Mitochondria-ER Contact Sites in CD8+ T Cell: How Organellular Distance Varies Between the CD8+ T Cell Subsets.

INTRODUCTION: The immune system, which encompasses the innate and adaptive immune system, protects against pathogens, maintains tissue homeostasis and promotes wound healing. CD8+ T cells, a type of lymphocyte belonging to the adaptive immune system, participate in the elimination of intracellular pathogens and tumor cells, and the maintenance of immunologic memory. The differentiation of CD8+ T cells involves several stages, from naïve T cells, over various memory subsets up to effector cells, each defined by distinct phenotypic and metabolic characteristics, which support their activation, differentiation, and specific function. Mitochondria-endoplasmic reticulum contact sites have been identified as pivotal regulators of various cellular processes, including calcium and lipid transfer, mitochondrial dynamics, and apoptosis. MERCs are therefore likely to also play a key role in CD8+ T cell differentiation and function. OBJECTIVE: The objective of this project is to characterize the MERC status across the different subsets of CD8+ T cells. Ultimately, we hope that this work will help in the development of new therapies for disease states such as viral infections and cancer or autoimmune conditions that remain unresolved because of a dysfunctional CD8+ T cell response, by tailoring CD8+ cell differentiation based on the modulation of MERCs. METHODS: We isolated CD8+ T cells from peripheral blood mononuclear cells and sorted them into five subsets using fluorescence-activated cell sorting based on surface markers (CD45RA, CXCR3, CD95). A Fluorescence Resonance Energy Transfer (FRET)-based probe was employed, utilizing energy transfer between two fluorophores to map the proximity between mitochondria and ER in in the different CD8+ T cell subsets. Conditions were optimized for effective high-content screening imaging in CD8+ T cells. RESULTS: We successfully established and optimized a protocol for MERC analysis in human CD8+ T cells, based on FRET. The characterization of the MERC status in the CD8+ T cell subsets indicated varying levels of mitochondria-ER proximities, with naïve and stem cell memory T cells exhibiting the highest ER-mitochondria interaction, as indicated by the highest FRET ratios, whereas central and effector memory T cells had the lowest, and effector T cells showed intermediate levels of mitochondria-ER proximity. CONCLUSION: Our findings demonstrate that CD8+ T cells exhibit distinct levels of proximity at the MERCs during their differentiation process. The ability to manipulate MERCs may hold potential for developing therapies aimed at optimizing CD8+ T cell responses, thus improving treatments for chronic viral infections, cancer, and autoimmune disorders.