| Background:About 30% of acute B lymphocytic leukemias involve Pax5 gene mutations.Pax5 gene mutation in children with ALL is a poor prognostic factor.At present,it is thought that tumor immunity tolerance caused by Pax5 mutation may be the cause of relapse,but the mechanism is still unclear.Objective: We consider using a mouse model of Pax5 knocked down to study the mechanism of Pax5 gene mutations involved in tumor immune tolerance.Methods:In this study,CRISPR-Cas9 was used to knock down the Pax5 gene in mouse B-cell lymphoma cell line A20 cells.After verifying the knock-down efficiency at the transcription level and protein level,we used the mutant and wild strains to test Balb/c mice subcutaneous tumor modeling respectively.After 2 weeks,the tumor masses were removed for immunohistochemistry,real-time quantitative PCR,and flow cytometry to observe the differences in T cells between the two groups of tumor masses,and further explore the differences in expression of chemokines related to T cell recruitment.Results:The Pax5 gene of A20 cell lines was successfully knocked down,and three mutant strains were obtained,named MUT1,MUT2,and MUT3.Sequencing results confirmed that the Pax5 gene had a mutation in the target position,and we further confirmed that the expression of Pax5 was decreased at the transcription and protein levels.Comparing the subcutaneous tumors constructed from wild-type A20 cells and mutant strain cells,we found that the transcription levels and protein levels of CD3,CD4,and CD8 in the three groups of mutant subcutaneous tumors were significantly lower than those of the wild-type control group,and have statistical difference.Further research found that the chemokines,such as Ccl2,Ccl3,Ccl4,Ccl5,Ccl6,Cxcl9,and Cxcl10 were significantly down-regulated,which cause effector T cells homing to the tumor microenvironment.Conclusions:The Pax5 mutation results in a decrease in effector T cells in the tumor microenvironment,leading to tumor immune tolerance. |
PDF Full Text Request