Effects Of Low Dose Splenic Irradiation On CD4~+T Cell Immune Function And The Expression Of Tim-3 In Lewis Lung Cancer Tumor-bearing Mice

Purpose:The purpose of this study was to observe whether low-dose spleen irradiation(LDSI)can affect the tumor growth of non-small cell lung cancer tumor bearing mice and whether LDSI can affect the effect of radiotherapy.Besides,this study was to explore the effect of LDSI on T cell immune and the expression of T cell immunoglobulin and mucin domain 3(TIM-3).Methods:C57/BL 6 mice aged 4-6 weeks were selected and adaptive fed for one week.Lewis lung cancer cells in logarithmic growth stage were resuscitated to about 1x10~7cells/ml.The right groin was selected as the site of subcutaneous tumor formation,and0.1ml tumor cells were injected into each mouse to establish the lung cancer mouse model.Body weight and tumor diameter were measured every 3 days after tumor formation.When the tumor diameter grew to 8-10mm,the mice were randomly divided into 4 groups(n=10):control group(CON group);Low-dose spleen irradiation group(LDSI group):10c Gy/d,50c Gy/min,continuous irradiation for 2 days.Tumor irradiation group(HDR group):300c Gy/d,200c Gy/min,continuous irradiation for 3 days.Low dose spleen irradiation after tumor area irradiation group(L+H group).On the 3rd day after radiotherapy,4 mice in each group were killed randomly.Spleen and tumor tissues of mice were taken under aseptic condition.The changes of spleen index of mice in each group were calculated and tumor diameter of each group was observed and compared.T lymphocyte subsets and the proportion of regulatory T cells(Treg cells)in spleen were analyzed by flow cytometry and the proportion of apoptotic cells in spleen were analyzed.Tim-3 expression in tumor tissues of each group was analyzed by immunohistochemistry.The other 6 mice in each group were kept until 2 weeks after radiotherapy for survival analysis.Results:Compared with the control group,the tumor diameter of HDR group and L+H group was significantly decreased,and the difference was significant(P<0.05).The tumor diameter in L+H group was smaller than that in HDR group(P<0.05).Compared with the CON group,the spleen index of mice in LDR group and L+H group was significantly increased(P<0.05).The apoptosis rate of spleen in HDR group was significantly higher than that in control group,LDSI group and L+H group(P<0.05).The apoptosis rate of spleen in L+H group was higher than that in control group and LDSI group(P<0.05).In terms of T cell immunity,the proportion of CD4~+T cells in spleen in LDR group and L+H group was significantly higher than that in CON group and HDR group(P<0.05).The proportion of CD4~+CD25~+Foxp3~+Treg cells in LDSI group and L+H group was significantly lower than that in control group and HDR group(P<0.05).The expression of Tim-3 in spleen was significantly lower in LDSI group and L+H group than in CON group(P<0.001).In tumor tissues,the positive expression rate of Tim-3 in LDSI group and L+H group was lower than that in CON group(P<0.05).Conclusion:Low dose spleen irradiation can reduce the decrease in CD4~+T cells and the increase in CD4~+CD25~+Foxp3~+Treg cells caused by HDR.LDSI could reduce the expression of Tim-3 both in spleen and tumor tissue.These indicate that LDSI can promote the immune response,reduce immunosuppression and improve the efficacy of radiotherapy.