Expression Of IDO/Kyn/AHR Signaling Pathway In CD20+B Cell Lymphoma Cells

Objective:Diffuse large B cell lymphoma(DLBCL)is the most common in all non Hodgkin's lymphoma,which is a group of highly heterogeneous and high proliferative activity of malignant tumor,and approximately 50% patients can be cured with the standard chemotherapy.The use of rituximab has led to a new era in the treatment of DLBCL,with a complete remission rate and overall survival rate of patients being further improved,but there are still about 40% of patients with disease progression and disease recurrence.The mechanism of tumor development and resistance is related to its complex signaling network.As we develop a more in-depth study of the mechanism of tumor development,we have found that the development of tumors may be related to the abnormal expression of certain genes involved in immune escape,in-depth understanding of the abnormal expression of these genes on the specific role of the anti-tumor immune system,and Targeted blocking leading to immune escape the key pathways,is an important way to improve the effectiveness of cancer treatment.IDO is a key enzyme in the metabolic pathway of tryptophan.The high expression and activity of IDO are closely related to immune escape.IDO is also a hotspot in tumor immunotherapy.IDO is highly expressed in a variety of human solid tumors and hemolymphatic system tumors.In lymphoma,a number of studies have shown that IDO is highly expressed in tumor cells of DLBCL patients,and high expression of IDO is associated with poor prognosis.In this team's previous experiments,one of the mechanisms of rituximab resistance was confirmed to be an overexpression of IDO in DLBCL cells.Many reports have showed that IDO / Kyn / AHR signaling pathway play an important role in the immune regulation system,the overexpression of IDO lead to local tryptophan depletion and metabolite kynurenine(Kyn)accumulation,aromatic hydrocarbon receptor(AHR)activation and the nuclear translocation,target gene transcription expression,to promote reg?latory T(Treg)cell differentiation and proliferation,inhibition of T cell proliferation activation,eventually leading to immune escape.But whether the IDO leading to tumor immune escape is through the initiation of IDO / Kyn / AHR pathway,remains to be further confirmed.The aim of this study was to observe expression of the each signal molecule in IDO / Kyn / AHR signaling pathway by 1-MT and CH223919 inhibit the expression of IDOand AHR in Raji cells and Je Ko-1 cells.Methods : 1.Raji cell line and Je Ko-1 cell line were cultured in vitro,The relative expression levels of IDOm RNA and AHR m RNA were detected by fluorescence quantitative PCR,and The changes of IDO m RNA and AHR m RNA expression level in the case of the using IDO inhibitor(1-MT)or AHR inhibitor(CH223191)at different concentrations after different time;Raji cells and Je Ko-1 cells were treated with different concentrations of 1-MT or CH223191 for 24 h.The supernatant was collected and the contents of tryptophan and kynurenine in the supernatant were detected by colorimetry.Res?lts: 1.fluorescence quantitative PCR results showed that:(1)1-MT 5,25?m/ L were treated with Raji cells and Je Ko-1 cells for 0.5h,1h,2h,measured IDOm RNA and AHRm RNA relative expression decreased,The expression of IDOm RNA and AHR m RNA decreased gradually with the increase of 1-MT concentration and the prolongation of the time of action.When the 25 mol/L concentration and the time of action was 2h,the decrease was the most obvious(P> 0.05).However,there was no significant difference in the expression of IDO m RNA between the 5?mol / L concentration group and the 25?mol / L concentration group(P> 0.05).(2)CH223191 5,25?m / L on Raji cells and Je Ko-1 cells for 0.5h,1h,2h,the relative expression of IDOm RNA and AHRm RNA decreased.With the increase of CH223191 concentration or the time of action,The relative expression level of IDOm RNA and AHR m RNA decreased more significantly at 25?mol / L concentration or after 2h(P <0.05).However,there was no significant difference in the expression of IDO m RNA between 0.5h group and 1h group after CH223191 5?m / L effect on Je Ko-1 cells(P> 0.05).(3)1-MT and CH223191 were treated with Raji cells and Je Ko-1 cells respectively,and the relative expression of IDOm RNA and AHRm RNA decreased with the increase of concentration or prolonged duration.The correlation analysis showed that there was a strong positive correlation Relationship(P <0.05).2.Determination of kynurenine and Tryptophan in Cell Supernatant:1-MT(5,25?m / L)and CH223191(5,25?m / L)were treated with Raji cells and Je Ko-1 cells for 24 h,The content of tryptophan in the supernatant of the cells increased,the content of kynurenine decreased,and the concentration of tryptophan increased with the increase of1-MT and CH223191 concentration,the decrease of kynurenine concentration was more obvious,the difference was statistically(P <0.05).Tryptophan(50,100?m / L)were treated with Raji cells and Je Ko-1 cells for 24 hours,the concentration of kynurenine increased significantly,and with the addition of tryptophan concentration increased,the increase in kynurenine concentration also increased(P<0.05).Conclusion: 1.B cell lymphoma cells have the expression and activation of IDO/Kyn/AHR signaling pathway;2.,the use of IDO or AHR inhibitors can block the IDO/Kyn/AHR signaling loop in B cell lymphoma cells.