Abstract
Purpose: Human papillomavirus (HPV)16 E6 and E7 oncoproteins that are required for cellular transformation represent candidate targets for HPV specific and major histocompatibility complex (MHC) class-I restricted CD8+ T-cell responses in patients with cervical cancer. In the present study, we identified HPV specific T-cells in different biological compartments (peripheral blood lymphocytes [PBL], tumor tissue, tumor-draining lymphnodes) using MHC-class I tetramers containing an appropriate HPV 16 E7 peptide. Material and Methods: FACS sorted (fluorescence activated cell sorting) CD8+T-cells isolated from healthy blood donors (n=10), patients with carcinoma in situ (n = 9) and invasive cervical cancer (n = 15) were obtained from PBL, tumor and pelvic nodes and evaluated for the presence of HPV 16 E7 specific effector CD3 + lymphocytes using the new tetramer technology. HPV DNA in tumor tissue was detected by PCR. All cervical cancer lesions could be identified as HPV 16 positive. PBL, tumor infiltrating lymphocytes (TIL), or T-cells from tumor-draining lymph nodes were isolated, gated on CD3 + CD8+T-cells as indicated and tested for the frequency of HPV 16 E7 using HLA-A2 tetramer complexes. All patients included showed an HLA-A2 haplotype. Results: 50% of healthy individuals, but less than 20% of patients (cervical intraepithelial neoplasia (CIN), invasive cancer) showed a specific cytotoxic CD8 + immune response to a defined HPV 16 E7 11-19 epitope (YMLDLQPET) in peripheral blood. HPV-reactive cytotoxic T-cells were detectable in tumor-draining lymph nodes in only one patient. Conclusions: The tetramer-sorting of CD8 + T-cells from different biological compartments in patients with HPV associated cancer lesions permits the determination of the target specificity of CD8 + effector T-cells without further need for in vitro manipulation. Furthermore, this new method helps to define the most appropriate target epitopes for novel vaccine designs.
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