Molecular Mechanism Responsible For Toll-like Receptor 8 Signaling-mediated Reversal Of T Cell Senescence Through Glucose Metabolism Induced By Tumors

Background:Reconstitution of the tumor microenvironment(TME)for enhanced tumor immunotherapy has been a hot research area for many years.The TME is the unique environment supporting a tumor,including the surrounding blood vessels,immune cells,fibroblasts,signaling molecules and the extracellular matrix(ECM).The tumor and the surrounding microenvironment are closely related and interact constantly.The TME determines tumor heterogeneity and plays a key role in controlling subsequent tumor growth and proliferation.Recent studies suggest that tumor cells and regulatory T cells(Treg)in the TME can induce T cell senescence in patients with different types of cancers and with different ages.T cell senescence acts as a dysfunctional status of T cells which is responsible for an immune escape and decreased efficiency of immunotherapy.The latest report has pointed out that enhanced efficiency of immunotherapy will depend on understanding of the molecular mechanisms and regulations of T cell senescence in the TME.It has been proved that Toll-like receptor 8(TLR8)signaling can reverse tumor-induced T cell senescence.However,the molecular mechanism responsible for the processes is unknown and needs to be further explored.TLRs have been recognized as critical components of the innate immune system,acting as a link between innate and adaptive immunity.Increasing evidence suggests that TLRs directly involve tumor pathogenesis,regulating both tumor cells and tumor-infiltrating DCs and T cells.However,very limited information is known about the molecular cross-talks between TLR signaling and tumor and/or immune cell metabolism although abundant TLRs express in these cells.INPP5B was recently identified as an anti-tumor gene regulated by TLR8 signaling by our group.However,it needs further exploration of whether this gene is involved in reversal of T cells senescence.Objective:The purpose of this research is to explore the molecular mechanism is responsible for TLR8 signaling-mediated reversal of reverse tumors induced T cell senescence.The study is further to dissect how TLR8 signaling regulates tumor glucose metabolism,resulting in reversal of tumor-induced T cell senescence.In addition,the current study is to investigate the causative relationship between TLR8 signaling,INPP5B signaling,and tumor metabolism regulation during T cell senescence mediated by tumor cells.Not only exploring the regulation of TLR8 signaling to an anti-tumor gene,especially,starting from the molecular cross-talks between TLR signaling and tumor metabolism,to confirm the TLR8 reverse the tumors induced T cells senescence.Improved understanding of the INPP5B and involved molecular processes should open new avenues for the development of novel strategies of reprogramming tumor metabolism and TLR-based cancer immunotherapy.Methods:1.Confirm whether TLR8 signaling can reverse tumors induced T cell senescence①Co-culture different human-derived tumor cell lines,including breast cancer:MCF-7,prostate cancer:PC-3,melanoma cancer:Me1586 and A375,with human CD4+/CD8+T cells(T cells were purified derive from different human peripheral blood with immunomagnetic bead positive selections).To determine the status of T cell senescence,the co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with staining and flow cytometry.②Different human tumor cells pretreated Poly-G3(TLR8 ligand)with different human derived tumor cells to activate TLR8 signaling,were co-cultured with human CD4+/CD8+T cells.The co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with flow cytometry.2.TLR8 signaling inhibits tumor metabolism to reverse tumors induced T cell senescence①Human derived tumor cells(MCF-7,PC-3,Me1586)were treated with Poly-G3(TLR8 ligand)to activate TLR8 signaling.Poly-T3 was used as a control group.Glucose uptake and glycolysis associated enzyme gene expressions were determined using 2-NBDG labeling assay and qPCR,respectively.②Glutl/Glut4 genes were transfect into tumors to improve glycolysis level in tumors,and then Poly-G3 was added into tumor culture medium,Glucose uptake and glycolysis associated enzyme gene expressions were determined using 2-NBDG labeling assay and qPCR,respectively.