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Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.