P16^INK4a is a tumor autoantigen in HR-HPV induced cancers

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The cellular tumor suppressor gene p^16INK4a is over-expressed in a large number of high-risk human papilloma virus (HR-HPV) induced tumors, especially in cervical cancers. The massive over-expression results from a disrupted feed back mechanism of the Rb/E2F pathway induced by the HPV oncoprotein E7. In contrast, p16INK4a is expressed only in few normal cells, mainly terminally differentiated cells with low antigen presenting capacity. We could show previously, that T-cells from healthy donors can be stimulated to react against p16^INK4a peptides presented by HLA-A2 positive cells. To further analyze if p16^INK4a acts as a tumor autoantigen in cervical cancer, humoral and cellular immune responses against p16INK4a were monitored in cervical cancer patients. To measure humoral immune responses, sera derived from 70 cervical cancer patients were analyzed by western blotting. Using recombinant p16^INK4a protein blotted on nylon membranes, five patients showed clear p16^INK4a specific signals that could be blocked by serum pre-incubation with recombinant p16INK4a. For the analysis of cellular immune responses, infiltrating T-cells (TILs) were isolated from ten freshly resected cervical cancers or highly dysplastic lesions (CIN3) and tested for their reactivity against p16^INK4a peptides. In two patients, TILs with a significant reactivity against the identical p16^INK4a peptide that showed strong stimulation of the healthy donor T-cells were isolated from the respective lesions. These findings indicate that cervical carcinoma patients can develop humoral and cellular immune responses against the cellular tumor suppressor protein p16^INK4a. P16^INK4a can therefore be added to the group of over-expressed tumor autoantigens (like HER2/neu, hTERT, MUC1) and might be used as a novel target in immunotherapy of HR-HPV induced cancer.