The goal of vaccination is to induce sustainable and functional T cell memory. To do so, it is essential to generate large numbers of memory T cell precursors during the acute phase of the immune response, which then form the pool of resting memory T cells which form the source for secondary immune responses. At the same time, cellular metabolism has a central impact on the fate and function of T cells, influencing their activation, differentiation, proliferation, effector function as well as long-term survival. However, the metabolism of T cells during the course of a human immune response is still poorly understood. In this regard, a long-standing unanswered question is whether distinct human T cell memory and effector subsets that were generated in vivo possess unique metabolic signatures that contribute to their complementary function.
An important reason for this lack of understanding is that, until recently, it has been methodologically challenging to analyze the metabolism of defined cell populations with subset or single cell resolution. Over the last years, novel technologies have emerged that could take metabolic profiling of cellular subsets to the next level. For T cells, analysis of single-cell energetic metabolism by profiling translation inhibition (‘SCENITH’) represents a particularly promising technology, since it is compatible with ex vivo investigation of human antigen-specific T cells via flow cytometry. With SCENITH, cellular incorporation of puromycin is investigated after different metabolic pertubations. Assuming that protein translation (as measured by puromycin incorporation) is one of the most energy-consuming cellular processes, this enables a direct assessment of cellular dependencies on distinct metabolic pathways.
This project aims at using SCENITH to perform, for the first time, ex vivo metabolic profiling of human antigen-specific T cell responses. To this end, we will investigate antigen-specific T cell responses induced by yellow fever and SARS-CoV-2 mRNA vaccination. The yellow fever vaccine YF-17D, which has been used since 1937, is perhaps the most effective vaccine available, with almost no reports of primary vaccine failure and long-lasting, if not life-long immune protection of immunocompetent vaccinees that is mediated by memory T cells. SARS-CoV-2 mRNA vaccination in turn uses a modern vaccine technology that has been crucial in the fight against the current SARS-CoV-2 pandemic in many countries.
The specific experimental plan for the project is as follows: T cells from vaccinees before, shortly (10-14 days) and long-term (6-12 months) after individual immunizations with YF-17D or SARS-CoV-2 mRNA vaccines will be isolated from peripheral blood and antigen-specific populations will be identified by activation markers or peptide major histocompatibility complex (pMHC) tetramers. SCENITH will then be applied to characterize cellular metabolism by short-term metabolic pertubations, followed by measurement of puromycin incorporation. In parallel, antigen-specific T cells will be phenotyped in-depth by flow cytometry and single-cell RNA sequencing. Further analyses will include assessment of energy-consuming processes in T cell memory and effector subsets - such as proliferation, survival and cytokine production – to provide a comprehensive picture of the energy sources that distinct T cell subsets require for their unique functions. Gained insights may provide a metabolic framework to instruct vaccine development for induction of long-lasting and functional T cell responses.