③Tumor cells that transfected with Glutl/Glut4 and then pretreated with Poly-G3,were co-cultured with human CD4+/CD8+T cells.To determine the status of T cell senescence,the co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with flow cytometry.3.mTOR-HIF-la signaling involves TLR8 signaling-mediated reversal of T cell senescence induced by tumor cells①Human tumor cells(MCF-7,PC-3,Me1586)were pretreated with Poly-G3(TLR8 ligand)to activate TLR8 signaling.Poly-T3 works as a control.Phosphorylation and activation of mTOR and down-stream molecules in mTOR signaling pathway were determined using western blot analysis.Gene and protein expression of HIF-1α was determined using both qPCR and flow cytometry,respectively.②Human tumor cells were transfected with plasmid-carrying Akt gene and infected retro-virus carrying RHEB gene to up-regulate mTOR signaling pathway.Glucose uptake and glycolysis associated enzyme gene expressions were determined using 2-NBDG labeling assay and qPCR,respectively.③Tumor cells that transfected with Akt and RHEB genes,and pretreated with Poly-G3,were co-cultured with human CD4+/CD8+T cells.To determine the status of T cell senescence,the co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with flow cytometry.It determined whether mTOR signaling pathway regulation is involved in TLR8 signaling-mediated reversal of tumors induced T cell senescence.④Human tumor cells were cultured in the presence of Hif-1α agonist DMOG and Poly-G3.Glucose uptake and glycolysis associated enzyme gene expressions were determined using 2-NBDG labeling assay and qPCR,respectively.It determined whether Hif-1α regulation is involved in TLR8 signaling-mediated regulation of glucose metabolism in tumor cells.⑤Tumor cells pretreated with by DMOG and Poly-G3 were co-cultured with human CD4+/CD8+T cells.The co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with flow cytometry.It determined whether HIF-1α regulation is involved in TLR8 signaling-mediated reversal of tumors induced T cell senescence.4.INPP5B is involved in TLR8 signaling-mediated reversal of tumor-induced T cell senescence①Human tumor cells(MCF-7,PC-3,Me1586)were treated with Poly-G3(TLR8 ligand)to activate TLR8 signaling or with Poly-T3 as a control.Gene and protein expression of INPP5B in tumor cells were determined using western blot and qPCR,respectively.②Tumor cells were transfected shRNA that carrying GFP to knock down TLR8 and its downstream molecules IRAK-4,MyD88.The transfected tumor cells were sorted based on GFP expression with Flow cytometry,and then treated with Poly-G3 into tumor culture medium to activate TLR8 signaling.INPP5B gene expression was determined using qPCR.③Tumor cells were transfected with the plasmid carrying INPP5B gene,and then co-cultured with human CD4+/CD8+T cells.Meanwhile,tumor cells were knocked down INPP5B expression with shRNA,and then treated with Poly-G3,and co-cultured with human CD4+/CD8+T cells.The co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with flow cytometry.It determined whether INPP5B regulation is involved in TLR8 signaling-mediated reversal of tumors induced T cell senescence.5.INPP5B regulates glycolysis in tumor cells.①Tumor cells were transfected with plasmid pcDNA3.1-INPP5B to over-express INPP5B expression.pcDNA3.1 plasmid transfection served as a control.Gene expression of key enzymes associated with glycolysis and Hif-1α was determined using qPCR.Activation and expression of key molecules in mTOR signaling pathway was determined using to Flow cytometry.②Tumor cells were knocked down INPP5B expression with shRNAs,and then cultured with or without Poly-G3.pLOKO.1 set as a control.Gene expression of key enzymes associated with glycolysis and Hif-1α was determined using qPCR.Activation and expression of key molecules in mTOR signaling pathway was determined using Flow cytometry.③Tumor cells pretreated with Rapamycin were treated with Poly-G3.INPP5B gene expression was determined with qPCR.6.INPP5B is methylated in tumor cells①INPP5B methylation status in MCF-7,PC-3,Me1586 tumor cells was evaluated using real-time PCR.Human non-tumorigenic epithelial cell MCF-10A and skin fibroblast cell HFF were included as control.