Cervical cancer is the possible outcome of genital infection with highrisk human papillomavirus (HPV) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Fortunately, the majority of genital HPV infections are cleared before the development of (pre)malignant lesions. Analysis of CD4؉ T-helper (Th) immunity against the E2, E6, and E7 antigens of HPV16 in healthy women revealed strong proliferative E2-and E6-specific responses associated with prominent IFN-␥ and interleukin 5 secretion. This indicates that the naturally arising virus-induced immune response displays a mixed Th1/Th2 cytokine profile. Of all HPV16؉ cervical cancer patients, approximately half failed to mount a detectable immune response against the HPV16-derived peptides. The other half of the patients showed impaired HPV16-specific proliferative responses, which generally lacked both IFN-␥ and interleukin 5. This indicates that the HPV16-specific CD4؉ T-cell response in cervical cancer patients is either absent or severely impaired, despite a relatively good immune status of the patients, as indicated by intact responses against recall antigens. It is highly conceivable that proper CD4؉ T-cell help is important for launching an effective immune attack against HPV because infection of cervical epithelia by this virus is, at least initially, not accompanied by gross disturbance of this tissue and/or strong proinflammatory stimuli. Therefore, our observations concerning the lack of functional HPV16-specific CD4؉ T-cell immunity in patients with cervical cancer offer a possible explanation for the development of this disease.

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16 + )-induced or HPV18 + -induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4 + , CD8 + , and regulatory T cells as well as by calculation of the ratio of CD8 + /CD4 + T cells and CD8 + / regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6-and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN À ) or presence (LN + ) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8 + T-cell tumor infiltration, a higher CD8 + /CD4 + T-cell ratio, and higher CD8 + /regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR + ) or absence (IR À

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNγ (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4 + CD25 + Foxp3 + T cells (P = 0.005) and displayed a lower HPV16-specific IFNγ/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4 + CD25 + Foxp3 + T cells was predictive of clinical success. Foxp3 + T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.human papilloma virus | immunomonitoring | therapeutic vaccine | regulatory T cells

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 ...

Human papillomavirus (HPV)-induced malignancies are frequently infiltrated by lymphocytes. To comprehend the contribution of HPV-specific T cells in this anti-tumor response we developed a method that allowed the analysis of the presence and specificity of cervix-infiltrating and draining lymph node resident T cells in a group of 74 patients with cervical malignancies, 54 of which were induced by HPV16 or HPV18. We detected the presence of HPV16 or HPV18-specific T cells in at least 23 of the 54 HPV-16 or -18 positive patients, and not in the 20 controls. Detailed studies resulted in the identification of 17 novel CD41 and CD81 T cell epitopes and their HLA-restriction elements, and also revealed that the HPV-specific immune response was aimed at both E6 and E7 and showed no preferential recognition of immunodominant regions. Unexpectedly, the vast majority of the CD41 T cell epitopes were presented in the context of the less abundantly expressed HLA-DQ and HLA-DP molecules. Since the identified T cell epitopes constitute physiological targets in the immune response to HPV16 and HPV18 positive tumors they will be valuable for detailed studies on the interactions between the tumor and the immune system. This is crucial for the optimization of cancer immunotherapy in patients with pre-existing tumor-immunity. ' 2007 Wiley-Liss, Inc.Key words: human papillomavirus; immunity; tumor-infiltrating lymphocytes; cervical cancer Cervical cancer is the second most common cancer in women worldwide. 1 High risk human papilloma virus (HPV) type 16 and 18 are the cause of cervical cancer in around two thirds of all patients. 2,3 The HPV genome encodes 2 oncoproteins, E6 and E7, which are constitutively expressed in high grade cervical lesions and cancer since they are required for the onset and maintenance of the malignant cellular phenotype. 4 The tumor-specific expression of these oncoproteins as well as the presence of low levels of circulating E6-and E7-specific T cells detected in the peripheral blood of almost half the patients with cervical cancer 5-11 suggested that they could serve as tumor rejection antigens. However, the existence of circulating HPVspecific T cells does not imply that they contribute to the anti-tumor response. To control the disease, these T cells should at least be able to home to the tumor sites. Indeed, a proportion of cervical carcinomas are infiltrated by lymphocytes 12-14 but in-depth knowledge on the specificity of the T cells infiltrating these cervical tumors is still lacking, probably due to relative difficulties in establishing T cell cultures from tumor tissue. Nonetheless, a few early pioneers were able to isolate HPV-specific tumor infiltrating lymphocytes (TIL) from tumors, resulting in the identification of 2 single CD81 T cell epitopes of HPV16 15,16 and 2 CD4 T cell epitopes specific for the less prevalent high risk subtypes HPV59 and HPV33. 17,18 However, larger studies on cervical tissue-infiltrating lymphocytes are urgently needed to comprehend the contribution and role of the...