Study On The Mechanism Of PD-1 Upregulation In Tumor Tissue Infiltration-specific CD8+ T Cells

Objective:The tumor treatment of immune checkpoint blockers represented by PD-1 has achieved unprecedented success.Compared with traditional chemo-radiotherapy,immune checkpoint treatment has greatly improved both in response rate and treatment effect.However,the mechanism of up-regulation of PD-1 expression in tumor infiltrating specific cytotoxic CD8+T cells has not been well understood.At the same time,antibody therapy against PD-1 often needs to face the problems of tolerance and tumor recurrence,which highlights the importance of further explaining the mechanism of PD-1 up-regulation.On the other hand,more and more evidence shows that tumor regenerative cells play an important role in mediating tumor immune escape,but the specific mechanism needs to be further demonstrated.At the same time,the research on the mechanism of PD-1 up-regulation in the academic circles today often focuses on T-cell receptor(TCR)-mediated signal pathways.Specifically,the expression of PD-1 is affected by several TCR-related transcription factors,such as Nfatc1,Notch,and STATs.However,in the tumor microenvironment,T cells are also affected by cytokines and metabolites secreted by tumor cells.These signaling molecules may bypass TCR and directly regulate PD-1 of T cells.This paper intends to use the homemade method of culturing Tumor-Repopulating Cells(TRCs)to analyze the mechanism by which TRCs induce CD8+T cells to up-regulate PD-1 through signal molecules secreted by themselves,and to further improve the CD8+T cell-based strategies for tumor immunotherapy.Methods:(1)In order to study the interaction between TRCs and specific T cells,we first cultured B16 and B 16-OVA TRCs derived from salmon fibrin gel with activated Pmel and OT-1 CD8+T cells.Analyzing function of CD8+T cells by inhibitory marker PD-1 and effector molecules including IFN? TNF-? and CD 107a as well as apoptosis of tumor cells with 7-AAD and annexin-V.(2)In order to further study the mechanism by which TRCs induced T cells to up-regulate PD-1 and down-regulate effector molecules,we used the supernatant after co-culture,to culture new CD8+T cells,and did further tests:1.Various cytokines(IFNy,TNF-?,IL2,IL10)in the co-culture supernatant;2.Expression of PD-1,IFN ?,TNF-?,and CD107a in CD8+T cells;3.in order to exclude the antigen-presentation pathway,we also analyzed the expression of H-2kb-ova(3)In order to explore the role played by various cytokines,we added neutralizing antibodies of IFN? or TNF-? to the co-culture system and analyzed PD-1 expression in T cells,meanwhile we also treated the TRCs and bulk tumor cells with IFN ? and furtherly used such treated supernatant to culture CD8+T cells and analyzed markers that related to the function state.(4)In order to investigate the cause of PD-1 up-regulation by IFN?,we measured the amount of Kyn producted after IFN? treatment in tumor cells,and furtherly treated T cells with Kyn and detected the effect of Kyn by analyzing PD-1 expression in T cells.In order to further confirm this conclusion,we used Trp-free medium for co-culture experiments,and analyzed the apoptosis of tumor cells and changes in various indicators of T cells(5)In order to further explore the cause of Kyn increasing,we analyzed the expression of the enzymes that catalyzed the reaction of Kyn conversion and transporters that transported Trp.(6)In order to confirm the function of IDO and SLC1A5 in TRCs in interacting with CD8+T cells.We added IDO inhibitor 1-MT or SLC1A5 inhibitor GPNA in the co-culture system and analyzed the apoptois of TRCs and T cell function.(7)In order to further confirm the function of IDO and SLC1A5,we constructed IDO and SLC1A5 knockdown cells.The knockdown efficiency was confirmed by PCR,WB and metabolite detection,and knockdown cell lines were co-cultured with T cells to analyze tumor cell apoptosis and T cell function.(8)In order to investigate the mechanism of Kyn causing PD-1 up-regulation in T cells,we further analyzed the role of its target AhR,including the expression of AhR in activated T cells,and the activation of AhR after Kyn treatment,and the role of AhR in PD-1 up-regulation.We also co-cultured AhR knockout OT-I T cells with tumor cells,and continued to analyze the apoptosis of tumor cells and the functional status of T cells.(9)In order to further confirm the direct regulation relation between AhR and PD-1,we constructed PD-1 promoter and detected its activation by Kyn with fluorescence intensity.Meanwhile we also treated T cells with another AhR agonist TCDD and analyzed PD-1 expression.