Peeking under the Hood of Naive T Cells

Abstract

Signaling and transcriptional regulation of metabolic reprogramming upon T cell activation has been studied intensively. In this issue of Cell Metabolism, Ricciardi et al. (2018) show that translational regulation of key metabolic enzymes GLUT1 and ACC1 plays a novel role in human naive CD4 T cell activation and subset differentiation.

CD4⁺ and CD8⁺ T cells constitute critical components of the adaptive immune system. Upon antigen recognition, naive T cells undergo prodigious proliferation and differentiate into effector subsets. These functions require robust and distinct bioenergetics and biosynthetic demands. Like most non-proliferating cells, naive T cells are generally considered metabolically quiescent and rely on fatty acid oxidation to maintain homeostasis. However, upon TCR engagement and co-stimulatory signals, naive T cells almost immediately switch from fatty acid oxidation to aerobic glycolysis. After an initial activation or priming process, they enter a rapid proliferating stage accompanied by increased glutaminolysis and fatty acid synthesis. Upon starting to proliferate, they are able to divide every 4 to 6 hr (Rathmell, 2011).

Efforts have been made to interrogate how naive T cells are able to achieve this rapid metabolic reprogramming. TCR ligation leads to coordinated upregulation of glucose and amino acid transporters, facilitating nutrient uptake and T cell blastogenesis. Following TCR activation, transcription factors such as Myc, Hif1a, IRF4, SREBP, and ERRα induce and sustain various metabolic genes’ expression necessary to support glycolysis, glutaminolysis, polyamine synthesis, sterol and lipid synthesis, and mitochondria metabolism (MacIver et al., 2013; Pearce et al., 2013).

Integrating environmental cues, including TCR signaling, growth factors, and nutrient availability, the mechanistic target of rapamycin (mTOR) plays an important role in regulating T cell metabolism and differentiation (Powell and Delgoffe, 2010). To this end, a major role of mTOR is to regulate the process of protein translation. Ricciardi et al. (2018) hypothesized that a translational regulation of T cell metabolism may exist in naive T cells. Sure enough, by analyzing transcriptomic and proteomic datasets of metabolic genes in human naive CD4⁺ T cells, they discovered genes with abundant mRNA presence that were essentially undetectable at the protein level. Among these genes were the critical metabolic components glucose transporter GLUT1 and acetyl-CoA carboxylase ACC1.

Surprisingly, by probing the transcriptomic and proteomic datasets further, Ricciardi et al. (2018) found abundant ribosomal and translational machinery in human naive CD4⁺ T cells. However, compared to TCR-activated cells, polysomal profiles of naive CD4⁺ T cells showed a high 80S peak, indicating an absence of translation initiation. 4E-BPs bind eIF4E and inhibit the formation of translation initiation complex. At the protein level, Ricciardi et al. (2018) found that 4E-BPs outnumbered eIF4E by 2-fold, indicating an inhibited translation state.

Using pharmacological inhibitors, Ricciardi et al. (2018) were able to show that in human naive CD4⁺ T cells, upon activation, GLUT1 and ACC1 were regulated at the translational level instead of transcriptional level. These observations suggested an elegant model to explain how naive CD4+ T cells could rapidly switch from fatty acid oxidation to glycolysis and fatty acid synthesis. That is, the "quiescent" naive T cells already had transcribed the genes necessary for such a switch and upon activation merely needed to translate these genes. By employing 4EGi-1, an inhibitor of eIF4E-eIF4G interaction and a biochemical luciferase assay, Ricciardi et al. (2018) further went on to show that ACC1 was translationally regulated via its 5'UTR. Importantly, both 4EGi-1, a broad translational inhibitor, and SorA, an ACC1-specific inhibitor, reduced pyruvate, lactate, and OCR of activated T cells, indicating that translational regulation of ACC1 is essential for metabolic reprogramming and crosstalk between glycolysis and mitochondria respiration. As the rate-limiting step in fatty acid synthesis, ACC1 catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Both glycolysis and the TCA cycle contribute to cytoplasmic acetyl-CoA pool, and this probably explains how ACC1 blockade slows down glycolysis and the TCA cycle. In addition, malonyl-CoA was shown to allosterically bind and inhibit Cpt1a, which is critical for fatty acid flux in mitochondria (Saggerson, 2008).

mTOR functions through two different complexes, mTORC1 and mTORC2. mTOR deficiency or inhibition blocks differentiation into Th1, Th2, or Th17 cells under their respective polarizing conditions and leads to T regulatory cells (Tregs) generation (Delgoffe et al., 2009). The observation that equivalent ACC1 mRNA levels were detected across all helper CD4⁺ T cell subsets while protein levels varied prompted Ricciardi et al. (2018) to examine the idea that translation might also play a role in regulating CD4⁺ T cell fate decision. In testing this hypothesis, they found that under Th17 skewing conditions, 4EGi-1 inhibited the generation of IL17-producing cells while increasing Foxp3⁺ T regulatory cells. Mechanistically, 4EGi-1 treatment decreased RORγt and increased Foxp3, both at the mRNA and protein level.

Overall, these findings certainly call into question the idea that naive T cells are quiescent. Indeed, while metabolically they are efficiently fueling themselves via a low level of OXPHOS and fatty acid oxidation, in fact their translational machinery is poised to rapidly fire up the key enzymes essential for robust metabolic reprogramming (Figure 1). To this end, these data shed a novel and exciting new light on T cell metabolic reprogramming. While Ricciardi et al. (2018) clearly show that ACC1 is one of the key enzymes that is translationally regulated, several questions remain to be addressed. First, how is the translational machinery poised at the molecular level? Second, TCR signaling induces AMPK activation that in turn phosphorylates and inhibits ACC1 (MacIver et al., 2011; Tamás et al., 2006). Ricciardi et al. (2018) concluded that in human CD4⁺ T cells, repression of ACC1 bypassed AMPK-driven control based on the observation that the ATP/AMP ratio didn’t change and phosphorylated AMPK was not detected 24 hr after activation. It would be interesting to know if, indeed, AMPK does provide another layer of regulation at earlier time points when AMPK is robustly activated. Third, Ricciardi et al (2018) identifiedGLUT1 as another translationally poised protein in addition to ACC1. Previous studies have shown that CD28 signaling is a key regulator of GLUT1 expression (Frauwirth et al., 2002; Jacobs et al., 2008). In this regard, it would be interesting to know the interplay between these two mechanisms of regulating GLUT1 expression upon T cell activation.

Figure 1. Translational Regulation of Pre-accumulated Acc1 and Glut1 mRNAs in Naive T Cells Accounts for Rapid and Full Metabolic Reprogramming upon T Cell Activation.

Human naive CD4⁺ T cells are poised to rapidly become activated by maintaining high levels of metabolic enzymes Acc1 and Glut1 mRNAs and translational machinery (left). Upon activation, translation repression is released and leads to rapid ACC1 and GLUT1 protein expression to fulfill fatty acid synthesis and glycolysis to support their function (right).

Overall, this work provides novel insight regarding metabolic regulation. It will be intriguing to see if translational regulation plays a role in naive CD8⁺ T cells, resting memory CD8⁺ T cells, or other cell types that are highly regulated by metabolism, like macrophages. Likewise, it will be of interest to determine whether this model of poised translational repression also plays a role in other genes and genetic programs that are critical for the rapid activation of cells.