| BACKGROUND AND OBJECTIVE:Immune inhibitory receptor LILRB4 is mainly expressed in normal monocytes,macrophages and some antigen presenting cells.Previous studies have shown that LILRB4 can be used as a diagnostic marker for acute myeloid leukemia(AML)M4 and M5 subtypes,and produce immunosuppressive microenvironment through apoE/LILRB4/SHP2/NF-kB/uPAR/Arginase-1 signaling pathway,mediating T cell proliferation inhibition and extramullary organ infiltration of AML cells.Blocking of LILRB4 signaling pathway by antibodies can significantly inhibit the development of AML.Whether LILRB4 is expressed in solid tumors has not been reported.Therefore,the main purpose of this study is to reveal the expression pattern of LILRB4 in solid tumors,to analyze the correlation between LILRB4 expression and pathological parameters,and to elucidate the function of LILRB4 in solid tumors.Methods:The expression of LILRB4 in multiple myeloma,prostate cancer,liver cancer,melanoma,breast cancer,brain cancer,head and neck squamous cell carcinoma,thyroid cancer,lung cancer,gastric cancer,colon cancer,pancreatic cancer,endometrial cancer and esophageal cancer tissues and cell lines was detected by immunohistochemistry and flow cytometry.The expression of LILRB4 was divided into four grades:strong(200-300),middle(100-200),weak(1-100)and negative(0).SPSS software was used to analyze the correlation between LILRB4 expression and pathological indicators.CRISPR-CAS9 was used to knock out the expression of LILRB4 in cell line RPM-8226.T cells were co-cultured by cell-cell contacting and Transwell phase isolation.Flow cytometry was used to detect the dilution ratio of CFSE to represent T cell proliferation.Result:According to TCGA and Protein Atlas database,LILRB4 is expressed higher than normal tissues in breast cancer,esophageal cancer,head and neck squamous carcinoma,kidney cancer,prostate cancer,gastric cancer,liver cancer,colon cancer and endometrial cancer.Immunohistochemical results showed that LILRB4 was mainly expressed in the cell membrane and cytoplasm,but not in the nucleus.The positive rates of LILRB4 were 40%(4/10),36.67%(33/90),16.67%(8/48),29%(28/95),19.5%(16/82)in multiple myeloma,breast cancer,prostate cancer,hepatocellular carcinoma and melanoma respectively.The expression of LILRB4 was not detected in gastric cancer,thyroid cancer,lung cancer,colon cancer,pancreatic cancer,head and neck squamous cell carcinoma,brain cancer,endometrial cancer and esophageal cancer.Pearson correlation analysis showed that LILRB4 had no significant correlation with clinical and pathological stages of any tumors except the grade of breast cancer.LILRB4 expression was detected in multiple myeloma cell lines RPM-8226?KMS26?OPM2?RPM1 and U266 cells;LILRB4 expression was not detected in breast cancer cell lines(SK-BR-3,MDA-MB-231,T47D,MCF7),prostate cancer cell lines(PC-3,DU-145,LnCap)and melanoma cell lines(SK-MEL-1,SK-MEL-2,SK-MEL-5,OCM1,OCM3,OCMMl,A375,M14,UACC257,UACC62,MDA-MB-435,MALEM-3M).The expression of LILRB4 was not detected in hepatocellular carcinoma cell line(HUH7,HePG2),lung cancer cell line(A549,H1299,H292)and colon cancer cell line(CACO2;SW620,SW480,HCT116).CRISPR-CAS9 knocked out the expression of LILRB4 in RPM-8226 cell line.LILRB4-KO could significantly reverse LILRB4-WT-mediated T cell proliferation inhibition through cell-cell contacting and transwell-isolated T cell co-culture system.Conclusion:In addition to normal monocytes/macrophages and AML cells,immune inhibitory receptor LILRB4 is also expressed in multiple myeloma,prostate cancer,hepatocellular carcinoma,melanoma and breast tumors.Knocking out the expression of LILRB4 can restore the proliferation of CD3+T cells. |
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