Experimental Study Of Immune Function On Radiotherapy Patients With Oral And Maxillofacial Cancer

ObjectiveIt’s still unclear about the regulation mechanism of the conventional fractionated radiotherapy on the immune system.Previous studies showed that radiotherapy can not only inhibit patients’immunity but also activate simultaneously.How to enhance the positive immune response and suppress the negative immune response of radiotherapy,has become one of the hotspots.We study the changes of immune cells numbers in peripheral blood of patients before and after radiotherapy to observe the immune effect by conventional fractionated radiotherapy on oral and maxillofacial cancer patients.Peripheral blood transcriptome and bioinformatics were used to study the molecular mechanisms of the activation and suppression effects of radiotherapy.The changes of the genes were validated by RT-PCR.We provide a new basis for radiotherapy combined with immunotherapy for the treatment of oral and maxillofacial cancer patients.Methods1.The lymphocyte subsets number count before and after radiotherapy:The number and percentage of CD3~+T、CD4~+T、CD8~+T、B and NK cells were examined before and after radiotherapy in 22 patients with oral and maxillofacial cancers.2.Microarray expression profiling of peripheral blood leukocytes before and after radiotherapy:14 squamous cell carcinoma cases of oral and maxillofacial tumor patients were selected,whose peripheral blood leukocytes RNA were extracted before and after radiotherapy and detected by microarray expression profiling.Bioinformatics methods were used for filtering the different genes.The gene ontology and signal transduction pathway were analyzed.Differences of co-expression were analyzed based on the number count changes and gene expression changes.3.RT-PCR validation:Based on the bioinformatics analysis,10 critical genes related to lymphocyte subsets number counts were selected for RT-PCR validation.The correlation of PCR results and lymphocyte subsets number counts were analyzed with statistical methods.Results1.After radiotherapy,peripheral blood lymphocytes,CD3~+T cells,CD4~+T cells,CD8~+T cells,B cells and NK cells were decreased than those before radiotherapy,the difference was statistically significant(P<0.05).The CD3~+T cell percentage increased after radiotherapy than that before radiotherapy,but the difference was not statistically significant(P>0.05).The percentage of CD4~+T cells and B cells and the CD4/CD8 ratio significantly decreased after radiotherapy,the difference was statistically significant(P<0.01),CD8~+T cells and NK cell percentage increased after rad iotherapy,the difference was statistically significant(P<0.01).2.1202 differentially transcripts(probe-level)mapped to 743 expressed genes were identified by microarray,in which 1041 transcripts mapped to 623 genes were down-regulated and 161 transcripts mapped to 120 genes were upregulated.GESA pathway enrichment analysis showed the differentially expressed gene pathways like ribosome pathway,T cell receptor signaling pathway,B cell receptor signaling pathways,primary immunodeficiency,cell-cell interaction,cell cycle and so on.Differences co-expression analysis showed that a total of 161 genes(corresponding to 235 probes)co-expressed with the cell count.The coexpression genes mainly concentrated in five pathways by KEGG pathway analysis.3.PCR validation showed CD3D,CD8B,FYN,GIMAP5,IL7R,KLPK1,LY9,MS4A1,PPP3CC and ZAP70 gene expression decreased after radiotherapy,and the difference was statistically significant(P<0.05).Conclusions1.The number of lymphocyte subclasses decreased significantly after radiotherapy,which meant radiotherapy had immune suppression effect,but the percentage CD8~+T cells and NK cells was increased,which meant radiotherapy might have some immune activation effect.2.By peripheral blood transcriptome,the signal pathway after radiotherapy mainly inhibited ribosomal pathway,T、B cell receptor signaling pathway,Natural killer cell mediated cytotoxicity pathway and so on.The ribosome signaling pathways inhibited by radiotherapy was rare reported in literature,so further study of the mechanism and function repairation may provide new ideas to study immune function in patients after radiotherapy.3.PCR results showed that the CD3 cell counts associated with four genes:CD3D,KLRK1,LY9,PPP3CC;the CD4 cell counts associated with genes IL7R and LY9;and CD8 cell counts associated with genes CD3D and GIMAP5.These genes are biomarkers which can reflect immune function effected by radiotherapy,and also new targets for immunotherapy of radiotherapy patients.