②Human tumor cells(MCF-7,PC-3,Me1586)pretreated with methylation inhibitor 5-aza(5-Aza-2’-deoxycytidine),and then treated with Poly-G3.INPP5B protein expression was determined using western blot,and gene expression of key enzymes associated with glycolysis was determined using real-time qPCR.Furthermore,survival rates of tumor cells were evaluated using trypan blue staining.In addition,tumor growth,proliferation and adhesion abilities were determined using tumor growth curve,MTT assay,and adhesion assay,respectively.③Human tumor cells(MCF-7,PC-3,and Me1586)pretreated with Poly-G3,were co-cultured with human CD4+/CD8+T cells.The co-cultured T cells were isolated,stained with SA-β-gal and analyzed the expression of co-stimulatory molecule CD28 with flow cytometry.,7.TLR8 signaling can reverse T cell senescence and enhance anti-tumor immunity in vivo in tumor models①Human derived melanoma A375 tumor cells were injected into immunodeficient mice.Poly-G3 and 5-aza were,intratumorally injected Poly-G3 and intraperitoneally injected 5-aza into tumor-bearing mice,respectively.Tumor growth and progression were evaluated every 3 days.②Human melanoma cell A375 that transfected with mouse MHC-class Ⅰ H2-Db gene and tumor antigen gp100 gene were injected into mice.mice.gp100 specific Pmel-T cells were injected into tumor-bearing mice through tail intravenous injection.In addition,intratumorally injected with Poly-G3 and intraperitoneally injected with 5-aza for different times.Tumor growth and mouse survival were evaluated every 3 days.At the end of time points,mice were sacrificed,and T cells were purified from blood,spleens,and tumor tissues.The purified T cells were further stained for SA-β-gal and analyzed functional markers such as IFN-γ and perforin production,using Flow cytometry.Results:1.TLR8 signaling reverses tumors induced T cells senescence Different types of human tumors induce both CD4+/CD8+T cells senescence,showing that SA-β-gal positive cells population is increased,and co-stimulatory molecule CD28 expression is decreased.Poly-G3 treatment with tumors to activate TLR8 signaling pathway can reverse both CD4+/CD8+T cells senescence induced by tumor cells,evidenced by decreases in SA-β-gal positive CD4+/CD8+T cells populations and increases in CD28 expression.However,Poly-T3 treatment has no effect on reversal of T cell senescence.2.TLR8 signaling reverses tumors-induced T cells senescence through glycolysis inhibition of tumor cells Poly-G3 can down-regulate glucose uptake of tumors and suppress key genes expression of key enzymes associated with glycolysis in tumor cells.Glut1/Glut4 transfection into tumors can increase the glycolysis level of tumors.Meanwhile,tumors that have higher level of glycolysis can induce more senescent T cells,SA-β-gal positive T cells population is more than other groups,and CD28 expression is lowest.Furthermore,Glut1/Glut4 transfection can prevent Poly-G3-induced suppression of glucose uptake and genes expression of enzymes associated with glycolysis in tumor cells.In addition,Glut1/Glut4 transfection into tumors plus Poly-G3 treatment increased SA-β-gal positive CD4+/CD8+T cells populations,and decreased CD28 expression compared with Poly-G3 treatment only group.3.TLR8 signaling reverses tumors-induced T cells senescence through regulating mTOR-HIF-la signaling pathway Poly-G3 treatment down-regulated activation of mTOR signaling pathway,including the phosphorylation of mTOR,p70S6K,4E-BP1,Akt and the expression of Hif-1α in different types of human tumor cells.Over-expression of Akt and RHEB genes in tumors significantly increased phosphorylation and activation of mTOR,p70S6K,4E-BP1,and Akt.Meanwhile,tumors have stronger mTOR signaling pathway can induce more senescent T cells,showing more SA-β-gal positive T cell populations and lower CD28 expression.Furthermore,over-expression of Akt and RHEB genes in tumors prevented Poly-G3-induced suppression of glucose uptake and gene expression of key enzymes associated with glycolysis in tumor cells.In addition,over-expression of Akt and RHEB genes in tumors can alleviate Poly-G3-induced reversal of T cell senescence mediated by tumor cells,showing more SA-β-gal+CD4+/CD8+T cell populations and decreased CD28 expression compared with Poly-G3 treatment only group.On the other hand,DMOG treatment can increase Hif-1α expression in tumors.Meanwhile,tumor cells with higher Hif-1α expression can induce more senescent T cells than tumor cells without DMOG treatment,evidencing by more SA-β-gal+T cell populations and lower CD28 expression.Furthermore,DMOG