(10)In order to further analyze the details of Kyn acting on T cells,we analyzed the expression of Kyn transporters.(11)In order to further determine the role of the Kyn transporters in T cell function,we treated the T cells with inhibitors of PAT4 and SLC7A8 and also constructed PAT4 and SLC7A8 knockdown T cells,and identified the knockdown efficiency by RT-PCR.At the same time,the inhibitory effect of the transporter was analyzed by detecting the amount of Kyn taken up by the cells and the changes in AhR nuclear translocation.The successfully knocked down T cells were treated with Kyn or co-cultured with tumor cells to analyze tumor apoptosis as well as T cell function.(12)In order to further determine the regulatory relationship of the transporters,we used ChIP-PCR to analyze the binding of AhR to PAT4 and SLC7A8 promoters after Kyn treatment,and also treated T cells with AhR inhibitors to analyze the expression of the transporter,and also constructed promoter reporting system of PAT4 and SLC7A8 to analyze the regulation of these two genes by AhR.(13)In order to distinguish the relationship between PD-1 up-regulation by TCR signals and the Kyn-AhR signaling,we treated naive T cells with Kyn or pre-activated T cells with CD3/28 antibodies,and treated with Kyn,and detected PD-1 expression in T cells.(14)In order to further analyze the TCR signal and the effect of Kyn-AhR on PD-1,we used the inhibitor Nfatc1 which was the core transcription factor of TCR signal to analyze the relationship between its activation and the two transporters PAT4 and SLC7A8,and also analyzed Nfatc1 binding to PAT4 and SLC7A8 promoter via ChIP-PCR.(15)In order to further verify the results of our in vitro experiments,we intended to conduct in vivo experiments,including:1.Injected Kyn into normal mice and AhR knockout mice,and analyze PD-1 expression in T cells.We also injected the same mice with CD3 antibody and analyzed the expression of PD-1 in different mice during activation;2.Constructed tumor bearing mice,and we conducted these experiments:1.Injected Kyn intratumorally and analyzed markers related to T Cell function;2.Using AhR inhibitor and transferring tumor specific T cells and analyzed T cell function,tumor volume and survival of the mice;3.Transferring PAT4 and SLC7A8 knock-down OT-1T cells and analyzed the in-vivo T cell function,tumor volume and survival of the mice.(16)Then we further analyzed whether the conclusions we obtained in animal models could also apply in human T cells.We performed the following experiments:1.Collecting peripheral blood and tumors from healthy people and patients with various types of tumors,and isolating the T cells and furtherly analyzed PD-1 expression.We also collected serum as well as tumor peripheral tissues and tumor tissue lysate,and analyzed kyn concentration.2.We Collected CD8+T cells from peripheral blood of tumor patients and activated them with CD3/CD28 then we added Kyn or AhR inhibitors to detect the expression of PD-1 and related transporters in T cells.We also Collected T-cells with high expression of PD-1 and low expression of PD-1 in the peripheral blood of patients and analyze the expression of the transporter and the effect of AhR on the transporter by ChIP-PCR and RT-PCR.Results:(1)1.When TRCs were co-cultured with specificCD8+T cells,they exhibited lower apoptosis;2.CD8+T cells,when co-cultured with TRCs,showed higher expression of PD-1,while T cell effector molecules including IFN-y,TNF-? and degranulating pointer CD107a all showed reduced expression.(2)1.There was no significant difference in the expression of H-2Kb-OVA between TRCs and bulk tumor cells;2.IFN?,TNF-?,IL2,and IL10 could be detected in the co-culture supernatant,among them,TNF-?,IFN-? secreted the most;3.Culturing the CD8+T cells with supernatant from TRCs induced higher expression of PD-1 and lower expression of TNF-?,IFN-? and CD107a.(3)1.Neutralization of IFNy could lead to a decrease in PD-1 expression in CD8+T cells in the co-culture system,while neutralization TNF-? did not show same effect;2.Supernatant from IFNy treatment to TRCs could lead to higher expression of PD-1 than from bulk tumor cells.(4)1.TRCs treated with IFN-y could produce more Kyn than bulk tumor cells;2.CD8+T cells treated with Kyn could lead to PD-1 up-regulation;3.When Trp was removed from the medium,the apoptosis of TRCs in the co-culture system was greatly improved,and the expression of PD-1 in CD8+ T cells was down-regulated,and related effector molecules,IFNy,TNF-? and CD107a were up-regulated.