Cycloxygenase-2 inhibition augments the efficacy of a cancer vaccine.

Tumor-derived cyclooxygenase-2 (COX-2) and its product, prostaglandin E2, exert strong immunoinhibitory effects that block dendritic cell function and CD4+ and CD8+ T-cell proliferation and function. We have shown previously that the addition of an oral COX-2 inhibitor to immunogene therapy using IFN-beta markedly augmented therapeutic efficacy in murine tumor models. In this study, we hypothesized that COX-2 inhibition might also augment an antitumor vaccination strategy. Mice bearing tumors derived from TC1 cells, a tumor line that expresses the human papillomavirus (HPV) E7 protein, were thus vaccinated with an adenoviral vector expressing HPV E7 protein (Ad.E7). This vaccine approach effectively generated E7-specific CD8+ cells and slowed the growth of small tumors but had little effect on large tumors. However, feeding mice with the COX-2 inhibitor, rofecoxib, restored the effectiveness of the vaccine against large tumors and prolonged survival. This effect was accompanied by a larger percentage of E7-specific CD8+ cells in the regional draining lymph nodes and a markedly increased number of tumor-infiltrating E7-specific CD8+ cells (as determined by flow cytometry) and total CD8+ T cells (as determined by immunohistochemical staining). Increased immunocyte trafficking was likely mediated by the generation of a Th1-type tumor microenvironment because COX-2 inhibition increased expression levels of mRNA for IFN-gamma, interleukin-12, IP-10, and MIG while lowering the expression of vascular endothelial growth factor within tumors. This study shows that the effectiveness of a cancer vaccine can be significantly improved by adding COX-2 inhibition.     

CD4+ T cell-mediated antigen-specific immunotherapy in a mouse model of cervical cancer

A major agenda for tumor immunology is the generation of specific immune responses leading to the destruction of incipient and frank neoplasia. In this report, we show that a novel HPV16 E7 fusion protein can produce objective therapeutic responses against incipient cervical cancer in genetically engineered mice that express in the cervix the HPV16 early region genes implicated as causative agents in human cervical cancer. Although nonresponsive toward the HPV16 E7 oncoprotein in the CD8+ T-cell compartment by virtue of MHC haplotype, the mice were capable of mounting an induced CD4+ T-cell response against E7, and in addition developed spontaneous anti-E7 antibodies. HPV16/CD4-/- mice showed increased tumor burden indicative of CD4-mediated immune surveillance. Seeking to enhance the CD4 response, we immunized mice bearing incipient cervical cancer with a recombinant protein fusing E7 with a mycobacterial heat shock protein. The incidences of cervical carcinoma and of high-grade dysplasia (CIN 3) were consequently reduced by comparison to control mice. Thus, an HPV16 E7 immunogen holds promise for noninvasive treatment and prevention of human cervical cancer.     

Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.     

Pretreatment with cisplatin enhances E7-specific CD8+ T-Cell-mediated antitumor immunity induced by DNA vaccination.

PURPOSE: Because the combination of multiple modalities for cancer treatment is more likely to generate more potent therapeutic effects for the control of cancer, we have explored the combination of chemotherapy using cisplatin, which is routinely used in chemotherapy for advanced cervical cancer, with immunotherapy using DNA vaccines encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 antigen (CRT/E7) in a preclinical model. EXPERIMENTAL DESIGN: We characterized the combination of cisplatin with CRT/E7 DNA vaccine using different regimen for its potential ability to generate E7-specific CD8+ T-cell immune responses as well as antitumor effects against E7-expressing tumors. RESULTS: Our results indicate that treatment of tumor-bearing mice with chemoimmunotherapy combining cisplatin followed by CRT/E7 DNA generated the highest E7-specific CD8+ T-cell immune response and produced the greatest antitumor effects and long-term survival as well as significant levels of E7-specific tumor-infiltrating lymphocytes compared with all the other treatment regimens. Furthermore, we found that treatment with cisplatin leads to the cell-mediated lysis of E7-expressing tumor cells in vitro and increased number of E7-specific CD8+ T-cell precursors in tumor-bearing mice. In addition, we observed that E7-specific CD8+ T cells migrate to and proliferate in the location of TC-1 tumors in mice treated with cisplatin. CONCLUSIONS: Thus, our data suggest that chemoimmunotherapy using cisplatin followed by CRT/E7 DNA vaccine is an effective treatment against E7-expressing tumors and may potentially be translated into the clinical arena.     

