The Effect Of HPV16E7 Peptide Vaccines On The Immune Function Of The Hu-PBL-SCID Mouse With SiHa Cells And The Mechanisms Of Immune Escape In Cervical Cancer

Cervical cancer is the second most common cancer among women. Even with optimum treatment for the cervical cancer, including radiotherapy, surgery and chemotherapy, a lot of patients are likely to relapse and die. It is essential to purse a new method of treatment. There is now compelling evidence for the causative role of human papilloma infection (HPV) in the disease, predominantly of the HPV 16. HPV DNA has been demonstrated in almost all of tumor biopsy specimens, and the HPV 16 E6 and E7 gene products are retained and highly expressed in cervical cancer cells, which is a tumor-specific antigen for cervical cancer. Development of immunotherapy directed against tumor-specific Ags encoded by HPV 16 could be desirable. Peptide-based vaccines could have several advantages compared with live or recombinant vaccines, because it should be possible to specifically direct the immune system towards specific epitopes, with safety and relatively easy clinical grade production and specific induction of responses to less immunodominant epitopes. Thus, vaccination strategies have been developed to enhance the cellular immune responses against E6 and E7 viral proteins and eliminate HPV-infected cells. But HPV cannot be cultured in vitro, with strictly specific to race, which restrict its study on animal model. Severe combined immunodeficiency (SCID) mouse makes it possible to study the relationship between human immune cells to human tumors.The role of the immune response in the pathogenesis of HPV-associated neoplasia is still poorly studied, although its significance has suggested by indirect and in vivo evidences:increased incidence of cervical neoplasia observed among immunosuppressed patients. Although several reports about the development of HPV vaccines, some of which have been tested in clinical trials, most of the effects are not promising. The reason may be associated with tumor immune escape from the host. This immune escape is the defense mechanism executed by tumor cells and/or the defect of host immune system function, which is consisted of two mechanisms, one is a passive immune escape and another is immune counterattack.The mechanisms of passive immune escape involve altered expression of cellular membrane molecules of tumor cells, which prevents recognition and elimination from host immune cells. The molecules affecting recognition by immune cells include membrane histocompatibility-related leukocyte antigens (HLA) Class I and II. HLA molecules are known to play a significant role in stimulating and regulating the immune response. CD99 can regulate the expression of HLA Class I molecules from Golgi's complex to cell surface. HLA-G is a kind of non-classical HLA-1 antigens, which may be one of the mechanisms for tumor immune escape. The significance of altered expression of MHC- II molecules have not yet determined.A prerequisite for neoplastic cells to expand their population and eventually form solid tumors is the development of strategies for escaping surveillance by the host immune system. The Fas-Fas ligand (FasL) pathway figures prominantly in the execution of apoptosis induced by the immune system. Fas is expressed in normal and neoplastic cells, and FasL expression is largely restricted to the immune system. On contact with FasL-positive cytotoxic lymphocytes, Fas-bearing tumor cells rapidly undergo apoptosis. However, tumors may mount a counterattack by expressing FasL, which represents a mode of tumor escape from immune surveillance.But the clinical effect and immune responses are not definite. It is necessary for us to study the clinical effect and immune responses of HPV peptide-based vaccines.Recent studies reveal that HLA-DR, HLA-G, and CD99 are also important in immune response. The aim of this study was to investigate the role of HLA-DR, HLA-G and CD99 during cervical carcinogenesis. We also compared the indices of apoptosis and expression patterns of apoptosis-related antigens, Fas, FasL and c-FLIP in the normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cancer, in order to investigate their role of Fas-mediated apoptosis in the pathogenesis of cervical neoplasia.PartiThe effect of HPV16E7 peptide vaccines on the immune function ofthe hu-PBL-SCID mouse with SiHa cellsObjective To evaluate the effect of HPV16E7 peptide vaccines on the immune function of the hu-PBL-SCID mouse with SiHa cells. Methods Thirty CB17SCID mice were randomly divided into 4 groups: group A intraperitoneally injected with human peripheral blood lymphocyte(PBL) for immune reconstruction, vaccinated with only incomplete Freund's adjuvant(IFA);group B intraperitoneally injected with PBL and subcutaneously injected with SiHa cells, vaccinated with HPV16E712-2o (MLDLQPETT) , T helper peptide and IFA;group C intraperitoneally