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The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allows scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.

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 with severity of COVID-19 and with 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 mo after hospitalization. Overall, in this work, we extend the current understanding of the distinct contribution of granulocyte subsets to COVID-19 pathogenesis., 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 a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (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 multiorgan 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 While broad efforts toward getting an overview of immune cell and soluble factor alterations in COVID-19 are under way, a deep and comprehensive understanding of the mononuclear phagocyte system, including circulating progenitors, is still largely lacking. This study provides a reference for the mononuclear phagocyte response to SARS-CoV-2 infection and unravels mononuclear phagocyte dysregulations associated with severe COVID-19., Dendritic cells (DCs) and monocytes are crucial mediators of innate and adaptive immune responses during viral infection, but misdirected responses by these cells may contribute to immunopathology. Here, we performed high-dimensional flow cytometry-analysis focusing on mononuclear phagocyte (MNP) lineages in SARS-CoV-2–infected patients with moderate and severe COVID-19. We provide a deep and comprehensive map of the MNP landscape in COVID-19. A redistribution of monocyte subsets toward intermediate monocytes and a general decrease in circulating DCs was observed in response to infection. Severe disease coincided with the appearance of monocytic myeloid-derived suppressor cell-like cells and a higher frequency of pre-DC2. Furthermore, phenotypic alterations in MNPs, and their late precursors, were cell-lineage–specific and associated either with the general response against SARS-CoV-2 or COVID-19 severity. This included an interferon-imprint in DC1s observed in all patients and a decreased expression of the coinhibitory molecule CD200R in pre-DCs, DC2s, and DC3 subsets of severely sick patients. Finally, unsupervised analysis revealed that the MNP profile, alone, pointed to a cluster of COVID-19 nonsurvivors. This study provides a reference for the MNP response to SARS-CoV-2 infection and unravels mononuclear phagocyte dysregulations associated with severe COVID-19.

Neutrophils play a key role in defence against infection and in the activation and regulation of innate and adaptive immunity. In cancer, tumour-associated neutrophils (TANs) have emerged as an important component of the tumour microenvironment. Here, they can exert dual functions. TANs can be part of tumour-promoting inflammation by driving angiogenesis, extracellular matrix remodelling, metastasis and immunosuppression. Conversely, neutrophils can also mediate antitumour responses by direct killing of tumour cells and by participating in cellular networks that mediate antitumour resistance. Neutrophil diversity and plasticity underlie the dual potential of TANs in the tumour microenvironment. Myeloid checkpoints as well as the tumour and tissue contexture shape neutrophil function in response to conventional therapies and immunotherapy. We surmise that neutrophils can provide tools to tailor current immunotherapy strategies and pave the way to myeloid cell-centred therapeutic strategies, which would be complementary to current approaches. This is the Author's Accepted Manuscript of Nature Reviewsarticle, as established by the editor's policies. &nbsp

Natural Killer (NK) cells represent a lymphocyte population of the innate immunity, involved in the control of viral infections and tumor cells. Among the wide number of functions that NK cells can mediate, the most important are the ability to lyse target cells via the release of cytotoxic granules, and the production of pro-inflammatory mediators, such as cytokines and chemokines. In mouse, according to the expression of the surface markers CD11b and KLRG1, within the NK cell population 4 discrete subsets can be distinguished, which represent different developmental stages and show heterogeneous functional capacities. Moreover, their tissue localization is dissimilar in primary and secondary lymphoid organs, and in non-lymphoid organs: the most immature populations are mainly represented in the bone marrow (BM), thymus and lymph nodes (LN), while the most mature ones preferentially distribute in blood, spleen, liver and lungs. Chemokines are chemotactic molecules of proteic nature, which bind specific GPCR receptors, and direct the migration of several cell types through the formation of a concentration gradient. NK cells express a great variety of chemokine receptors, which regulate their organ distribution and their trafficking to inflamed tissues. The first part of this work dealt with the study of the function of CX3CR1 receptor, specific for the chemokine CX3CL1, in the NK cell positioning in the BM microenvironment. To this aim, a murine knock-in model, in which one or both copies of cx3cr1 gene were replaced by GFP cDNA, was used. Through the analysis of GFP expression it was possible to track CX3CR1+ NK cells in heterozygous mice, or the cells that should express CX3CR1, but express only GFP, in the homozygous mice. Data obtained by adoptive transfer experimental approaches, single or mixed BM chimeras and the phenotyping of NK cells in BM compartments, demonstrated that CX3CR1 is expressed by a terminally differentiated subset, which mainly resides in the vascular compartment of the BM, and displays reduced effector functions. Moreover, CX3CR1 was associated to a positive regulation in the exit of most mature NK cells from the BM towards the periphery, similarly to what was previously demonstrated for S1P5. Data regarding these studies have been published in two international peer-reviewed journals. The second part of this thesis work was centered on the influence of multiple myeloma (MM) growth on the functional and trafficking capacities of BM NK cells, in a murine model of this pathology. MM was followed at different growth stages, and we observed the selective exclusion from BM of the most functional NK cell subpopulation (KLRG1-), at an intermediate stage of tumor progression. Adoptive transfer experiments on healthy controls and MM-bearing mice have elucidated that the KLRG1- NK cell reduction was attributable to a trafficking alteration within BM. We analysed the mechanism of this phenomenon, by studying the production of several chemokines and the expression of many chemokine receptors on BM NK cells in presence or absence of MM. At the same stage of MM growth we observed a decrease of CXCR3 expression, while its ligands CXCL9 and CXCL10 were augmented in tumor-bearing mice. Furthermore, CXCL9 concentration was higher as compared to healthy controls also in the sera in MM-bearing mice.In parallel, we highlighted that CXCR4/CXCL12 axis was altered too, as CXCL12 level was significantly lower in the BM of tumor-bearing mice. With competitive adoptive transfer experiments, we demonstrated that CXCR3+ NK cells are selectively excluded from BM, and accumulate in liver and spleen. Globally these data suggest a strategy for the tumor cells to evade the NK-cell mediated effect in the tumor microenvironment, by altering directly or indirectly different chemokine/receptor axes, important for BM NK cell migration.