A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcome were previously correlated with Notch4 expression on regulatory T (Treg) cells, here we show that the Treg cells in MIS-C are destabilized through a Notch1-dependent mechanism. Genetic analysis revealed that MIS-C patients were enriched in rare deleterious variants impacting inflammation and autoimmunity pathways, including dominant-negative mutations in the Notch1 regulators NUMB and NUMBL leading to Notch1 upregulation. Notch1 signaling in Treg cells induced CD22, leading to their destabilization in a mTORC1-dependent manner and to the promotion of systemic inflammation. These results establish a Notch1-CD22 signaling axis that disrupts Treg cell function in MIS-C and point to distinct immune checkpoints controlled by individual Treg cell Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.

Erroneous immune responses in COVID-19 could have detrimental effects, which makes investigation of immune network underlying COVID-19 pathogenesis a requisite. This study aimed to investigate COVID-19 related alterations within the frame of innate and adaptive immunity. Thirty-four patients clinically diagnosed with mild, moderate and severe COVID-19 disease were enrolled in this study. Decreased ILC1 and increased ILC2 subsets were detected in mild and moderate patients compared to healthy controls. NK cell subsets and cytotoxic capacity of NK cells were decreased in severe patients. Moreover, CD3+ T cells were reduced in severe patients and a negative correlation was found between CD3+ T cells and D-dimer levels. Likewise, moderate and severe patients showed diminished CD3+CD8+ T cells. Unlike T and NK cells, plasmablast and plasma cells were elevated in patients and IgG and IgA levels were particularly increased in severe patients. Severe patients also showed elevated serum levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8, reduced intracellular IFN-γ and increased intracellular IL-10 levels. Our findings emphasize that SARS-CoV-2 infection significantly alters immune responses and innate and acquired immunity are differentially modulated in line with the clinical severity of the disease. Elevation of IL-10 levels in NK cells and reduction of CD3+ and CD8+ T cells in severe patients might be considered as a protective response against the harmful effect of cytokine storm seen in COVID-19.

Objectives: In this preliminary study, the in vitro effect of C-Vx in human PBMCs and the in vivo effect of C-Vx in rats were investigated. Methods: The human part was analyzed in PBMCs isolated from healthy subjects. Apoptotic index, cytotoxic activity of CD8+ T and NK cells, and cell proliferation of CD3+, CD4+, CD8+ T and NK cells in response to different doses of C-Vx were investigated. Also the hematological and biochemical parameters of the rats administered subcutaneously in three different doses of C-Vx were monitored for 14-days. Results: Increased CD107a expression in response to C-Vx on NK cells but not on CD8+ T cells support the increasing of NK cell cytotoxicity. C-Vx alone was capable of triggering proliferation of T and NK cells. The PHA-induced proliferation of CD3+ and CD4+ T cells was diminished in response to C-Vx, while PHA-induced CD8+ T cell proliferation was up-regulated. PHA-triggered proliferation of total NK cells was enhanced with the existence of C-Vx. C-Vx was well tolerated in rats with no serious adverse effects or mortality (death) after 14-days of follow-up. Biochemical-parameters (creatinine, blood urea nitrogen, etc.) were not significantly different among treated and control groups. The levels of white blood cells and lymphocytes were increased up to two-folds in the C-Vx group (especially 0.25 ml/day) as compared to the control group. Conclusion: Taken together, these preliminary findings support the immunomodulatory effects of C-Vx. But these findings should cautiously be evaluated due to the low numbers of subjects in both human and experimental arms.

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