| Background and PurposesAccording to cancer data reported by the World Health Organization in 2020,the global new cases of hepatocellular carcinoma(HCC)were 910,000 accounting for the sixth in cancers and the mortality cases of HCC were 830,000 accounting for the third leading cause of cancer-related death.The treatment of HCC remains a major global medical challenge,and new treatments are urgently needed.The inhibited antitumor immunity of regulatory T cells(Treg cells)makes inhibiting Treg become a potential strategy for immunotherapy against HCC.It has been reported that IL-28B belonging to IFN-λs family down-regulates Treg cells and shows anti-tumor effect.However,the anti-tumor immune mechanism of IL-28B is still unclear.The aim of the research is to evaluate the therapeutic effect of IL-28B on hepatoma carcinoma-bearing mice by down-regulating Treg cells,to clarify its effects on T cell subsets and cytokines in the immune microenvironment and to study the effect of IL-28B on the generation of Treg cells in vitro.Methods(1)The adenovirus rAd-mIL-28B was amplified by HEK 293 cells and identified the target gene by PCR.The rAd-mIL-28B titer was measured by the 50%tissue culture infective dose(TCID50)method.(2)MTT assay was used to detect the inhibitory effect of IL-28B on cervical cancer TC-1 cells proliferation in vitro.(3)On day 0,1 × 106 H22 cells were injected subcutaneously into female BALB/c mice,and 1 × 108 PFUs rAd-mIL-28B were injected intratumorally at day 4,7 and 10,respectively.The diameter of the tumor was measured.On the 11th day,mice were sacrificed and the wet weight of tumor tissues,spleen,lung,liver and thymus was weighed.The tumor tissues were stained with HE to observe the cells morphology.The lymphocytes from spleen and tumor tissue were isolated and the expression of CD4,CD8,Foxp3 and PD-1 on T cells were detected by flow cytometer(FCM).The levels of Th1,Th2,Th17,IL-10 and monocyte related cytokines in serum and tumor tissue homogenate were detected by protein microarray.(4)The splenocytes separated from BALB/c mice stimulated with anti-CD3/CD28,IL-2 and TGF-β,and induced the generation of Treg cells.After the addition of IL-28B under the stimulated condition and culture for 3 days,the expression of CD4,CD25 and Foxp3 were detected by FCM,the mRNA levels of Foxp3 and PD-1 were detected by RT-PCR,and the levels of IL-10 in cell culture supernatant were detected by ELISA.(5)The H22-beaing mice were established and treated with 100 mg/kg/day intragastrically and 1 × 108 PFUs rAd-mIL-28B injected intratumorally at day 4,7 and 10.The diameter of the tumor was measured.On the 11th day,mice were sacrificed and the wet weight of tumor tissues was weighed.The tumor tissues were stained with HE to observe the cells morphology.The lymphocytes from spleen and tumor tissue were isolated and the expression of CD4,CD8,Foxp3 and PD-1 on T cells were detected by FCM.The levels of Th1,Th2,Th17,IL-10 and monocyte related cytokines in the serum were detected by protein microarray.Results(1)The amplified adenovirus vector rAd-mIL-28B was confirmed to contain IL-28B gene by PCR.The titers of rAd-mIL-28B and rAd-EGFP measured by the 50%tissue culture infective dose(TCID50)method were 1010.6 PFU/mL and 109.55 PFU/mL,respectively.(2)MTT assay showed that after treated with TC-1 cells 50 μg/mL IL-28B and 100 μg/mL IL-28B for 72h,the OD values were 0.80±0.09 and 0.85 ± 0.08,respectively,which were lower than that of negative control group 0.92 ± 0.06(p<0.05).(3)In H22-beaing mice,the tumor volume of rAd-mIL-28B group(796.74±447.58 mm3)was lower than rAd-EGFP group(1415.27±513.37 mm3)(p<0.05).The average tumor weight of rAd-mIL-28B group(0.63 ± 0.30 g)was reduced than that of rAd-EGFP group(1.16 ± 0.34 g)(p<0.05),and the tumor inhibition rate was 45.69%.HE staining showed tumor tissue necrosis in rAd-mIL-28B group.The proportion of CD8+T cells in lymphocytes in the tumor microenvironment(TME)of rAd-mIL-28B group(8.65 ± 5.49%)was higher than the untreated group(3.21 ±1.45%)(p<0.05).Compared with the rAd-EGFP group,the percentages of Foxp3+T and CD4+Foxp3+cells in TME in the rAd-mIL-28B group were not different significantly(p>0.05).The proportion of splenic CD4+Foxp3+ cells in CD4+T cells in rAd-mIL-28B group(16.78 ± 2.93%)was lower than rAd-EGFP group(21.47 ±4.17%)(p<0.05).The percentage of splenic CD4+PD-1+T cells in lymphocytes in rAd-mIL-28B group(1.46±0.08%)was significantly lower than rAd-EGFP group(2.40±0.31%)(p<0.05).The serum levels of CXCL13,ICAM-1,MCP-5 and IL-7 in rAd-mIL-28B group were decreased compared to rAd-EGFP group(p<0.05).Meanwhile,compared with rAd-EGFP group,rAd-mIL-28B decreased the expression of IL-15 and sFasL in tumor tissues(p<0.05).(4)Compared with the unstimulated splenocyte group,the proportion of Foxp3+-producing cells in lymphocytes increased in the splenocyte stimulation group(500 ng/ml anti-CD3/CD28,5 ng/ml IL-2 and 1.25 ng/ml TGF-β)after cultured for 3 days(p<0.05).Compared with the splenocyte stimulation group,the percentage of Foxp3+-producing cells in lymphocytes decreased(p<0.05),the level of Foxp3 mRNA was significantly decreased(p<0.05),the level of PD-1 mRNA was not different significantly(p>0.05),and the level of IL-10 in the supernatant of the medium decreased significantly(p<0.05)in the splenocyte stimulated group in the presence of 50 ng/ml IL-28B.(5)rAd-mIL-28B combined with E2-a inhibited tumor volume in H22-beaing mice(p<0.05).Compared with the untreated group,the percentages of Foxp3+,CD4+Foxp3+,CD8+Foxp3+and PD-1+Foxp3+in TME in the rAd-mIL-28B group increased significantly(p<0.05).The serum cytokine IL-1α,IL-12p40 and TNF RI in rAd-mIL-28B group were decreased significantly,while the serum cytokine MIP-1γ was significantly increased compared to rAd-EGFP group(p<0.05).Conclusion(1)rAd-mIL-28B inhibited tumor growth by down-regulating Treg in H22 tumor-bearing mice.(2)rAd-mIL-28B modulated the tumor immune microenvironment in H22 tumor-bearing mice.(3)IL-28B shown inhibitory effect on iTreg cells directly in vitro.(4)rAd-mIL-28B combined with E2-a inhibited tumor growth in H22 tumor-bearing mice.(5)rAd-mIL-28B combined with E2-a did not enhance the antitumor effect of E2-a. |
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