Study Of Anti-tumor Immunity By L-1-methyl Tryptophan Combined With DC Vaccine On Breast Cancer-bearing Mice

Object: To determine whether dendritic cells vaccine sensitized by 4T1 antigen in mice breast cancer cells can cause anti-tumor immune response in vitro and vivo;In order to explore the inhibition degrees of IDO expression in breast cancer-bearing mice’s tumor-draining lymph node(TDLN),and it’s mechanisms for enhancing immune responses in tumor-bearing mice after therapy by L-1-MT(the chemical inhibitor of IDO)combined with DC vaccine.Methods: 1.To prepare DC vaccine,we have used repeated freezing and thawing method to extract 4T1 antigen of breast cancer cells in mice and cultured DC from BALB/c mice in vitro.Flow cytometry detected mature DC phenotype;MLR detected the efficacy of DC stimulate t-lymphocyte proliferation and cytotoxic t-lymphocyte(CTL)kill tumor effect;4T1 were target cells,DC sensitized T cells as effector cells detection of cytotoxic t lymphocytes(CTL)kill tumor in vitro.2.To solid tumor model,1×1054T1 cells were injected subcutaneously on the fourth fat pad female BALB/c mice.Those tumor-bearing mice randomly divided into four groups: control group,DC vaccine group,L-1-MT group,L-1-MT combined with DC vaccine group,and given normal saline,DC vaccine,L-1-MT,L-1-MT combined with DC vaccine treatment.Respectively measuring tumor size and depicting tumor growth curve,calculate each group average volume.The mice were killed on the 21 th day.Take out tumor tissues,TDLN and spleen.Western blot detected the expression of IDO in TDLN;Flow cytometry detected the proportion of CD4 + CD25 + Foxp3 + Treg cells in TDLN and spleen;Determined by MTT test a tumor-burdened specific cytotoxic t-lymphocytes in mice.Results:1.4T1 freeze-thawing antigen sensitization of DC(4T1-DC)group on the surface of co-stimulating molecules CD40,CD80,CD86 expression is significantly higher than no sensitization DC(DC)(P<0.01),4T1-DC has a stronger stimulation of t-lymphocyte proliferation ability.The stimulation index of 4T1-DC and DC group were 8.45±0.24 and 6.56±0.29 on the rate of 1:10,which is maximum(P <0.01).Comparing the killing rate of 40∶1 and 20∶1,4T1-DC group sensitization of CTL specific effect of kill tumor proliferation in vitro were higher than DC group,there were 35.03±1.01,27.02±1.33 and 31.00±1.06,23.96±1.06(P <0.01).2.After inoculation of 4T1 breast cancer cells,the tumor growth curve showed that L-1-MT combined with DC vaccine group tumor growth is slowest than the other three groups.On the 21 days,the volume and weight of tumor results of 4 groups lined as follows: CON group>DC vaccine group>L-1-MT combined with DC vaccine group > L-1-MT group(P < 0.05).L-1-MT combined with DC vaccine group compared with CON group,inhibitory rate of 43%.3.Western blot detected each groups’ relative expression of IDO in TDLN and flow cytometry detected the CD4+CD25+ Foxp3+ Treg cells proportion of TDLN and spleen results of 4 groups lined as follows: DC vaccine group>CON group> L-1-MT combined with DC vaccine group>L-1-MT group(P < 0.05).2Conclusion:1.Application of DC vaccine to treat breast cancer-bearing mice can significantly increase IDO expression in tumor-draining lymph node,and increase the number of CD4+CD25+ Foxp3+ Treg cells of TDLN and the spleen,which may be one of the important factors that influence breast cancer immunotherapy effect.2.L-1-MT can effectively inhibit activity protein expression of IDO in breast cancer-bearing mice.At the same time can be downregulated the number of CD4 + CD25 + Foxp3 + Tregs in tumor-bearing mice’s TDLN and spleen,thereby enhancing the DC vaccine antitumor effect effectively inhibit tumor growth.