Mitochondria and effector functions of human CD8* T cells

Naïve CD8+ T cells become activated if they recognize their specific antigen in the presence of co-stimulatory signals and inflammatory cytokines. This activation is followed by a rapid clonal expansion and differentiation into effector cells. These energetically demanding processes require significant changes in cellular metabolism. T cell activation induces metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis, proving both energy and metabolic intermediates. When the pathogen is cleared, the majority of antigen-specific CD8+ T cells undergo apoptosis. Only a small subset switch back to oxidative phosphorylation and survives this contraction phase to form a long-lived memory T cell pool with the ability to respond faster and stronger after reinfection. Although both naïve and memory CD8+ T cells use oxidative phosphorylation to generate ATP for their housekeeping functions, there are fundamental metabolic differences between these two subsets. Our group recently demonstrated that memory CD8+ T cells are able to rapidly induce glycolysis upon reactivation. This immediate-early glycolytic switch is required for memory recall response and it is regulated by the mTORC2-Akt axis. Interestingly, memory CD8+ T cells have higher mitochondrial mass and functionality than their naïve counterparts. Whether the rapid increase in glucose metabolism in memory T cells also involves oxidation in the mitochondria via TCA cycle remains to be elucidated. How and when mitochondrial biogenesis occurs in the naïve to effector transition is still unknown. The kinetics, regulation and functional implications of mitochondrial biogenesis in naïve CD8+ T cells are the main focus of manuscript 1. Naïve CD8+ T cells were activated in vitro and mitochondrial mass and functions were assessed. We found that mitochondrial mass was increased in naïve CD8+ T cells early after activation and before the first division. In parallel to glycolysis, both mitochondrial respiration and mROS generation were increased in early-activated naïve CD8+ T cells. Inhibition of mitochondrial biogenesis diminished activation-induced mROS generation and IL-2, TNF and IFN-γ secretion. Notably, direct inhibition of mROS had a similar effect on early-effector cytokine secretion. The study presented here assigned additional roles for mitochondrial biogenesis early after activation, and that this event is mROS dependent, which results in increased IL-2 secretion and the modulation of TNF and IFN-γ production. How mitochondrial differences contribute to early effector response in memory CD8+ T cells is still elusive. In manuscript 2, we studied the impact of glucose oxidation in mitochondria on early effector functions in both naïve and memory CD8+ T cells. Metabolomic and glucose tracing experiments assessing metabolic intermediates in both subsets revealed that glycolysis and TCA cycle intermediates were elevated in activated memory CD8+ T cells upon activation. Moreover, we demonstrated that mitochondrial respiration was increased early after activation in memory CD8+ T cells. Blocking mitochondrial respiration diminished early-recall response in memory CD8+ T cells. This suggests that both glycolysis and glucose flux into the TCA are important for rapid IFN-γ secretion. Additionally, we also demonstrated that key components of the mTORC2-Akt axis are present in the mitochondria associated membrane of the endoplasmic reticulum in CD8+ T cells, suggesting a close association between mTORC2-Akt signaling and mitochondrial function. These results further established metabolic differences between naïve and memory CD8+ T cells. Thus, the resulting metabolic plasticity in memory CD8+ T cells could be a requirement to support effector functions in competitive microenvironments. Lastly, to mimic a CD8+ T cell - APC interaction in vitro we established a protocol using dual antibody coupling on polystyrene beads and assessed distinct antibody ratios loaded on the beads. This is the topic of manuscript 3 (technical notes). We found that input ratios of antibody were not always directly reflected by the final ratio loaded on the beads. Moreover, differently loaded beads showed differences in CD8+ activation, in vitro. All together, the studies presented here contribute to a better understanding on how T cell metabolism supports immune function and could have implications for future strategies aiming to therapeutically manipulate CD8+ effector and memory functions.

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