Expression Of MHC-I Of Lymphocytes In Breast Cancer Patients And Influence On CTL’s Cytotoxicity

BackgroundBreast cancer is one of the most frequent malignant tumors in female. And immune function of the organism is intimately correlative with generation and development of breast cancer, that is depressed immune function creates favorable conditions for generation and development of breast cancer, and instead malignant growth and increment of tumor enhance inhibition of immune function. Over a long period of time, studies of tumor immunology in breast cancer focus on fields of major histocompability classⅠmolecule (MHC-Ⅰ) on tumor cells, tumor antigens, co-stimulatory molecules, cytokines and antigen presenting cells. Lots of researches support that the expression of MHC-Ⅰmolecule in tumor tissues of breast cancer decreases and is even lost, and the function of infiltrating lymphocytes is inhibited. Cell mediated immunity is the supreme mechanism of anti-tumor immune effect, and T lymphocytes are the major effector cells which circulate substantially in peripheral blood. It is benefit for the acquirement of novel immune diagnosis marker and immune therapy target to study the changes and effects of phenotype and function of peripheral T lymphocytes during the generation and development of breast cancer.MHC is a closely linked gene cluster located in one chromosome of mammals and codes a group of proteins, which are the key molecules participating in antigen presenting and activation of T lymphocytes. And MHC-Ⅰmolecule play important roles in reject reaction, antibodies production, cytolysis and antigen presenting to CD8 positive T lymphocyte. It is notable that T lymphocyte is one of the cells bearing most abundant MHC-Ⅰmolecules. Some researches argued that HLA-A and HLA-B transcription decrease with ageing in peripheral blood leucocytes and might be a monitor of failed immune function in aged person; MHC-Ⅰmolecule play a protective role for memory T cell; MHC-Ⅰmolecule prolong the survival time of CD4 positive T cell. It could be presumed that MHC-Ⅰmolecules on T lymphocytes, different from ones on tumor cells, affected the function of T lymphocytes and played a vital role in anti-tumor immune.Objective1. To study the expression change of MHC-ⅠmRNA and protein of peripheral lymphocytes in breast cancer, its correlation with treatment and stages. To establish mouse model rumor-bearing, observe the dynamic changes of MHC-ⅠmRNA in PMLs and TILs, and discuss the function in valuing immune level of cancer patients.2. To block MHC-Ⅰgene by RNAi, and MHC-Ⅰprotein with anti-MHC-Ⅰantibody, and observe changes of cytotoxicity of CTLs.To discuss the influence of MHC-Ⅰon CTLs.3. To establish tumor+skin transplanting mouse model, induce graft rejective reaction, and observe the pattern of PBLs MHC-Ⅰexpression.To discuss influence of MHC-Ⅰon tumor growth invivo.Method1. To detect the mRNA expression levels at different locus of MHC-Ⅰ(HLA-A, HLA-B, HLA-Ⅰ) by FQ-PCR and HLA-ⅠMFI on different subtypes of lymphocytes (CD4+T, CD8+T, B, NK) by FCM.2. To establish breast cancer model with injecting 4T1 cell to BALB/C mice, detect the relative expression of H-2K. H-2D mRNA at different time, and observe the correlation between PMLs and TILs.3. To block MHC-Ⅰgene of CTLL-2 cell line by H-2K siRNA, and link the anti MHC-Ⅰantibody and MHC-Ⅰprotein on CTL induced by DC bearing 4T1 antigen.To observe cytotoxicity of CTLL-2 and CTL by LDH releasing method.4. To establish skin-transplanting mouse model, induce graft rejective reaction, and observe the pattern of PBLs MHC-Ⅰexpression. To establish tumor+transplanting model, discuss relationship of acute rejective reaction and tumor growth.Result1. Expression of MHC-Ⅰmolecules on peripheral blood cell was detected by flow cytometry. In breast cancer groupⅢandⅣ, down-regulation of MHC-Ⅰmolecules on CD4+T 1ymphocyte,CD8+T 1ymphocyte, B 1ymphocyte, natural killer cell (NK) were all detected comparing with health group. CD8+T 1ymphocyte decreased most obviously not only in groupⅢ,Ⅳ, but also in groupⅠ,Ⅱ. Expression of MHC-Ⅰmolecule showed no change after surgery and chemotherapy. There was no correlation between age and MHC-Ⅰ.2. Real-time RT-PCR analysis. Down expression of HLA-Ⅰ, HLA-A and HLA-B mRNA in PBMC was observed in cancer groupⅢ. Expression of HLA-Ⅰ, HLA-A and HLA-B mRNA showed no changes after surgery and chemotherapy. HLA-B mRNA showed more significant changes, even in groupⅡ,Ⅱ.3. MHC-Ⅰgene of CTLL-2 cell line was successfully inhibited by H-2K siRNA, and the cytotoxicity of CTLL-2 decreased. Ligand of anti MHC-Ⅰantibody and MHC-Ⅰprotein on CTL induced by DC bearing 4T1 antigen influenced the function of CTL.4. Expression of MHC-ⅠmRNA decreased with acute graft rejective reaction. But there was no changes observed in tumor growth between tumor-only group and tumor-tansplanting group.Conclusion1. MHC-ⅠmRNA and protein of peripheral lymphocytes were down regulated in advanced stage patients suffering breast cancer. Expression of mRNA at HLA-B locus decreased at early stage, and the change tendency was correlated with staging. CD8+T also showed earlier decline in MHC-Ⅰexpression.The levels of MHC-Ⅰwere not correlated with stages and not affected by chemotherapy and surgery. MHC-Ⅰof PBL might prompt the disorder of immune balance in tumor patients.2. Blocking MHC-Ⅰgene by RNAi or MHC-Ⅰprotein with anti-MHC-Ⅰantibody depressed cytotoxicity of CTLs. MHC-Ⅰof CTL might become a new target of anti-tumor immune.