treatment with tumors alleviated Poly-G3-indued inhibition of glucose metabolism with increases in glucose uptake and gene expression of enzymes associated with glycolysis compared with Poly-G3 treatment only group.In addition,DMOG treatment can block Poly-G3-induced reversal of T cell senescence mediated by tumor cells,showing more SA-β-gal+CD4+/CD8+T cell populations and decreased CD28 expression compared with Poly-G3 treatment only group.4.TLR8 signaling reverses tumors-induced T cells senescence through regulating INPP5B TLR8 signaling activation can increase INPP5B expression in MCF-7,PC-3 and Me1586 tumor cells.Furthermore,knockdown of gene expression of TLR8,IRKA-4 and MyD88 with shRNAs in tumor cells prevented Poly-G3-indued increase in INPP5B expression in tumors.In addition,knockdown of gene expression of TLR8,IRKA-4 and MyD88 with shRNAs in tumor cells prevented Poly-G3-indued reversal of T cell senescence mediated by tumor cells,showing more SA-β-gal+CD4+/CD8+T cell populations and decreased CD28 expression compared with Poly-G3 treatment only group.5.INPP5B and mTOR-Hif-1α signaling pathway cooperatively regulate tumor cell metabolism mediated by TLR8 signaling Over-expression of INPP5B can decrease expression of glycolysis related genes and Hif-1α.Knock-down of INPP5B expression in tumors not only can increase glycolysis related gene expression,but also can block Poly-G3-induced downregulation of mTOR signaling and Hif-1α expression.However,rapamycin treatment in tumors have no effect on Poly-G3-mediated increase in INPP5B expression.6.TLR8 signaling activation combined with INPP5B demethylation markedly increases INPP5B expression in tumors Compared with human epithelial cell MCF-10A and skin fibroblast HFF cells,methylation level of INPP5B is increased in MCF-7,PC-3,Me1586 tumor cells.Treatment with methylation inhibitor 5-aza can increase INPP5B expression in tumors,while co-treatments of Poly-G3 and 5-aza can result in higher INPP5B expression.Meanwhile,co-treatments with Poly-G3 and 5-aza result in stronger suppression of gene expression of key enzymes related with glycolysis in tumor cells than Poly-G3 or 5-aza only treatment group.In vitro culture system,Poly-G treatment has no obvious effect on tumor growth,proliferation and adhesion abilities;while 5-aza treatment slightly decreases tumor growth,proliferation and adhesion abilities.However,combined treatments of tumor cells with Poly-G3 and 5-aza markedly decreased tumor growth,proliferation,and adhesion abilities compared with single treatment groups.7.TLR8 signaling activation combined with INPP5B demethylation can significantly enhance anti-tumor immunity and decrease tumor growth in vivo Tumor can grow progressively in vivo.Poly-G3 treatment did not significantly affect tumor growth and progression;while 5-aza treatment slightly decreased tumor growth in vivo.However,combined treatments with Poly-G3 and 5-aza can markedly decrease tumor growth and progression in vivo and show low tumor weights.From adoptively transferred T cell immunotherapy mouse models,T cell adoptive transfer combined with single treatment of Poly-G3 or 5-aza can suppress tumor development and growth and decrease tumor weights.T cell adoptive transfer combined with co-treatments of Poly-G3 and 5-aza has significant effect on anti-tumor efficacy,showing more inhibition on tumor growth and longer survival than those of other double combination groups.In addition,in the triple combination group,tumor-specific T cells purified from blood,spleens,tumors of tumor-bearing mice had lowest SA-β-gal+T cells populations,and highest production of IFN-y,perforin,and granzyme B in T cells compared with any other groups.Conclusion:In the current study,we identified that that TLR8 signaling pathway can reverse both CD4+and CD8+T cell senescence induced by different types of human tumor cells.Molecularly,TLR8 signaling pathway can increase INPP5B expression that inhibits glucose uptake and glycolysis in tumors through further regulation of mTOR-Hif-1α signaling pathway,resulting in reversal of tumor-induced T cell senescence.Meanwhile,combined treatments with TLR8 signaling activation and INPP5B demethylation can markedly decrease tumor growth and development,prevent senescence in tumor-specific T cells induced by tumors,resulting in enhanced T cell functions and anti-tumor immunity.