The Role And Mechanism Of Glycogenolysis In The Early Recall Response Of CD8~+ Memory T Cells

When the body is invaded again by pathogens,CD8⁺memory T（CD8⁺Tm）cells can bind with the major histocompatibility complex class I（MHC-I）antigenic peptide molecules,and rapidly activate the recall response in a short period of time,and secrete cytokines such as IFN-γand TNF-αto eliminate invading pathogens,which is the key mechanism to the body’s adaptive immunity.It is also the theoretical basis for the development of T-cell vaccines.Changes in cell differentiation or functional status require metabolic reprogramming to match corresponding energy shift.Current studies have shown that CD8⁺Tm cells are mainly powered by fatty acid oxidation（FAO）and can up-regulate the glycogen synthesis by phosphoenolpyruvate carboxykinase 1（Pck1）through gluconogenesis for energy storage.Glycolysis meets the rapid increase in energy demand during recall response,but the underlying mechanism of the metabolic mode switch during the rapid transition from memory to effect has not been fully elucidated.In this study,the CD8⁺OT-I Tm model was constructed by inducing CD8⁺Tm cells with IL-15 in vitro,adopting transfered with CD8⁺OT-I T cells to mice in vivo,and infected with ovalbumin specific Listeria monocytogenes（LM-OVA）.The characteristics of metabolic transition in the recall response were further studied.It’s different from naive T cells,the activation of CD8⁺Tm cells is not dependent on exogenous glucose.Further studies confirmed that glycogenolysis is a key factor in maintaining the early activation of CD8⁺Tm cells,because the level of glycogen decreased rapidly in the early activation stage and blocking glycogenolysis led to the failure of CD8⁺Tm activation.Subsequent metabolite tracing by ¹³C glucose showed that the metabolite of glycogenolysis glucose 1 phosphate（G-1-P）was converted into glucose 6 phosphate（G-6-P）,most of which was then fueled by glycolysis.The other G-6-P was diverted into the pentose phosphate pathway（PPP pathway）,the former provided the energy supply for the rapid activation of CD8⁺Tm cells.The latter provides reducing power by producing NADPH to resist the cumulative reactive oxygen species（ROS）damage caused by rapid activation,which provides the material basis for rapid and efficient activation of CD8⁺Tm cells.In order to explore the molecular mechanism,the phosphorylation sites of glycogen phosphorylase（PYG）was analyzed by high performance liquid chromatogram-mass spectrometry（LC-MS）,which is a key enzyme of glycogenolysis,and three tyrosine sites（Y75,Y280,and Y767）of brain glycogen phosphorylase（PYGB）were found highly expressed in CD8⁺Tm were phosphorylated in the early stage of the recall response.It was further confirmed that the phosphorylation of these three sites was related to the activity of PYGB,and the ZAP70/LCK complex downstream of TCR activation signal combined with PYGB at the early stage of the recall response,thereby activating PYGB to initiate glycogen decomposition.In conclusion,this study identified the unique energy metabolism pattern of CD8⁺ Tm cells in the early stage of recall response,and elucidated the molecular basis of glycogenolysis driven by phosphorylation of PYGB by TCR signal in the early stage of CD8⁺Tm cells’transition from memory to effector,thus providing a deeper understanding of the role of metabolic reprogramming in immune cell differentiation.It provides a theoretical basis for enhancing vaccine efficacy.