[Show abstract][Hide abstract] ABSTRACT: Cervical cancer is caused by Human papillomavirus (HPV) in virtually all cases. These HPV-induced cancers express the viral oncogenes E6 and E7 and are therefore potentially recognized by the immune system. Despite the abundant presence of these foreign antigens, the immune system is unable to cope with the tumor. Due to the constant immunological pressure, cervical cancers can evolve different immune evasion strategies, which will be described in the current review. Several approaches for immunotherapy of cervical cancer are currently under development, which aim at inducing strong HPV-specific immunity. Besides the reinforcement of potent anti-tumor immune responses, immunotherapy could also enhance HPV-specific T regulatory cells. Supplementary strategies that neutralize an immunosuppressive milieu may have great potential. These strategies are discussed as well.
Preview · Article · May 2011 · Cancer Microenvironment
[Show abstract][Hide abstract] ABSTRACT: Control and termination of infection with Influenza A virus is associated with increased IL-10 production in mouse models. Notably, IL-10 can be produced by Treg. Therefore, we investigated whether the population of IL-10-producing influenza-specific CD4(+) T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza-specific IL-10-producing T cells were detected in all human donors displaying influenza-specific immunity. Isolation of Matrix 1 protein-specific IL-10-producing T-cell clones revealed that a substantial proportion of these T-cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3(+) and FOXP3(-) CD4(+) Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus-infected cells. In vitro suppression was not mediated by IL-10 but involved interference with the IL-2 axis. The isolated Treg suppressed amongst others the IL-2 production of influenza-specific T-helper cells as well as partially prevented the upregulation of the high-affinity IL-2 receptor on CD8 effector cells. So far the induction of virus-specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus-specific Treg can also be induced by viruses that are rapidly cleared in humans.
Full-text · Article · Nov 2010 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: 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 IFNgamma (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 IFNgamma/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.
Full-text · Article · Jun 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The diversity and extent of the local tumor-specific T-cell response in a given individual is largely unknown. We have performed an in-depth study of the local T-cell repertoire in a selected group of patients with cervical cancer, by systematic analyses of the proportion, breadth, and polarization of human papillomavirus (HPV) E6/E7-specific T cells within the total population of tumor-infiltrating lymphocytes (TIL) and tumor-draining lymph node cells (TDLNC). Isolated T cells were stimulated with sets of overlapping E6 and E7 peptides and analyzed by multiparameter flow cytometry with respect to activation, cytokine production, and T-cell receptor Vbeta usage. HPV-specific CD4+ and CD8+ T-cell responses were detected in TIL and TDLNC and their relative contribution varied between <1% and 66% of all T cells. In general, these HPV-specific responses were surprisingly broad, aimed at multiple E6 and E7 epitopes and involved multiple dominant and subdominant T-cell receptor Vbetas per single peptide-epitope. In most patients, only few IFNgamma-producing T cells were found and the amount of IFNgamma produced was low, suggesting that these are poised T cells, rendered functionally inactive within the tumor environment. Importantly, stimulation of the TIL and TDLNC with cognate antigen in the presence of commonly used Toll-like receptor ligands significantly enhanced the effector T-cell function. In conclusion, our study suggests that within a given patient with HPV-specific immunity many different tumor-specific CD4+ and CD8+ T cells are locally present and poised for action. This vast existing local T-cell population is awaiting proper stimulation and can be exploited for the immunotherapy of cancer.
[Show abstract][Hide abstract] ABSTRACT: The interaction between programmed cell death 1 (PD-1), expressed by activated effector or regulatory T cells, and B7-H1 (PD-L1) and B7-DC (PD-L2) results in the inhibition of T-cell function. The aim of this study was to determine B7-H1, B7-DC, and PD-1 expression in cervical carcinoma.
A tissue microarray of a well-defined group of 115 patients was stained with antibodies against B7-H1 and B7-DC. Three-color fluorescent immunohistochemistry was used to study the number and phenotype of tumor-infiltrating T cells expressing PD-1. Additional analyses consisted of in vitro T-cell suppression assays.
B7-H1 was expressed in 19%, and B7-DC was expressed by 29% of the 115 tumors. PD-1 was expressed by more than half of both the infiltrating CD8+ T cells and CD4+Foxp3+ T cells, irrespective of B7-H1 or B7-DC expression by tumors. The expression of B7-H1 did not show a direct impact on patient survival. However, subgroup analysis revealed that patients with a relative excess of infiltrating regulatory T cells displayed a better survival when the tumor was B7-H1 positive (P = 0.033). Additional studies showed that the presence of B7-H1 during the activation of CD4+Foxp3+ regulatory T cells impaired their suppressive function in a functional in vitro assay.
B7-H1 is expressed on only a minority of cervical cancers and does not influence the survival of patients with cervical cancer. PD-1 is expressed by a vast number of infiltrating CD8 T cells, suggesting that blocking of PD-1 could have therapeutic potential in cervical cancer patients.
No preview · Article · Oct 2009 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: To characterize HPV16 E6- and E7-specific T-cell immunity in patients with high-grade squamous intraepithelial lesions (HSIL).