(5)1.The enzyme IDO that catalyzed Kyn and the transporter SLC1A5 were higher expressed in TRCs;2.After treated with IFNy,the expressions of IDO and SLC1A5 were both higher induced in TRCs than bulk cells;3.Other enzymes or transporters that may be involved showed no significant changes.(6)Adding IDO inhibitor 1-MT or SLC1A5 inhibitor GPNA could increase the killing of TRCs by CD8+T cells,reduce PD-1 expression and enhance the secretion of related effector molecules.(7)After confirming the knockdown effect of IDO and SLC1A5,we further confirmed the previous inhibitors' conclusion.(8)1.With the activation of T cells,the protein level of AhR was significantly up-regulated;2.Adding Kyn induced further up-regulation of AhR as well as enhanced AhR Nuclear translocation;3.In T cells treated with AhR inhibitor DMF and Kyn,PD-1 up-regulation could be suppressed;4.Co-culture of tumor cells with OT-1 T cells knocked out AhR showed increased tumor cells apoptosis,while PD-1 expression of T cells was down-regulated,and the expression of effector molecules increased.(9)1.Kyn could enhance the luciferase signal of PD-1 promoter;2.TCDD could increase the expression of PD-1 in T cells.(10)We found that Kyn treatment could induce the expression of Kyn transporters PAT4 and SLC7A8 in T cells.(11)1.When using inhibitors or knocking down PAT4 or SLC7A8,Kyn was blocked from entering T cells,and nuclear translocation of AhR was also inhibited;2.Inhibitors or PAT4 or SLC7A8 knocked-down T cells cultured with tumor cells increased the apoptosis of tumor cells,as well as down-regulated PD-1 expression and increased expression of effector molecules.(12)1.When Kyn and AhR inhibitors were added,the induction effect of Kyn on PAT4 and SLC7A8 was inhibited;2.ChIP-PCR showed that Kyn could increase the binding of AhR to PAT4 and SLC7A8;3.Kyn-AhR promoted stronger luciferase signal of PAT4 and SLC7A8 promoters.(13)1.Kyn could induce naive T cells to up-regulate PD-1 expression;2.Kyn could induce pre-activated T cells to express higher PD-1.(14)1.Inhibited Nfatc1 activity could induce decreased expression of PAT4 and SLC7A8;2.ChIP-PCR showed Nfatcl binding to the promoters of PAT4 and SLC7A8 and the activity became stronger with T cell activation.(15)1.Injecting Kyn into wild-type mice can cause T cells to up-regulate PD-1 expression,while injecting into AhR knockout mice showed no such response;2.Compared to AhR knockout mice,wild-type mice showed higher PD-1 expression after T cell activation in vivo;3.Injecting Kyn into tumors of tumor-bearing mice,PD-1 expression of infiltrating T cells was also Up-regulated;4.Adoptive transfer of PAT4 or SLC7A8 knock-down T cells in tumor-bearing mice,tumors were smaller than transferring normal T-cells,and mice have a longer life span;5.Intratumoral injection of tumor-bearing mice with DMF,the intratumoral infiltrating T cells showed decreased PD-1 and increased effector molecules;6.the combination of specific T cell adoptive and AhR inhibitor treatment could increase the T cell tumor killing effect,extended mouse survival.(16)1.Tumor infiltrated T cells showed higher PD-1 expression than in peripheral blood in tumor patients;2.Kyn concentrations in the serum of tumor patients were higher than in healthy peopleThe concentration of Kyn in the tumor was higher than that in the healthy tissue around the tumor,and for breast cancer,the concentration of Kyn in the peripheral blood is directly proportional to the expression of PD-1 in T cells,but for healthy people,there is no such correlation;3.Treating human T cells with Kyn could up-regulate PD-1 expression and abrogated while inhibiting AhR.Similarly,PAT4 and SLC7A8 are also regulated by Kyn and AhR in human T cells;4.PD-1 high-expressing T cells had higher AhR?PAT4 and SLC7A8 expression.On the other hand,in PD-1 high-expressing T cells,AhR binded stronger to PAT4,and SLC7A8 promoter.Conclusion:Through the above experiments,we found that when specific T cells kill tumor cells,a group of stem-cell like tumor cells are difficult to kill.This group of TRCs activated SLC1A5 and IDO to greatly activate their own Trp metabolic pathways and generate a large amount of Kyn into the microenvironment.Exogenous Kyn enters T cells through Kyn transporters PAT4 and SLC7A8 on the surface of T cells,and activates the transcription factors AhR,AhR,which then binds to the PD-1 promoter,up-regulates PD-1 expression,and also down-regulates the relevant effector molecules like IFN?,TNF-?and GranzymeB.By intervening the above metabolite-based metabolic pathways,we can increase the function of T cells and achieve better tumor control.This study revealed the direct connection between IDO and T cell function inhibition,and provided a theoretical basis for proposing new tumor immunotherapy strategies.