The Kinase MTORC2 Regulates Memory CD4~+T Cells Via Balancing Oxidative Stress

Adaptive immunity plays a crucial role in the body’s resistance to infection and tumorigenesis,and immunological memory is a specialized feature of the adaptive immune response.Memory CD4⁺ and CD8⁺ T cells synergistically orchestrate a sophisticated T cell immunity,which are long-lived,self-renewal,and can rapidly differentiate into effector cells upon rechallenge.Over the past decades,researchers have comprehensively investigated the mechanisms of memory CD8⁺ T cell formation and maintenance.However,the limited characteristic surface markers of memory CD4⁺ T cells have impeded its research process.Therefore,exploring the mechanism of memory CD4⁺ T cell establishment and maintenance is an important scientific question to be investigated soundly.Immunological memory formation is accompanied with biological alterations including transcriptional modulation,epigenetic modification and metabolism remodeling.In this process,mammalian target of rapamycin（mTOR）protein regulates T cell immune function as a hub of intracellular metabolic pathways.mTOR is an evolutionarily conserved serine/threonine protein kinase,constituting two functionally different complexes,mTORC1 and mTORC2.It was unknown that mTORC2 regulates the formation and maintenance of memory CD4⁺ T cells,although previous studies have reported that it plays an important role in the early differentiation and functional maturation of TFH.To this end,we utilized a bone barrow chimera（BMC）infected with Lymphocytic choriomeningitis virus（LCMV）,and acutedly delete Rictor to disrupte mTORC2 signaling during the formation（Day 21）or long-term maintenance（Day 41）phase,and we found impaired mTORC2 signaling markedly reduced memory CD4⁺ T cells in either situation.Meawhile,the application of adoptive transferred model of SMARTA CD4⁺ T cells,which specifically recognize LCMV GP_66-77 antigenic epitopes,combined with mTOR inhibitors（Torin1 & Rapamycin）further validated that mTORC2 signaling is required for memory CD4⁺ T cell persistence.Intriguingly,mTORC2 ablation appeared not to influence the homeostatic proliferation of memory CD4⁺ T cells,but led to enhanced cell death,which are not related to apoptosis,necroptosis and pyroptosis.It turned out to be ferroptosis,which was further comfirmed by several means including lipid peroxidation accumulation（Bodipy C11）detected by flow cytometry and morphological characteristics of ferroptosis observed via transmission electron microscopy.In addition,the gene knockout and overexpression of GPX4,a key enzyme antagonizing ferroptosis,strongly demonstrated that mTORC2 deficiency induced ferroptosis in memory CD4⁺ T cells.To futher probe the molecular mechanisms of ferroptosis in memory CD4⁺ T cells,we took advantage of multiple biochemistry and immunological techniques and approaches.We confirmed impaired mTORC2 signaling inhibits the phosphorylation of downstream signals AKT and GSK3β through Western Blot and flow cytometry.Besides,we found that the mTORC2-p-AKT_Ser473-p-GSK3β_Ser9 cascade plays a dual role in preventing ferroptosis in virus-specific memory CD4⁺ T cells.First,the mTORC2-p-AKT_Ser473-p-GSK3β_Ser9 signaling likely maintains the interaction between HK2 and VDAC to balance mitochondrial ROS production,thereby preventing ferroptosis in memory CD4⁺ T cells.Moreover,the mTORC2-p-AKT_Ser473-p-GSK3β_Ser9 axis modulates nuclear accumulation of NRF2,a key regulator of oxidative stress,to ensure the expression of NRF2 downstream target genes（slc3a2,gclc,gclm）.Among them,SLC3A2 encoded by slc3a2,is a a subunit of the antitransporter xc-system,which exports glutamate in exchange for cystine,and the imported cystine is reduced to cysteine as an essential substrate of GSH synthesis in the cytosol.GSH is essential cofactor of GPX4,an enzyme antagonizing ferroptosis to exert anti-peroxidative lipase activity,which is catalyzed by GCLC and GCLM（encoded by Gclc and Gclm）.Meanwhile,it has been previously reported that pathogenic CD4⁺ T cells with memory cell characteristics were detected in multiple sclerosis（MS）patients.We therefore introduced an experimental autoimmune encephalomyelitis（EAE）model to investigate whether ferroptosis of pathogenic CD4⁺ T cells could be triggered by inhibiting mTORC2 signaling to alleviate clinical symptoms in the murine model of EAE.Consistent with previous results,we found that the mTORC2 signaling deficiency also endogenously induced ferroptosis of pathogenic CD4⁺ T cells,accompanied with alleviating clinical symptoms in the EAE model.In summary,our study reveals the molecular mechanism that mTORC2-p-AKT_Ser473-p-GSK3β_Ser9 signaling pathway antagonizes the ferroptosis in memory CD4⁺ T cells.What’s more,targeting ferroptosis to pathogenic CD4⁺ T cells can dramatically alleviate clinical symptoms of EAE models,providing a new therapeutic strategy for the treatment of autoimmune diseases.