PD-1 Couples Glucose Starvation with Autophagy and Survival Through AMPK-Mediated Phosphorylation of Ulk1

Abstract 836 PD-1 is a potent inhibitor of T cell proliferation and can compromise anti-viral and anti-tumor T cell responses. PD-1 inhibits expression of Glut1 via which T cells uptake glucose, which is utilized to generate energy by glycolysis. Recently, we determined that CD4+ primary human T cells receiving PD-1 signals are blocked at the G1 phase and do not progress to the S and G2/M phases. Although PD-1-stimulated T cells did not proliferate, they remained viable at levels comparable or even better than T cells stimulated by CD3 and CD28. Here, we sought to determine whether T cells receiving PD-1 signals remain metabolically active and to identify the mechanisms via which PD-1 might lead to generation of energy and promotion of cell survival. We used primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies (mAbs) against CD3 and CD28 with or without agonist mAb against PD-1. Stimulation of T cells via TCR/CD3 and CD28 resulted in prominent upregulation of Glut1 by 48 hours, which remained elevated for five days of culture and gradually declined. In contrast, T cells receiving signals via TCR/CD3, CD28 and PD-1 did not upregulate Glut1 at any time point. T cells stimulated via TCR/CD3 and CD28 but not those receiving PD-1 signals displayed glucose uptake as determined by incorporation of 14C(U)}-deoxy-D-glucose. To determine whether T cells receiving PD-1 signals were metabolically active, we examined mitochondrial homeostasis by assessing membrane potential using the potentiometric dye TMRE. At all time intervals of culture, mitochondrial membrane potential was comparable between T cells stimulated via TCR/CD3/CD28 and T cells stimulated via TCR/CD3/CD28 and PD-1. Thus, despite the lack of glucose uptake, T cells receiving PD-1 signals were metabolically active. In other cell types glucose deprivation activates AMPK (AMP activated protein kinase), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. The substrate of AMPK that connects energy sensing to cell survival during starvation is Ulk1, the mammalian homologue of the yeast kinase ATG1, which has an essential role in autophagy, survival and longevity under limited nutrient supplies. AMPK-mediated phosphorylation of Ulk1 is required for Ulk1 activation and initiation of autophagy, thereby maintaining mitochondrial homeostasis and cell survival during starvation. Because by suppressing glucose uptake PD-1 induces a state of glucose starvation in T cells, we examined whether PD-1 might induce AMPK activation. In CD4+ T cells activated via TCR/CD3 and CD28, AMPK activation was transient and insignificant. In contrast, T cells receiving PD-1 signals displayed robust and sustained activation of AMPK, which mediated a prominent phosphorylation of Ulk1 on the AMPK-specific site Ser555. Furthermore, T cells receiving PD-1 signals displayed robust phosphorylation of Raptor on Ser752, an AMPK-specific site, leading to inactivation of mTORC1. These biochemical events were in complete contrast to those observed in T cells receiving TCR/CD3 and CD28-mediated signals without PD-1, which displayed no AMPK-mediated phosphorylation of Ulk1 or Raptor and had increased mTORC1 activity. Because mTORC1-mediated phosphorylation of Ulk1 disrupts Ulk1-AMPK interaction and prevents Ulk1 activation we examined whether PD-1 induced differential AMPK-Ulk1 interaction and Ulk1 activation. Immunoprecipitation with AMPK-specific mAb revealed a prominent Ulk1-AMPK interaction and Ulk1 activation in T cells stimulated via TCR/CD3, CD28 and PD-1 but not in T cells stimulated via TCR/CD3 and CD28 in the absence of PD-1 signals. To determine the biological consequences of these signaling events we examined whether, via AMPK-Ulk1 activation, PD-1 might induce autophagy in T cells. During autophagy, carboxy-terminal lipid modification of LC3 is a well-characterized phenomenon required for autophagosome formation which can be readily detected by an increased electrophoretic mobility (LC3-II). PD-1 ligation induced a prominent increase of LC3-II, whereas no LC3-II increase was observed during stimulation via TCR/CD3 and CD28. These results uncover an unexpected biochemical mechanism coupling glucose starvation, autophagy and survival downstream of PD-1 and suggest that therapeutic targeting of AMPK might be a novel approach to modulate PD-1-mediated signaling. Disclosures: No relevant conflicts of interest to declare.