CD4+ T cells are able to promote tumor growth through inhibition of tumor-specific CD8+ T-cell responses in tumor-bearing hosts

Modulation of the immune response by established tumors may contribute to the limited success of therapeutic vaccination for the treatment of cancer compared with vaccination in a preventive setting. We analyzed the contribution of the CD4+ T-cell population to the induction or suppression of tumor-specific CD8+ T cells in a tumor model in which eradication of tumors crucially depends on CD8+ T cell-mediated immunity. Vaccine-mediated induction of protective antitumor immunity in the preventive setting (i.e., before tumor challenge) was CD4+ T cell dependent because depletion of this T-cell subset prevented CD8+ T-cell induction. In contrast, depletion of CD4+ cells in mice bearing established E1A+ tumors empowered the mice to raise strong CD8+ T-cell immunity capable of tumor eradication without the need for tumor-specific vaccination. Spontaneous eradication of tumors, which had initially grown out, was similarly observed in MHC class II-deficient mice, supporting the notion that the tumor-bearing mice harbor a class II MHC-restricted CD4+ T-cell subset capable of suppressing a tumor-specific CD8+ T-cell immune response. The deleterious effects of the presence of CD4+ T cells in tumor-bearing hosts could be overcome by CD40-triggering or injection of CpG. Together these results show that CD4+ T cells with a suppressive activity are rapidly induced following tumor development and that their suppressive effect can be overcome by agents that activate professional antigen-presenting cells. These observations are important for the development of immune interventions aiming at treatment of cancer.     

Both E7 and CpG-oligodeoxynucleotide are required for protective immunity against challenge with human papillomavirus 16 (E6/E7) immortalized tumor cells: involvement of CD4+ and CD8+ T cells in protection

An important goal of immunotherapy against human papillomavirus (HPV) infection and the cervical cancer is to control viral infection and the cancer cell growth. Here we investigate the utility of HPV 16 E7 along with CpG-oligodeoxynucleotide (ODN) for protection against HPV-immortalized tumor cells using an animal model. E7+ODN coinjection showed a significant suppression of tumor growth at both prophylactic and therapeutic levels. However, no such effect was observed without addition of both E7 and ODN. We additionally evaluated levels of immune responses by E7+ODN coinjection. E7+ODN resulted in E7-specific antibody (IgG1, IgG2a, IgG2b, and IgG3) and T-helper cell proliferative responses significantly higher than E7 alone. However, CTL responses were induced only by E7+ODN. Moreover, IFN-gamma production was detected only in E7+ODN immunized groups in which IFN-gamma releasing CD4+ (T-helper 1 type) and CD8+ T cells (CTL) were induced only by E7+ODN. Moreover, tumor protection appears to be mediated by CD4+ and in most CD8+ T cells, as determined by in vivo T-cell subset depletion. Taken together, these data suggest that E7+ODN codelivery could be an effective approach to induce E7-specific protective immune responses as a possible immunotherapeutic strategy for cervical cancer.     

Induction of human papillomavirus oncogene‐specific CD8 T‐cell effector responses in the genital mucosa of vaccinated mice

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV‐16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70–80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7_1–98 polypeptide showed induction of 100% tumor protection against s.c. TC‐1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7‐specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7‐specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high‐avidity E7‐specific INF‐γ secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV‐tumors reside.     

Naturally processed and HLA-B8-presented HPV16 E7 epitope recognized by T cells from patients with cervical cancer

Several major histocompatibility complex (MHC) alleles have been reported to present peptides derived from the HPV16 E7 oncoprotein to T cells. We describe an overrepresentation of the HLA-B8 allele (28.44%) in cervical cancer patients as compared to the MHC class I allele frequency in a local healthy control population (18.80%) and the identification of an HLA-B8-binding peptide TLHEYMLDL (HPV16 E7(7-15)), which is able to drive HPV16 E7-specific and MHC class I-restricted T-cell responses in peripheral blood lymphocytes from healthy individuals. TLHEYMLDL-specific T cells recognize the naturally processed and presented peptide on HPV16+ cervical cancer cells transfected with the HLA-B8 gene defined by IFN-gamma production. This peptide epitope is also recognized by freshly harvested tumor-infiltrating T cells or T cells from tumor-draining lymph nodes from patients with cervical cancer determined by flow cytometry as well as by tetramer in situ staining. HLA-B8-restricted HPV E7(7-15)-specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7- T-cell population.     

