Altered Expression Of HLA Class I And APM Components In Esophageal Squamous Cell Carcinoma And Experimental Immunotherapy

Esophageal carcinoma is one of the most common and highly aggressive malignant neoplasms in the world, particularly in China. More than 90% of esophageal cancers are ESCC. Despite advances made in clinical treatment during the past several decades for the treatment of ESCC, the prognosis of ESCC remains poor with the 5-year survival rate of approximately 15%. There is an urgent need to develop a novel therapeutic strategy for this disease, and immunotherapy may be a promising approach for improvement of ESCC treatment. However, the tumor associated antigens that can be used as vaccine(s) for successful ESCC immunotherapy have not been well identified, and it has been suggested that the HPV16 E7 protein could be a potential target for ESCC vaccines.The discovery of tumor antigens has led to research and development of therapeutic vaccines designed to generate antitumor responses with the aim of controlling metastasis. The HLA ClassⅠrestricted antigen presentation pathway consists of a number of genes. The pathway generates peptides from endogenous proteins by the degradative action of proteasome units LMP2 and LMP7. The peptides are transported from the cytoplasm into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP), a heterodimer that is composed of 2 subunits, TAP1 and TAP2. Within the ER, the peptide may be loaded onto an HLA ClassⅠmolecule, which consists of HLA heavy chain and beta-2-microglobulin (β2M). The assembly of this complex is aided by a number of chaperone proteins including calreticulin and calnexin, which are responsible for the folding of HLA ClassⅠheavy chains and stabilization of their association withβ2M, and tapasin, which is responsible for anchoring the HLA molecules to TAP and loading the peptides onto the HLA ClassⅠmolecules. The properly assembled complex is then transported to the cell surface via the Golgi. The function of these molecules offers a ligand to the TCR of CD8+T-cells. If the TCR binds with high enough affinity, activation of the T-cell occurs and the target cell can be destroyed. Any alterations or deficiencies in the antigen presentation pathway can lead to variants, which give rise to nonimmunogenic tumors.Among these genes, TAP1 is a critical factor in HLA classⅠantigen deficiencies and has been associated with disease progression and death in human malignancies of distinct histology. Its restoration or re-expression is able to enhance HLA classⅠantigen and a number of APM components in tumor cells, as a result the tumor cells can be recognized and eventually killed by the CTL. It has been suggested that TAP1 re-expression is a general method for cancer immunotherapy.Here we investigated the expression of HLA classⅠantigen and TAP1, the key APM molecule in esophageal cancer lesions by tissue microarray assay and cell lines, and assessed the role of defects in the expression of these molecules during the progression and immunotherapy of ESCC. The expression of HLA-Ⅰ,β2m, TAP1 and HPV infection were examined by immunohistochemical staining and in situ hybridization in 200 formalin-fixed and parafin-embedded esophageal lesions (120 primary ESCC,40 matched adjacent squamous epithelium and lymph node metastases) in a tissue microarray, respectively.We found that TAP1 expression was positive in all adjacent squamous epithelium, and positive in 55.0% and 15.0%, reduced in 22.5% and 27.5%, or absent in 22.5% and 57.5% of ESCC primary and metastatic lesions, respectively. The difference of expression for TAP1 among the 3 groups was statistically significant (P<0.001). Primary ESCC lesions with lymph node metastasis had significantly lower expression of TAP 1 than those without lymph node metastasis (P<0.05). Moreover, TAP1 expression was significantly associated with tumor grade (P<0.05). These results suggested that TAP1 could be a factor of esophageal cancer progression. HLA-Ⅰwas positive in 50%, reduced in 20%, and absent in 30% of adjacent squamous epithelium, whileβ2m was positive in 27.5% and absent in 72.5% of these cases, which suggested that downregulation of HLA-Ⅰandβ2m expression is frequent in the pathogenesis of ESCC. Significant downregulation of HLA-I andβ2m was also observed in primary and metastatic lesions, and was not associated with tumor grade. Moreover, HLA-I andβ2m expression was more frequently down-regulated in primary ESCC lesions with lymph node metastasis than those without lymph node metastasis (P<0.05). All lymph node metastases and 114 of 120 ESCC lesions exhibited deficient expression of at least one of the three molecules. The positive rate of HPV type 16/18 in 40 matched adjacent squamous epithelium, primary tumors and lymph node metastases was 52.5%,57.5% and 42.5%, respectively. No significant difference was found among them. And there was no direct correlation between the infection of HPV DNA and altered HLA-I, (32m and TAP1 expression.The results showed that there was a high frequency of down-regulation expression of HLA classⅠantigen and APM molecules in ESCC, which was one of the major hurdles for ESCC immunotherapy. The development of a suitable tumor model with downregulated HLA classⅠantigen and APM molecules expression is critical for assessing the relationship of HLA classⅠantigen and APM molecules expression and immunotherapy of ESCC. Flow cytometry analysis of four esophageal cancer cell lines demonstrated that HLA classⅠsurface expression was significantly reduced. RT-PCR and Western blot showed that TAP2, LMP7, LMP10, PA28a and PA28βmRNA were expressed in KYSE510 constitutively, while at least one of these genes was down-regulated or lost in the other three cell lines (EC9706, KYSE150 and NEC). TAP1,β2m and LMP2 protein expressions were downregulated or lost in all of the 4 cell lines, but the decreased level of tapasin only existed in KYSE150. IFN-γtreatment could correct the abnormal expression of these genes, indicating that the lost or markedly reduced level of these molecules in the cell lines is not due to structural change of the genes. Immunoblotting revealed that the EC9706 cell line exhibits the highest level of HPV16-E7 protein among the four esophageal cancer cell lines. Thus, EC9706 was chosen to establish an E7-expressing ESCC/Hu-PBL-SCID mouse model, which is characterized by downregulation of many of the components of the APM including HLA classⅠheavy chain,β2m, TAP1 and 2, LMP2, and PA28β. Next, we tested fusion protein vaccine for the ability to control tumor growth in vivo and found that vaccination (HHP) generated a significant antitumor effect against EC9706, and prolonged the survival time of tumor-bearing mice as compared with the control groups (P<0.05). CTL assay and IFN-y Elispot assay revealed that splenocytes from vaccinated-mice exhibit a significant increase in tumor-specific lysis of EC9706 cell line (P<0.05). Moreover, much more activated lymphocytes (CD8+) were infiltrated into the tumor site, and significant up-regulation of IFN-y, HLA classⅠand APM molecule TAP1 was only detected in the tumors from vaccinated-mice. The lysed rate of IFN-y treated EC9706 cells by CTL from vaccinated-mice was further enhanced in in vitro experiment as compared with the untreated ESCC cells. In conclusion, our results suggest a high frequency of TAP1, HLA-I and p2m down-regulation and/or loss was found in ESCC lesions and in ESCC cell lines studied, probably by providing ESCC cells with a mechanism to escape from CTL recognition during disease progression. The high HPV infection rate in esophageal carcinoma of Shanxi province is consistent with former studies in other high-risk areas of ESCC, which may be attractive targets for immunotherapeutic strategies. Deficient TAP1 and HLA classⅠsurface expression in the cell lines can be corrected by IFN-γtreatment. To assess the relationship between these molecules expression and immunotherapy, E7-expressing ESCC Hu-PBL-SCID mouse model was established. A significant antitumor effect of HHP vaccine was observed. Significant up-regulation of TAP1 and HLA classⅠantigen was only detected in the tumors from vaccinated-mice. These results could contribute to optimize T cell-based strategies against esophageal cancer.