MiRNA Regulates T Cell Exhaustion In Antitumor Immunotherapy And Transplantation Tolerance

Chapter 1 IntroductionExhausted T cells were initially observed in chronic LCMV infection in mice and characterized by sustained expression of inhibitory receptors(IRs),such as PD-1,TIM-3,BTLA,CTLA-4,2B4 and TIGIT.T cell exhaustion describes a state of late-stage differentiation usually associated with active prevention of functionality via ligation of negative signaling receptors on the cell surface,and which can be reversed by blocking these interactions.T cell exhaustion is a mechanism of immune tolerance,although the mechanisms are poorly understood,it has been claimed that its induction can promote self-tolerance and transplant tolerance.However,the impact of T cell exhaustion on both graft rejection and transplant tolerance remains to be evaluated.In our research,a novel therapeutic approach using miRNA to regulate inhibitory receptors both in enhancing anti-tumor immune response and prolong allograft survival after transplantation in vivo.Chapter 2 miR-149-3p reverses CD8 T cell exhaustion by repressing inhibitory receptors and promoting cytokines secretion in breast cancerObjective: To investigate whether miRNA can improve anti-tumor immune response by regulating immune checkpoints on CD8+ T cells in breast cancer.Methods: miRNA array was used to screen downregulated miRNAs in PD-1 overexpressed CD8+ T cells from 4T1 breast cancer mouse model.RT-q PCR and Flow cytometry were performed after miRNA transfection in vitro.Results: In this study,our results reveal that miR-149-3p had potential to bind to 3'UTR of some immune inhibitory receptors and forkhead box P1(Foxp1)gene.Apoptosis and exhausted T cells phenotype and gene transcriptional level of PD-1,TIM-3,BTLA and Foxp1 were downregulated with miR-149-3p mimic treatment.While the secretion of effector cytokines IL-2,TNF-?,IFN-? and proliferation were upregulated under the same condition.Moreover,cytotoxicity of CD8+ T cells targeting 4T1 breast tumor cells was promoted with miR149-3p mimic treatment.When the cells were treated with miR149 inhibitor,we acquired opposite results.Conclusions: Collectively,our results show that miR-149-3p can reverse CD8 T cell exhaustion and have potential in antitumor immunotherapy in breast cancer.Chapter 3 micro RNA-5119 regulates ligands of IRs in dendritic cells for enhancing cancer immunotherapyObjective: To demonstrate whether the micro RNA-5119 transfected DC vaccine can improve the anti-tumor immunity by reversing T cell exhuastion in breast cancer.Methods: We evaluate the expressions of ligands of IRs and miR-5119 in DCs from 4T1 breast cancer tumor-bearing mice,and examine the molecular targets of miR-5119.miR5119 mimic or inhibitor transfecting DC vaccines were achieve to determine the recovery of exhausted CD8+ T cells and the efficiencies of anti-breast cancers in vitro and in vivo.Results: We firstly found that miRNA-5119 was significantly downexpressed in splenic DCs from breast cancer-bearing mice.The in silico and q-PCR results showed miRNA-5119 targeted such negative immune regulatory molecules as PD-L1 and IDO2,while those ligands of IRs as PD-L1,Galectin-9 and HVEM in splenic DCs were highly overexpressed in breast cancer-bearing mice to induce T cell exhaustion.Secondly,the miRNA-5119 to engineer DC vaccines for treating breast cancer-bearing mice can effectively restore the functions of exhausted CD8+ T cells in vitro and in vivo,resulting in much higher anti-breast cancer efficiencies.In addition,the miRNA-5119 engineering DC vaccines inhibited the generation of Treg cells in vivo as well due to targeting the Fox P-3 molecule.Conclusions: This study evidences miRNA-5119 engineering DC vaccines as an effectively novel strategy to enhance the efficacy of DC-based breast cancer immunotherapeutic.Chapter 4 Prolonging allograft survival in heart transplantation using miR-5119 repressed DCsObjective: To demonstrate whether the miR-5119 inhibitor transfected DC will prolong heart transplantation allograft survival and improve T cell exhaustion.Methods: We evaluated the expressions of iR ligands in DCs from mice that tolerated allogeneic heart transplants for long periods(>100 days post-surgery).A miRNA microarray was used to screen up/downregulated miRNAs in PD-L1+ tolerogenic DCs from the allograft-tolerant mice.Mimics or inhibitors of miR-5119 were transfected into DCs for in vitro and in vivo experiments,followed by treatment of allogeneic(C57BL/6 to BALB/c)heart transplantation.Results: We found that miRNA-5119 was significantly downregulated in rapamycin-treated mice tolerant to heart transplantation,by miRNA array analysis and RT-q PCR validation.In silico analysis indicated miR-5119 targets immune regulatory molecules,such as PD-L1 and IDO2.Treatment of heart graft recipients with miRNA-5119 repressed DCs significantly prolonged allograft survival which is associated with an upregulation of PD-L1 and IDO2 expression,promotion of T cell exhaustion and the generation of Treg cells.Conclusions: micro RNAs such as miR-5119 can target multiple immune molecules in DCs and generate immune tolerance,thus prevent allograft rejection after heart transplantation.