Ketogenesis-generated β-hydroxybutyrate Is An Epigenetic Regulator Of CD8~+T-cell Memory Development

Objective:Glycogen has been considered to play a key role in energy metabolism for a long time.However,our recent studies have shown that glycogen metabolism mediated by cytoplasmic phosphoenolpyruvate carboxykinase Pck1 regulates the formation and maintenance of CD8⁺memory T(T_mem)cells by regulating redox state.However,a question is raised about this unusual metabolic pathway,that is,how Pck1is up-regulated in CD8⁺T_memcells.Here,we will explore the specific mechanism of Pck1 up-regulation in CD8⁺T_mem cells.Methods:（1）In vitro,T cells were induced into T_eff and T_mem:obtain spleen of OT-I mice and made into a single fine suspension,and respectively cultured by IL-2 or IL-15.The cells were planted in a 6-well plate with a cell concentration of 1×10⁶/mL,and spleen cells were stimulated with 10 ng/mL SIINFEKL Peptide（OVA257-264）.The spleen cells were cultured with 10 ng/mL IL-2 for 3 days,and after 3 days,90%of the surviving cells were CD8⁺OT-I T cells,and the SIINFEKL Peptide was washed away.The activated T cells were respectively cultured with IL-2 or IL-15 for 4 days,and the cells cultured by IL-2 were T_eff,by IL-15,which was called T_mem.（2）In order to determine whether ketone bodies were produced in CD8⁺T_mem cells,we first transferred1×10⁵ CD45.1⁺OT-I T cells to C57BL/6（CD45.2⁺）mice,and then injected Listeria monocytogenes（Lm-OVA）expressing OVA protein in 5×10⁵ colony forming units（CFU）into the tail vein of mice to infect and activate T cells.Flow staining was performed on the 6th and 30th day after infection,and OVA-specific CD8⁺effector T cells and T_mem cells were sorted.The levels of AcAc and BHB in CD8⁺T_mem cells were analyzed by liquid chromatography-mass spectrometry（LC-MS）and fluorescence.（3）In order to verify the active metabolism of ketone body in CD8⁺T_mem cells,q-PCR and Western Blot were used to detect the expression of enzymes related to ketone body metabolism,such as Acat,Hmgcs2,Hmgcl and Bdh1.（4）To study whether and how ketone metabolites affect CD8⁺T_mem cells.In vitro experiment:physiological concentration of BHB（2-10 m M）or AcAc were added to the culture medium to treat CD8⁺T_mem respectively.In vivo experiment:CD45.1⁺CD8⁺OT-I T cells were transferred to C57BL/6 mice,and on the seventh day after Lm-OVA infection,mice were injected intraperitoneally with BHB and normal saline.On the 30th day,the mice were killed and the cells in eyeball blood,spleen and lymph nodes were analyzed.（5）In order to verify the effect of BHB on the memory development of CD8⁺T cells,we used shRNA to knock down the Bdh1 in OT-I T cells in vitro to block the transition from AcAc to BHB.In vivo,CD8⁺OT-I T cells transfected with shNC or shBdh1 were transferred to C57BL/6 mice,and on the seventh day after Lm-OVA infection,mice were intraperitoneally injected with BHB and normal saline.On the 30th day,the mice were killed and the cells in eyeball blood,spleen,lymph nodes,liver,lung and bone marrow were analyzed.（6）Tumor immunotherapy model:first,CD45.1⁺CD8⁺OT-I T cells were transferred to C57BL/6 mice,and on the seventh day after Lm-OVA infection,mice were injected intraperitoneally with BHB and normal saline.On the 30th day,five days after subcutaneous inoculation of OVA-B16,the survival time of mice was observed and the tumor volume was recorded.（7）CD8⁺T_n,T_eff or T_memcells were treated with ¹³C-BHB in vitro.The proportion of ¹³C of intermediate metabolites（citric acid,fumaric acid,α-KG and malic acid）in TCA cycle was detected by LC-MS.（8）Detection of oxygen consumption rate（OCR）of CD8⁺T_eff or T_mem cells cultured in vitro by Seahorse.（9）To explore how BHB regulates the expression of Pck1.The effect of BHB on Pck1 of CD8⁺T_mem cells was analyzed by Western blot and ChIP-qPCR.Results:（1）Through liquid chromatography-mass spectrometry（LC-MS）analysis and fluorescence quantitative analysis,it was found that there were a large number of AcAc and BHB in CD8⁺T_memcells compared with T_ncells or T_effcells.（2）Through q-PCR and Western Blot analysis,it was found that the up-regulated expression of Acat,Hmgcs2,Hmgcl and Bdh1 in CD8⁺T_memcells was higher than that in T_nand T_effcells.（3）Through in vitro and in vivo experiments,it was found that BHB increased the number of CD8⁺T_mem cells and up-regulated the expression of memory-related transcription factors Tcf7,Lef1 and Bcl6,indicating that ketogenic metabolite BHB was involved in the memory development of CD8⁺T cells.（4）Through qPCR and Western blot detection and analysis,it was found that the content of BHB in T cells decreased after knocked down Bdh1,at the same time,the formation of T_mem cells was inhibited,and this phenomenon can be remedied by providing BHB.It is suggested that ketogenic metabolite BHB plays an important role in regulating the formation of CD8⁺T_mem cells.（5）By observing the survival time of mice and recording the tumor size,it was found that intraperitoneal injection of BHB could enhance the destruction of OVA-B16melanoma mediated by OT-I T cells and prolong the survival time of mice.In contrast,mice that inherited shBdh1 OT-I T cells showed uninhibited tumor growth and shortened survival,but this could be alleviated by injection of BHB.（6）Through LC-MS analysis,it was found that the ratio of ¹³C-α-KG,¹³C-fumaric acid and ¹³C-malic acid in T_mem cells was slightly higher than that in T_eff cells,which indicated that BHB could be used as the energy source of metabolism in T_mem cells.（7）The oxygen consumption rate was measured by hippocampal XF analyzer and（OCR）showed that the oxygen consumption rate（OCR）of CD8⁺T_eff or T_memcells treated with BHB did not change compared with the untreated group.Moreover,knocked down Bdh1 does not affect the OCR,of CD8⁺T_mem cells,indicating that the main role of BHB in CD8⁺T_mem cells does not include its function as an energy molecule.（8）Through Western blot and ChIP-qPCR analysis,it was found that BHB upregulated the expression of Pck1 by modifying H3K9 on FoxO1.Conclusion:Acetyl-CoA in the mitochondria of CD8⁺T_memcells enters the ketogenic metabolic pathway to produce BHB,and epigenetic modification of FoxO1 by BHB,resulting in the upregulation of FoxO1 and its target gene Pck1.