 101 and