PD-1 Induces Metabolic Reprogramming Of Activated T Cells From Glycolysis To Lipid Oxidation

Programmed death (PD)-1 is a potent T cell inhibitor and can compromise anti-viral and anti-tumor T cell responses. PD-1 abrogates activation of the PI3K/Akt pathway but the mechanism remains unclear. The main negative regulator of PI3K/Akt is the lipid phosphatase PTEN, which dephosphorylates PIP3 at the 3′ position to generate PI(4,5)P2. By limiting the amount of PIP3, PTEN opposes PI3K activation and blunts the survival and proliferative signal of the PI3K/Akt pathway. A critical regulatory component of PTEN is the 50-amino-acid C-terminus domain (aa residues 354 to 403), which undergoes phopshorylation by CK2 (casein kinase 2) on three residues S380, T382 and T383, thereby decreasing PTEN phosphatase activity while stabilizing PTEN protein. To examine the effects of PD-1 on PTEN we used primary human CD4⁺ T cells and magnetic beads conjugated with monoclonal antibodies against TCR/CD3 and CD28 with or without PDL-1-Ig fusion protein to induce PD-1-mediated signals. During TCR/CD3/CD28-mediated stimulation, PTEN was phosphorylated in the Ser380/Thr382/Thr383 cluster within the C-terminus regulatory domain, resulting in suppressed PTEN phosphatase activity and increased protein stability. PD-1 inhibited phosphorylation of the Ser380/Thr382/Thr383 cluster resulting in decreased PTEN stability but increased phosphatase activity. These effects were secondary to inhibition of CK2 and were recapitulated by pharmacologic CK2 inhibitors upon TCR/CD3/CD28-mediated stimulation without PD-1. PI3K/Akt has a central role in regulating expression of Glut1 that is required for glucose uptake and utilization for glycolysis, which is the main metabolic pathway for energy generation upon T cell activation. Because PD-1 activated PTEN and inhibited PI3K/Akt, we examined the effects of PD-1 on cell survival and energy generation. Surprisingly, T cells receiving PD-1 signals remained viable at levels comparable to T cells stimulated by TCR/CD3 and CD28, although failed to express Glut1 and did not display uptake and incorporation of ¹⁴C(U)}-deoxy-D-glucose. Moreover, T cells receiving PD-1 signals were metabolically active, as determined by mitochondrial membrane potential. We determined that PD-1 induced a sustained activation of the key energy sensor AMP-activated protein kinase (AMPK) and phosphorylation of the AMPK substrate Ulk1, the mammalian homologue of the yeast kinase ATG, which is essential for autophagy, survival and longevity under limited nutrient supplies. Under these conditions, T cells also displayed phosphorylation of Raptor on the AMPK-specific site, leading to inactivation of mTORC1. Because Ulk1 activation is essential for autophagy and survival under limited nutrient supplies, we analyzed markers of autophagy. Compared to TCR/CD3/CD28-stimulated cells, T cells receiving PD-1 signals had higher LC3B-II/LC3B-I ratio and decreased expression of SQSTM1/p62, indicating a higher degree of autophagy. Besides autophagy, AMPK has a central role in lipid metabolism by stimulating fatty acid oxidation (FAO). In the liver, the rate of FAO is controlled by allosteric regulation of carnitine palmitoyltransferase I (CPTI), which is located on the outer mitochondrial membrane and esterifies long chain fatty acids to carnitine, thereby initiating mitochondrial import. In contrast, in hematopoietic cells, FAO is controlled by CPT1A abundance, and metabolic reprogramming is regulated by alterations of CPT1A expression. Assessment of CPT1A in T cells stimulated via TCR/CD3 and CD28 revealed decrease of CPT1A expression and parallel suppression of FAO compared to base line levels of unstimulated cells. In contrast, T cells receiving PD-1 signals displayed a dramatic upregulation of CPT1A mRNA and protein, which temporally coincided with a robust increase of FAO as determined by assessment of fatty acid β-oxidation using [9,10-³H] palmitate. Our results uncover an unexpected biochemical and metabolic mechanism coupling PD-1-mediated PI3K/Akt inhibition to AMPK activation leading to a metabolic shift from glycolysis to autophagy and lipid oxidation in T cells receiving PD-1 signals. Furthermore, our findings provide a mechanistic explanation for the survival and persistence of PD-1-exhausted T cells in patients with chronic infections and tumors and identify novel targets that can be exploited therapeutically to overcome PD-1-mediated T cell exhaustion.