Orphan Nuclear Receptor TLX Promotes Immunosuppression Via Its Transcriptional Activation Of PD-L1 In Glioma

High-grade gliomas are rapidly progressing tumors of the central nervous system(CNS)with a very poor prognosis and novel treatment are urgently needed to improve patient outcomes.Clinical trials of immunotherapy for glioma including PD-1/PD-L1 inhibition have performed but the outcome was not optimistic.In the present study,we found that orphan nuclear receptor TLX and PD-L1 were significantly upregulated in glioma lesions and the expression of TLX and PD-L1 in glioma was positively correlated.Moreover,the expression pattern of TLX was also associated with a suppression microenvironment in glioma.Functional study revealed that suppression of TLX inhibited in vivo growth of glioma allografts and rescued antitumoral immune response,which accompanied with the decrease of PD-L1+cancer cell population and M2 population and increase of TILs.We also characterized that TLX could bind directly to CD274 gene promoter and activate CD274 transcription.Together,our present study shows,for the first time,that TLX contributes to glioma malignancy and immune escape via promoting the expression of PD-L1,targeting the druggable TLX may have a potential therapeutic significance in glioma immune therapy.Objective1.To verify that the expression of TLX is associated with the heterogeneity of immunoenvironment in glioma.2.To clarify wheather TLX can mediate the immune escape of glioma by regulating the expression of PD-L1.3.To explore the mechanism of TLX regulating PD-L1.Methods1.To study the relationship between TLX expression and immune environment heterogeneity in gliomaTLX and PD-L1 expression patterns,and their association with clinicopathological parameters and immune phenotypes of glioma were analysed using CIBERSORT algorithm and single-sample gene-set enrichment analysis(ssGSEA)from TCGA(n=695)and CGGA(n=1018)databases.Protein expression and cellular localization of TLX,PD-L1,and PD-1,and the cytotoxic tumor-infiltrating lymphocytes(TILs)and tumor-associated macrophages(TAMs)in the glioma immune microenvironment were analysed in a tissue microarray(TMA)by immunohistochemistry and multiplex immunofluorescence.2.Study on the function and mechanism of TLX in glioma mediating immune escape by regulating the expression of PD-L1Through bioinformatics analysis in the database,we know that TLX and PD-L1 expression are positively correlated,so it is speculated that TLX can mediate tumor immune escape by regulating the expression of PD-L1.1)Glioma allografts and xenografts with TLX manipulation(by knockdown/knockout or reverse agonist)were inoculated subcutaneously,or orthotopically into the brains of immuno-deficient and immuno-competent mice to study tumour growth by in vivo imaging and the immune microenvironment by flow cytometry(FCM).2)Chromatin immunoprecipitation(ChIP)and luciferase reporter assays were used to investigate PD-L1 transcriptional regulation by TLX.Results1.TLX and PD-L1 expression levels were positively associated with macrophage-mediated immunosuppressive phenotypes in gliomas.2.TLX showed significant upregulation and positive correlation with PD-L1.3.Suppression of TLX by knockout/knockdown or reverse agonist methods inhibited in vivo growth of glioma allografts and xenografts significantly(p<0.05),rescued anti-tumoral immune response,accompanied with a significant decrease of PD-L1+population and glioma-associated macrophages,and increased cytotoxic lymphocyte infiltration(p<0.05).4.Mechanistically,TLX could bind directly to CD274(PD-L1)gene promoter and activate CD274 transcription.ConclusionThis study shows,for the first time,that TLX contributes to glioma malignancy and immunosuppression through transcriptional activation of PD-L1 ligands that bind to PD-1 expressed on both TILs and TAMs.Thus,targeting the druggable TLX may have a potential therapeutic significance in glioma immune therapy.