A 40 colors spectral panel to decipher human NK cell involvement in the establishment of NeuroCOVID-19

Natural Killer (NK) cells are innate lymphocytes endowed with cytotoxic and immune regulatory features, independently from a prior antigen sensitization. NK cells properties are directed against cells downregulating self MHC class I molecules, including viral infected cells. In this context, it has been shown that NK cells are relevant the pathophysiology of Coronavirus disease 2019 (COVID-19): several groups reported a transient reduction in circulating NK cell counts, exacerbated in lethal cases and coherent with their sequestration in the lungs. Unfortunately, despite bearing an hyperactivated phenotype that can contribute to worsening lung tissue damage, NK cells from severe COVID-19 patients show an hyporesponsive/exhausted behavior, being uncapable of controlling the viral spread in vitro. Aside from acute respiratory manifestations, COVID-19 shows systemic sequelae, known as post-COVID conditions, which could include cognitive and psychiatric symptoms. These neurologic events associated to COVID-19 (NeuroCOVID) represent a major concern in the healthcare sector worldwide, with an expected increase in the incidence of neurodegenerative diseases. However, to date the causes underlying the establishment of NeuroCOVID-19 symptoms constitute a gap in the knowledge and the role of NK cells has been poorly investigated. Given these premises, in this study we aim at investigating the immunological landscape, particularly focusing on NK cells, of patients manifesting post-COVID and especially NeuroCOVID, by means of extensive spectral flow cytometry. To this aim, we enrolled 150 subjects: 107 subjects (67=M, 40=F; mean age 44) who experienced neurological manifestations after COVID-19 persisting at least 12 weeks after the acute infection, 9 subjects affected by post-COVID without neurological/psychiatric symptoms (5=M, 4=F; mean age 56), and 34 healthy controls without post-COVID sequelae (19=M, 15=F; mean age 43). We collected peripheral blood samples and isolated mononuclear cells. In parallel, we designed and optimized a 40-colors spectral flow cytometry panel including several NK cell receptors, markers of NK cell activation, as well as regulatory and cytolytic mediators to deeply investigate the phenotype and the functions of circulating human NK cells under homeostatic conditions and to disclose their alterations in post-COVID syndrome, especially NeuroCOVID. The application of this panel to our cohort will provide us with a rounded understanding of NK cell involvement in the establishment of NeuroCOVID syndrome, thus paving the way for new therapeutic options.