Human papillomavirus (HPV)-associated vulvar intraepithelial neoplasia (VIN) has serious sequelae for the sufferer. Current treatments are associated with poor response and high relapse rates. The development of HPV-specific T cell immunotherapies offers a new approach to treatment. This will require a detailed understanding of the spectrum of T cell responses induced by HPV antigens, and how effectively viral antigens can be accessed by the immune system. We have investigated the frequency and spectrum of HPV16-specific CD8+ T cell responses to three HPV 16 antigens in 9 women with high grade VIN (VIN3). CD4-depleted populations of responder cells were screened against overlapping 30-35mer peptides covering the sequences of HPV16 E6, E7 and E4 using ELISPOT assays of IFN-gamma release. We demonstrated CD8+ T cell reactivity to one or more of the proteins in 6 of 9 patient samples. All 6 of these responders recognised peptides covering the E7 protein, 3 of 9 women responded to E6 peptides, but no reactivity was seen to E4. Our results suggest that HPV16-specific cytotoxic T cells (CTLs) are relatively common in women with persistent VIN3. The HPV-specific CTL response, however, seems to be ineffective. There is some evidence that there are problems associated with the processing and presentation of HPV antigens by the infected vulvar epithelium. It will be crucial to address this in the design of any T cell based therapy, for HPV-associated VIN and vulval cancer. (C) 2003 Wiley-Liss, Inc.