injected with PBL and subcutaneously injected with SiHa cells, vaccinated with T helper peptide and IFA;and group D intraperitoneally injected with PBL and subcutaneously injected with SiHa cells, vaccinated with HPV16E7n-20 (YMLDLQPETT) , HPV16E786-93 (TLGIVCPI) ,T helper peptide and IFA. Human immunoglobulins G(IgG) in mouse serum, the percentage of human CD3"\ CD4+ and CD8+ T cells in peripheral blood, spleen weight, tumor infiltrating lymphocytes and CD8+ T cells, and cytotoxicity test of spleen cells were detected. Results The rate of successful tumor transplantation was 100%. Human IgG level rised significantly in the plasmas of all humanized SCID mice within 8 weeks following immunoreconstituted time elongating( p < 0.05). There is no significant difference of human IgGlevel in all groups ( p > 0.05) and so does the percentage of human CD3+,CD4+ and CD8+ T cells in peripheral blood. Compared with group A , the weight of spleen in other three groups were markedly increased( p < 0.05). TIL infiltrating in the tumor , tumor bleeding and necrosis wasremarkable but no human CD8+ T cells was detected by immunohistochemistry in all groups. Thespleen cells in group B^ C and D displayed lower cytotoxicity to target cells than that in group A(i><0.05) . Conclusion The reconstructed human immune function of the hu-PBL-SCIDmouse with SiHa cells is destroyed with the time ongoing , which is probably relieved by peptidevaccination.Part IIAltered expression of cellular membrane molecules of HLA-DR, HLA-G and CD99 in cervical intraepithelial neoplasias and invasive squamous cell carcinomaObjective. The aim of this study was to investigate the role of HLA-DR, HLA-G and CD99 during cervical carcinogenesis. Methods. Using specific antibodies for HLA-DR, HLA-G and CD99, we examined protein expressions in 19 normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive sqamous cell carcinoma (SCC) by immunohistochemistry. And we detected the expression of Ki67 in the same specimens. Results. None of normal cervix and CINs except three cases of CIN III expressed HLA-DR. HLA-DR expression increased progressively with the grade of the tumor, and significant differences could be observed between grade 1 and grade 2 (PO.01) and between grade 1 and grade 3 (PO.05). In all normal epithelial control samples, HLA-G expression was seen in ectocervical squamous and endocervical columnar epithelium and the staining was strong and uniform. Only a small proportion of CINs and SCCs showed reduced expression of HLA-G. Compared with the results in the control samples, CINs and SCCs showed significantly reduced expression of HLA-G (PO.001). SCCs showed significantly increased expression of CD99 when compared with normal cervix and CINs (P<0.05). Ki67 was expressed in all specimens. Significant differences were observed between CINs and normal cervix (PO.001) and SCCs and controls (P<0.001), but no significant differences could be observed between SCCs and CINs. Conlusions. These results indicate that increased expression of HLA-DR and loss of CD99expression may be related to the evolution of cervical cancer. Part IIIFas-mediated pathway and apoptosis in normal cervix, cervicalintraepithelial neoplasia and cervical squamous cancerObjective The aim of this study was to examine the expression patterns of apoptosis-related antigens such as Fas, FasL, and c-FLIP in the cervical squamous cells, and to investigate their role of Fas-mediated apoptosis in the pathogenesis of cervical neoplasia. Method Using specific antibodies for Fas, FasL, and cFLIP, we examined protein expressions in 19 normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive squamous cell carcinoma (SCC) by immunohistochemistry. And we detected the apoptotic indices by TUNEL in the same specimens. Results Fas expressions were quite similar in CIN I, CIN II and normal cervix. Though Fas expression tended to increase in Grade 2 cancers than in Grade 1 cancers and CIN III, and a slight decline was present in Grade 3 compared with Grade 2 cancers, these differences did not reach statistical significance. Almost all cases of CINs did not express FasL, while FasL expression increased with the grade of the tumor. Statistically significant differences could be observed between Grade 1 and Grade 2 (P<0.01) and between Grade 2 +Grade 3 and Grade 1 (PO.001). All cases of normal cervix and CINs except two cases of CIN III did not express cFLIP. cFLIP expression tended to increase with the grade of the tumor. Apoptosis could be determined in all samples by TUNEL. There was a tendency to decrease of apoptotic cells from normal cervix to cancers. A tendency to negative correlation between cFLIP and apoptosis (r=-0.499;P=0.000) was observed. Conclusions Deregulated Fas/FasL system and constitutive expression of cFLIP in the SCCs could be an important mechanism by which SCCs escape apoptosis during malignant transformation and progression of these tumors.