Because of their important role in the maintenance of selftolerance, CD4 ؉ regulatory T cells prevent autoimmune diseases but also curtail the efficacy of T cell immune responses against cancers. We now show that this suppressive action of CD4 ؉ regulatory T cells is not limited to cancers displaying tumorassociated self antigens, such as melanomas, but also extends to human papillomavirus (HPV)-positive cervical cancers that express foreign tumor antigens. HPV-specific CD4 ؉ T cells isolated from lymph node biopsies of cervical cancer patients were found to suppress proliferation and cytokine (IFN-␥, IL-2) production by responder T cells. The capacity of HPV-specific CD4 ؉ T cells to exert this suppressive effect depended on their activation by cognate HPV antigen and on close-range interactions with responder T cells. HPV-specific CD4 ؉ regulatory T cells were also retrieved from cervical cancer biopsies, suggesting that they interfere with the anti-tumor immune response at both the induction and effector levels. Our findings offer a plausible explanation for the observed failure of the tumor-specific immune response in patients with cervical carcinoma.suppression ͉ Treg ͉ vaccine ͉ immunotherapy C ervical carcinomas arise as result of an uncontrolled persistent infection with a high-risk type of human papillomavirus (HPV), in particular, types 16 (HPV16) and 18 (HPV18), which account for approximately two-thirds of these cancers (1, 2). The HPV E6 and E7 proteins play a pivotal role in carcinogenesis and are expressed in both premalignant and advanced cervical lesions (3). Because HPV proteins are foreign to the body, one would expect the immune system to mount a response against these antigens when expressed in the cervical epithelium. Indeed, HPV16 E6-, E7-, and E2-specific Th1-and Th2-type CD4 ϩ T cell responses were frequently detected in peripheral blood mononuclear cell (PBMC) cultures of healthy individuals (4-6), showing that successful defense against HPV16 infection is commonly associated with the installment of a systemic effector T cell response against these viral antigens. In contrast, PBMC cultures from patients with HPV16-positive genital lesions either lacked detectable responses against HPV16 E6, E7, and E2 or displayed antigen-specific proliferative responses exhibiting a noninflammatory cytokine profile (5,7,8). Similarly, effective HPV18-specific T cell responses are only found in healthy controls but not in HPV18-positive patients (9). Taken together, these findings indicate that the development of high-risk HPVpositive cervical cancer is associated with failure of the HPVspecific T cell response.Studies in mouse models demonstrated that CD4 ϩ regulatory T cells play a critical role in curtailing effective immune responses against tumors (10, 11) and that these T cells can be primed in the same tumor-draining lymph nodes as their tumoricidal counterparts (12). Furthermore, analysis of tumor biopsies from melanoma patients revealed the presence of CD4 ϩ regulatory T cells recognizing the ...

BackgroundHuman papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma.MethodsPatients with HPV16-positive advanced or recurrent gynecological carcinoma (n = 20) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT.ResultsNo systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient’s immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease.ConclusionsThe HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore, plan to use this vaccine in combination with chemotherapy and immunomodulation.

This study investigates the clinical course of low grade squamous intraepithelial lesions (LSIL), HPV status and HPV16-specific immune response in a large prospective study of 125 women with LSIL followed cytologically, virologically and histologically. Women with low-grade abnormal smears were recruited and followed-up for one year. Colposcopy, cervical biopsy for histology and brushings for HPV typing was performed at recruitment, 6 months (no biopsy) and upon completion of the study at one year. HPV16-specific T-cell responses were analysed by interferon-c ELISPOT at entry, 6 and 12 months. Infection with multiple HPV types was detected in 70% of all patients, HPV16 was found in 42% of the patients. LSIL lesions progressed to HSIL in 24%, persisted in 60% and regressed to normal in 16% of the patients. No difference was observed in the clearance rate of infections with single or multiple HPV types among the groups with a different histological outcome. HPV16-specific type 1 T-cell responses were detected in only half of the patients with an HPV161 LSIL, and predominantly reactive to HPV16 E2 and E6. Interestingly, the presence of HPV16 E2-specific T-cell responses correlated with absence of progression of HPV161 lesions (p 5 0.005) while the detection of HPV16 E6 specific reactivity was associated with persistence (p 5 0.05). This large prospective study showed that the majority of LSIL persisted or progressed within the first year.This was paralleled by immune failure as most of the patients with an HPV161 LSIL failed to react to peptides of HPV16 E2, E6 or E7.Cervical cancer is preceded by a spectrum of epithelial atypia known as cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL) characterized by disturbances of cellular maturation, stratification and cytological atypia of increasing severity. CIN/SIL are caused by persistent infection with one of a subset of genital human papillomaviruses (HPV), in particular HPV type 16. Natural history studies 1 show that most (90%) low grade cervical intraepithelial neoplasia (CIN 1) regress spontaneously and this is attributed to the development of HPV antigen specific cellular immune responses 2 but 30-40% of CIN3 progress to invasive cervical cancer 3 and spontaneous regression is relatively uncommon. The key role of the adaptive cellular immune system in protection against HPV-induced lesions is indicated by the high incidence of persistent HPV-infections and subsequent HPV-related malignancies in immunosuppressed individuals 4 and the observation that only a small fraction of infected non-immunosuppressed subjects develop progressing epithelial lesions or cancer. 5 Composite data indicate the importance of CD4þ T-cells in the control of HPV-induced diseases as more severe lesions are observed in patients with low numbers of circulating CD4þ cells 6,7 and the increase in CD4 count, after anti-retroviral treatment, correlates with the regression of HPV-induced CIN lesions in HIV þ patients. 8 At the time of spontaneous regression of HP...

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n 5 11) and healthy individuals (n 5 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV161 patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD41 Th1/Th2 and CD81 T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity. ' 2008 Wiley-Liss, Inc.Key words: HPV16/delayed type hypersensitivity; cellular immunity; ELISPOT; skin testThe main cause of cervical carcinoma and cervical intraepithelial neoplasia (CIN) is a persistent infection with one of the highrisk, oncogenic human papilloma viruses.1,2 Anogenital infection with a high-risk HPV type is very common.3-5 The rate of incidence of infection is estimated to be 80-85% worldwide.6 Fortunately, the majority of infected subjects will clear the virus infection within a year 7,8 , and only a small proportion of women will become persistently infected and are at risk to develop HPVrelated malignancies.9,10 Cell-mediated immune responses play an important role in controlling HPV infections. 11,12Delayed Type IV hypersensitivity (DTH) reactions are used as a general measure of cell-mediated immunity in vivo.13 A local inflammatory reaction (induration and erythema), orchestrated by activated cytokine releasing CD41 memory T cells, usually appears within 24-72 hr after intradermal injection of antigen. [14][15][16] Delayed-type hypersensitivity reactions have been used to demonstrate an encounter with pathogens (e.g., Mantoux Test), vaccineinduced immune responses, [17][18][19][20] and in particular to show HPVspecific immune responses in various animal models.21-23 H€ opfl et al. were the first to study HPV16-specific cellular immunity by skin test in humans 24,25 and showed that a skin reaction, appearing within 2-6 days after intradermal injection with HPV16 E7, was correlated with regression of HPV-induced CIN lesions.We have previously studied the HPV16-specific T-cell responses in great detail in vitro, and the results of these studies suggested that the HPV16 E2, E6 and E7 specific Type 1 and Type 2 T-cell response ...