Peripheral blood mononuclear cells isolated from 38 patients with HPV16+ HSIL were used to determine the magnitude, breadth, and polarization of HPV16-specific T-cell responses by proliferation assays and cytokine assays. Furthermore, HSIL-infiltrating T cells isolated from 7 cases were analyzed for the presence of HPV16 E6- and/or E7-specific T cells, phenotyped, and tested for the specific production of IFN-gamma and interleukin-10 as well as for their capacity to suppress immune responses.
HPV16-specific T-cell responses were absent in the circulation of the majority (approximately 60%) of patients who visit the clinic for treatment of a HPV16+ HSIL lesion. Notably, HPV16-specific T-cell reactivity was predominantly detected in patients returning to the clinic for repetitive treatment of a persistent or recurrent HPV16+ HSIL lesion after initial destructive treatment. The majority (> 70%) of these HPV16-specific T-cell responses did not secrete proinflammatory cytokines, indicating that most of the subjects, although in principle able to mount a HPV16-specific immune response, fail to develop protective cellular immunity. This notion is sustained by our observation that only three HSIL-infiltrating T-cell cultures contained HPV16-specific T cells, one of which clearly consisted of HPV16 E7-specific regulatory T cells.
The presence of HPV16-specific T cells with a non-Th1/Th2 cytokine and even suppressive signature in patients with HSIL may affect the outcome of vaccine approaches aiming at reinforcing human papillomavirus-specific immunity to attack human papillomavirus-induced lesions.
Full-text · Article · Nov 2008 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy against cancer does not result in impressive clinical responses. Failure of vaccine-induced T-cell-mediated immunotherapy to improve therapeutic results occurs for many reasons, including regulatory networks imposed by growing tumours, intrinsic properties of cancer cells, suboptimal design of therapeutic vaccines and subversion of the immune system. Most vaccines are incapable of inducing strong immunity, the vaccine's capacity to induce effector T cells is blocked by regulatory T cells (Tregs) in the lymph nodes, the vaccine-induced T cells are not able to infiltrate the tumour, tumour-infiltrating T cells cannot exert their effector function due to locally present Tregs and/or vaccines may induce/boost Tregs.
We focus on the negative effects of Tregs in cancer immunotherapy and highlight potential therapeutic options to counteract these.
Interplay and balance between Tregs and effector cells determines the efficacy of vaccines and thereby clinical outcome.
No preview · Article · Oct 2008 · Expert opinion on biological therapy
[Show abstract][Hide abstract] ABSTRACT: A large body of evidence indicates that the presence of regulatory T cells (Tregs) in tumors is associated with a dampened tumor-specific immune response and a clear negative impact on survival. Many different subsets of Tregs have been identified, which all act through similar or distinct pathways to suppress tumor-specific effector cells. The observation that tumor-infiltrating Tregs are able to recognize tumor-derived antigens and can be expanded by vaccines that primarily aim at reinforcing the effector arm of the antitumor response stresses the need to study Tregs for each type of cancer targeted by immunotherapy. Current protocols enable us to isolate and culture tumor-infiltrating Tregs. Ultimately, this will not only lead to a full comprehension of the specificity and working mechanisms of Tregs but also facilitate the development of successful interventions strategies for the immunotherapy of cancer.
[Show abstract][Hide abstract] ABSTRACT: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients.
Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFN gamma-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3.
Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4(+) and CD8(+) T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype.
The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4(+) and CD8(+) T cells to a broad array of epitopes in all patients. The expansion of CD4(+) and CD8(+) tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4(+)CD25(+)Foxp3(+) phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.
Full-text · Article · Feb 2008 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: 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 CD4+ and CD8+ 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 CD4+ 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.
Full-text · Article · Feb 2008 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Because of their important role in the maintenance of self-tolerance, 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 tumor-associated 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-gamma, 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.
Full-text · Article · Aug 2007 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: 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-) 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-) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN- patients displaying a concomitant systemic tumor-specific immune response (LN-IR+). CD8+ T-cell infiltration in LN-IR- patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN- patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis.
[Show abstract][Hide abstract] ABSTRACT: Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.
[Show abstract][Hide abstract] ABSTRACT: Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.
[Show abstract][Hide abstract] ABSTRACT: Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in sensitivity to Diphtheria Toxin. This decrease was almost immediate, indicating that this phenomenon was not caused by a change in membrane structure or protein expression. We investigated the effect of SFM on Diphtheria Toxin in order to determine the cause of the decrease in sensitivity. Our results show that oligopeptides, which are often used in SFM as part of the replacement of foetal calf serum, are the most likely cause.
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer is induced by HPV in virtually all cases, but it is still unknown how these virus-positive tumors arise in the face of immunity. In this thesis we examined the role of different arms of the adaptive immune response on cervical cancer. In previous studies, it was shown that cancer patients do have a detectable immune response against the HPV16 E6 and E7 antigens. However, in general this response was not associated with cytokine production. In the present study, the mechanism underlying this dysfunctional immune response was investigated by studying the presence, specificity and function of the local T-cell response in patients with HPV-induced cervical malignancies at different stages of disease. Low CD8/Treg ratio is a significant independent unfavorable prognostic factor in cervical cancer patients. Frequently, the tumor is also infiltrated with HPV-specific CD4+ and CD8+ T-cells which can potentially attack the tumor. The infiltrating T-cells also include HPV-specific regulatory T-cells which are able to suppress the reactivity of neighboring T-cells. Their presence may explain the failure of the immune system to control HPV-induced tumors.