Intracellular Cytokine Profiling by High-Dimensional Flow Cytometry Demonstrates Highly Augmented Cytotoxicity and Overly Activated NK cells in COVID-19 Patients

ABSTRACT

Background: COVID-19 pandemic has caused more than 4.7 million deaths worldwide to date and still continues globally unabated. Numerous studies have linked the mortality in COVID-19 to aggressive immune response and cytokine storm. However, little is known about the cytokine profiles of individual immune cells that are directly involved in tissue damage. Here we investigate intracellular cytokines in individual T and NK cells of COVID-19 patients. Design: We studied 50 blood samples from 22 COVID-19 patients, 4 with mild, 6 moderate and 12 severe disease. There were 6 healthy controls. We performed high-dimensional 30-color spectral flow cytometry to characterize the immune cell subsets. For cytokine study, cells were stimulated for 6 hours, and stained for surface antigens and intracellular cytokines (IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17a, IL21, INFg, GnzB, TNFa, and GMCSF). Data ware acquired on FACSymphony 50-parameter analyzer and analysis performed using FlowJo. Results: Our studies revealed significant differences in lymphocyte cytokine profiles between COVID+ and healthy controls (Fig 1). CD4+ and CD8+ T-cells exhibited increased percentages of IL2+ and IFNg+ cells, indicating a shift towards Th1 reaction. Granzyme B is highly upregulated in all T and NK cell subsets, demonstrating highly armed cytotoxic cells in COVID patients. The most prominent changes were noted in NK cells, 7 cytokines were highly expressed, most are proinflammatory cytokines. Of particular interest are IL-21 and GMCSF, both are known to play important roles in inflammatory cell recruitment, activation and renewal, which can lead to augmented tissue inflammation and injury. These changes were already evident in patients with mild disease, but there is heightened cytokine production in severe cases. Conclusions: Using high-dimensional flow cytometry we demonstrated for the first time significantly increased production of multiple proinflammatory cytokines and cytotoxic molecules in individual T and NK cells of COVID-19 patients. NK cells are most drastically activated. It is conceivable that when recruited to the target tissue such as lung, these highly primed cells will play a major role in tissue injury and ultimately organ failure via their direct cytotoxicity and cytokine secretion. This is consistent with previous reports of increased NK cells in the COVID lungs. Analysis of NK cell cytokine profiles may serve to predict disease progression, and reveal new targets for immune-therapy for severe COVID patients. (Table Presented).