Immunological Landscape And Immunostimulatory Gene Therapy For H3.3-G34R High Grade Gliomas

Objective Pediatric high grade glioma(PHGG)represents the most aggressive form of glioma and is a major threat to those patients.Among them,H3.3-G34 R mutation,formed by the substitution of arginine(R)from the 34 th glycine of H3 histone family member 3A(H3 histone family member 3A,H3F3 a or H3.3),is an important molecular subtype of PHGG.H3.3-G34 mutant PHGG has obvious clinical manifestations and molecular characteristics,but there is still a lack of research on its biological characteristics.The purpose of this study was to explore the biological characteristics of H3.3-G34 mutant PHGG and the immune characteristics of the tumor microenvironment(TME),and to evaluate the efficacy of immunostimulation therapy on H3.3-G34 R mutant PHGG.Methods The primary animal model of H3.3-G34 mutant PHGG was constructed using the transposable enzyme system of Sleeping Beauty(SB).The tumor characteristics of the primary animal model were identified by hematoxylin-eosin staining and immunohistochemistry,and the biological characteristics of H3.3-G34 mutant PHGG were identified by survival experiment.After the glioma cells in the TME of the primary animal model were isolated by magnetic beads,the transcriptional,epigenetic and chromatin status characteristics of immune-related genes in H3.3-G34 mutant PHGG cells were analyzed by RNA extraction-sequence analysis and chromatin immunoprecipitation-sequencing methods.The response of H3.3-G34 R glioma cells to IFNγ stimulation was assessed by IFNγ sensitivity assay and apoptosis induction assay in vitro.After the immune cells in the TME of mouse model were isolated by magnetic beads,the effects of H3.3-G34 mutation on the transcription level of immune-related genes in the immune cells were analyzed by RNA extraction-sequence analysis.H3.3-G34 mutant p HGG primary tumor cell lines or neurospheres were isolated from primitive animal model and cultured for the generations,and used in striatum stereotactic cultivation method to construct H3.3-G34 mutant p HGG mouse model.Flow cytometry and T cell proliferation in vitro experimental were used to detect the influence of H3.3-G34 R mutations on immune cells,immune suppression,immune activity and the secretion of cytokines in the TME;H3.3-G34 mutant PHGG mice were treated with immunostimulant-gene therapy based on TK/ Fl T3 L.The efficacy of the therapy was evaluated by survival test and Rechallenge test,and the safety of the therapy was evaluated by tissue sections and hematoxylin-eosin staining.Finally,flow cytometry was used to evaluate the immunopositivity of the therapy against H3.3-G34 mutant PHGG.Results In this study,a mouse glioma model with typical characteristics of H3.3-G34 R mutant PHGG was successfully constructed by SB transposable enzyme system.Based on the model analysis,it was found that the presence of H3.3-G34 R mutation can change the immune-related molecular characteristics of glioma cells,and promote the up-regulation of the expression of immune-related genes at the transcriptional level through epigenetic regulation.H3.3-G34 R mutation has important effects on a variety of signaling pathways,especially on the immune-response process and IFNγ signaling pathways.In vitro,H3.3-G34 R glioma cells were more sensitive to IFNγ stimulation than H3.3-WT glioma cells,and were more prone to apoptosis induced by IFNγ.H3.3-G34 R mutation can also affect a series of cytokines secreted by glioma cells.These differentially secreted cytokines can promote the proliferation of effector T cells,reduce the generation of myeloid suppressor cells(MDSC)with immunosuppressive function or infiltration to tumor sites,and ultimately reduce the immunosuppression of TME.Immunostimulant-gene therapy based on TK/ Fl T3 L can effectively prolong the survival time of H3.3-G34 R mutant PHGG mouse model,and effectively improve the anti-tumor response and anti-tumor immune memory formation in H3.3-G34 R mutant PHGG mouse model,without causing damage to other organs and tissues.Conclusions In this study,we developed an animal model with characteristics typical of human H3.3-G34R＿PHGG,which enables us to understand how H3.3-G34 R mutations affect the molecular characteristics and biological behavior of glioma cells at the epigenetic and transcriptome levels.We observed that the presence of H3.3-G34 R would affect the interaction between PHGG and the immune system,making H3.3-G34R＿TME have weaker immune suppression and stronger immune activity than H3.3-WT＿TME.The immune characteristics of H3.3-G34R＿TME provide a good biological basis for the application of TK/ FLT3L-GT therapy.TK/ Fl T3L-GT therapy increased the survival of H3.3-G34 R tumor-bearing animals and promoted the development of tumor-specific immune response.We believe that the preclinical data in this study can serve as evidence to support TK/ FLT3L-GT as a possible immunotherapy for patients with H3.3-G34R＿PHGG.