Expression Of Immune Checkpoint Molecules TIM-3,PD-1 And Its Ligand PD-L1 In DLBCL And Their Bioinformatics Analysis In Tumor Immunity

Objective:The structures and functions of T cell immunoglobulin domain and mucin domain protein-3(TIM-3),programmed cell death protein 1(PD-1)and its ligand programmed cell death ligand 1(PD-L1)were analyzed,and their expressions and functions were verified in diffuse large B cell lymphoma(DLBCL).It is expected to provide a basis for the application of corresponding inhibitors in DLBCL.Methods:1.The protein number was queried by Uniprot,the amino acid sequence was searched by NCBI,the physical and chemical properties of the protein were analyzed by Prot Param,the hydrophobic properties of the protein were analyzed by Prot Scale,the signal peptide was analyzed by Signal P-5.0,the transmembrane domain was predicted by TMHMM,the secondary structure was predicted by SOPMA,and the protein interaction network was analyzed by String.GO and KEGG were analyzed by David and R.2.The expression of TIM-3,PD-1 and PD-L1 genes in DLBCL was analyzed by GEPIA2 database.TIMER database was used to analyze the relationship between TIM-3,PD-1 and PD-L1 gene expression and immune infiltration in tumor microenvironment of DLBCL.3.Immunohistochemical technique was used to detect the expression of TIM-3,PD-1 and PD-L1 in different cells of DLBCL,and the relationship between their expression and clinicopathological features was analyzed.K-M method was used for survival analysis.Results:1.TIM-3 protein consists of 301 amino acids,a stable hydrophilic protein with regular secretory pathway and transmembrane region,and its secondary structure is mainly random curl.Protein interaction network analysis showed that TIM-3 protein was closely related to LGALS9,LAG3,CEACAM1,CTLA4,IL-2,PD-L1,TBX21,FOXP3,PD-1,CD160 and other proteins.GO and KEGG found that most of the biological processes and signal pathways involved in TIM-3 protein were related to immune response.2.PD-1 protein is composed of 288 amino acids and belongs to unstable hydrophilic protein.It has conventional secretory pathway and transmembrane region.The secondary structure is mainly random curl.Protein interaction network analysis shows that PD-1 protein is closely related to PD-L1,PD-L2,PTPN11,PTPN6,CD3 E,LCK,HLA-DRB1,CD3 D,CD4,HLA-DQB1 and other proteins.GO and KEGG found that most of the biological processes and signal pathways involved in PD-1protein are related to immune response.3.PD-L1 protein consists of 290 amino acids,a stable hydrophilic protein with conventional secretory pathway and transmembrane region.The secondary structure is mainly random curl.Protein interaction network analysis shows that PD-L1 protein is closely related to PD-1,PTPN11,PD-L2,CTLA4,CD80,CD3 E,LCK,HLA-DRA,HLA-DQB2,CD4 and other proteins.GO and KEGG found that most of the biological processes and signal pathways involved in PD-L1 protein are related to immune response.4.GEPIA 2 showed that the expression of TIM-3,PD-1 and PD-L1 genes in DLBCL was significantly higher than that in normal tissues,but PD-1,PD-L1 and TIM-3 genes had no effect on the prognosis of DLBCL.5.TIMER showed that the expression level of TIM-3 gene was positively correlated with neutrophil and dendritic cell(DC)infiltration,the expression level of PD-1 gene was positively correlated with the infiltration of CD8+T and CD4+T cells,and the expression level of PD-L1 gene was positively correlated with neutrophil infiltration.6.Immunohistochemistry showed that the expression of TIM-3 and PD-1 in DLBCL tissue was lower than that in reactive hyperplastic lymph node tissue,while the expression of PD-L1 was on the contrary.The positive rates of TIM-3 in tumor cells and vascular endothelial cells were 48.0%(59/123),39.0%(46/118),respectively.The positive rates of PD-1 in tumor cells and tumor infiltrating lymphocytes(TIL)were 26.4%(32/121),8.3%(10/120),respectively.The positive rates of PD-L1 in tumor cells and TIL were 12.5%(15/120),1.7%(2/120).respectively.7.In terms of tumor cell expression,there was no significant difference in all clinicopathological features between TIM-3 positive group and TIM-3 negative group,while in vascular endothelial cell expression,there were differences in ethnic and IPI scores between TIM-3 positive group and negative group.In tumor cells,the expression of PD-1 in tumor cells was negatively correlated with primary site and Ki-67 index,while on TIL,the expression of PD-1 was positively correlated with nationality,negatively correlated with primary site,and negatively correlated with Ki-67 index.In the expression of tumor cells and TIL,there was no significant difference in clinicopathological features between PD-L1 positive group and PD-L1 negative group.8.There was no correlation between the co-expression of TIM-3 in tumor cells and vascular endothelial cells and the clinicopathological features.The co-expression of PD-1 in tumor cells and TIL was negatively correlated with age and nationality,positively correlated with primary location and Ki-67 index,and had no correlation with other characteristics.There was no correlation between the co-expression of PD-L1 in tumor cells and TIL and the clinicopathological features.9.The co-expression of TIM-3,PD-1 and PD-L1 in tumor cells,vascular endothelial cells and TIL had no difference in all clinicopathological features.10.The expression of TIM-3 in tumor cells was positively correlated with that of TIM-3 in vascular endothelial cells,the expression of TIM-3 in tumor cells was positively correlated with the expression of PD-L1 in tumor cells,and the expression of TIM-3 in tumor cells was positively correlated with the expression of PD-L1 in TIL.The expression of PD-1 in tumor cells was positively correlated with the expression of PD-1 in TIL,and the expression of PD-L1 in tumor cells was positively correlated with the expression of PD-L1 in TIL.11.The survival time was negatively correlated with age,positively correlated with Hans classification,and positively correlated with the expression of BCL-6.The prognosis of patients with age ≤ 60 years old was better than that of patients age > 60 years old.The prognosis of CD3 negative group was better than that of CD3 positive group,and that of PD-L1-TIL group was better than that of PD-L1+TIL group.Conclusions:1.TIM-3,PD-1 and PD-L1 proteins are hydrophilic and highly lipophilic secretory proteins,and their secondary structures are mainly random curls,which are conducive to binding with other proteins.There is a close interaction among2.TIM-3,PD-1 and PD-L1 proteins,and they mainly play a role in the process of immune response.3.TIM-3,PD-1 and PD-L1 genes showed no statistical significance in the prognosis,but the follow-up experiment only found that the prognosis was better in the PD-L1-TIL group.4.TIM-3,PD-1 and PD-L1 genes may promote DLBCL immune escape by regulating immune cell infiltration in tumor microenvironment.