Designing a combined Activation-Induced Marker and Intracellular Cytokine assay for comprehensive characterization of antigen-specific CD4+ and CD8+ T cells in SARS-CoV-2-infected children and adults

Abstract

The emergence of the novel coronavirus has prompted tremendous research effort towards the understanding of protective immune mechanisms following SARS-CoV-2 infection. Broadly speaking, while multiple branches of the human immune system contribute to protection against SARS-CoV-2, most knowledge to date concerns the behavior of SARS-CoV-2-specific antibodies. This understanding has come to show that while antibodies generated post-infection are well-protective, they naturally diminish over time, and therefore recovered individuals are often left with insufficient levels of antibodies to altogether prevent re-infection. Thus, scientists have turned their attention towards other immune cell subsets which are better suited to contribute to protection against severe infection outcomes (that is, clinical protection) rather than altogether prevent reinfection. In this regard, T cells are a highly diverse immune cell subset with various roles in antiviral immunity; and while T cells are more difficult to study, SARS-CoV-2-specific T cells could offer the possibility of long-lasting protection following SARS-CoV-2 infection, and could even provide some degree of protection against emerging viral variants. Consequently, establishing the quantity and quality of SARS-CoV-2-specific T cells needed for clinical protection, as well as which disease settings (e.g., disease severities, patient groups, etc.) lead to the generation of these T cells have become important research avenues in recent times. In this context, early studies throughout the pandemic have relied on more traditional techniques of identifying SARS-CoV-2-specific T cells, with the important drawback of a less precise assessment of the quantity of T cell responses. More recently, the technique called the Activation-Induced Marker (in short, “AIM”) assay has been preferentially used because it allows a more accurate identification of SARS-CoV-2-specific T cells. However, this assay is rather laborious and costly, and offers only information regarding the phenotype (that is, the surface characteristics of cells) but not of the functional properties of the T cells analyzed. Therefore, in this study we used a hybrid version of the AIM assay, named in short “AIM+ICS”, which allows the simultaneous assessment of both phenotype and function of SARS-CoV-2-specific T cells, thus allowing us to gain better insight into this immune cell subset. Importantly, we optimized and applied this technique to allow the in-depth characterization of SARS-CoV-2 specific T cells of not only adult but also pediatric samples. Interestingly, children appear to be better protected against severe infection outcomes compared to adults. Still, the study of pediatric immune responses has so far been hindered by the limited blood volumes that can be obtained, which in turn allows relatively limited scientific insights to be obtained. In this context, our combined AIM+ICS assay is a very well-suited experimental technique to thoroughly analyze pediatric T cell responses following SARS-CoV-2 infection. Specifically, we inquired whether the quantity, quality, temporal development, or long-term maintenance of SARS-CoV-2-specific T cells are distinct in children with mild or asymptomatic SARS-CoV-2 infection compared to disease severity-matched adults. Thus, here we explored the link between SARS-CoV-2-specific T cells and the superior clinical protection of children following SARS-CoV-2 infection.