Establishment of an HLA-A*0201 human papillomavirus type 16 tumor model to determine the efficacy of vaccination strategies in HLA-A*0201 transgenic mice

With the increasing generation of new cancer vaccine strategies, there is also an increasing demand for preclinical models that can carefully predict the efficacy of these vaccines in humans. However, the only tumor models available to study vaccines against human papillomavirus (HPV) 16 have been developed in C57BL/6 mice. To test the HLA-restricted capabilities of vaccination strategies, it is important to establish a tumor model in HLA-A*0201 transgenic mice. By transfecting heart lung fibroblasts from HLA-A*0201 mice with HPV16 E6 and E7 oncogenes and H-Ras V12, we have generated a transgenic cell line that is tumorigenic in HLA-A*0201 mice. The dominant H-2D(b) HPV16 E7 epitope was removed from the E7 construct to ensure that all antitumor responses were mediated through the HLA-A*0201-restricted epitopes. We used this tumor model to test the efficacy of two genetic vaccines: a plasmid DNA multi-epitope vaccine encoding human epitopes of HPV16, and a Venezuelan equine encephalitis (VEE) virus-based vector to deliver HPV16 E6 and E7 RNA. We show that both our multi-epitope DNA- and VEE-based vaccines protect 100% of HLA-A*0201 transgenic mice from tumor challenge and elicit a specific T-cell response against multiple HLA-A*0201-restricted HPV16 epitopes. Furthermore, both vaccines significantly decreased tumor burden when tested therapeutically. In conclusion, this is the first tumor model that allows for the assessment of the potential of a vaccine to induce HPV-directed, HLA-A*0201-restricted, antitumor responses in mice. These results pave the way for the clinical evaluation of these vaccines.     

High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer

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.     

Enhancement of DNA vaccine potency by sandwiching antigen-coding gene between secondary lymphoid tissue chemokine (SLC) and IgG Fc fragment genes

DNA vaccine has become an attractive approach for generating antigen-specific immunity. Targeting antigens to FcRs for IgG (FcgammaRs) on dendritic cells (DCs) has been demonstrated to enhance antigen presentation. Secondary lymphoid tissue chemokine (SLC) has been shown to increase immune responses not only by promoting coclustering of T cells and DCs in the lymph nodes and spleen but also by regulating their immunogenic potential for the induction of T cell responses. In this study, using HPV 16 E7 as a model antigen, we constructed a chemotactic-antigen plasmid DNA vaccine (pSLC-E7-Fc) by linking SLC and Fc gene sequences to each end of E7 and evaluated its potency of eliciting specific immune response. We found that immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte proliferative and cytotoxic T lymphocyte (CTL) responses than control DNA. All the mice receiving pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous inoculation of TC-1 cells, while those given control DNA all developed tumors. These tumor-free mice were also protected against TC-1 rechallenge. Complete tumor regression with long-term survival occurred in 72% of mice given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis model wherein TC-1 cells were intravenously injected, therapeutic vaccination with pSLC-E7-Fc significantly reduced the number of tumor nodules in the lung. In vivo depletion with antibodies against CD4+or CD8+ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced immunotherapeutic effect. Our data indicate that the DNA vaccine constructed by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is an effective approach to induce potent anti-tumor immune response via both CD4+ and CD8+ T cells dependent pathways.     

Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination,CpG-oligodeoxynucleotide adjuvant,and CTLA-4 blockade

Although peptide immunization often leads to the induction of strong T-cell responses, it is seldom effective against established tumors. One possibility is that these T-cell responses are not strong enough or do not last sufficiently long to have an effect in tumor eradication. Here, we examined the role of synthetic oligodeoxynucleotide (ODN) adjuvants containing unmethylated cytosine-guanine motifs (CpG-ODN) and CTLA-4 blockade in enhancing the antitumor effectiveness of peptide vaccines intended to elicit CTL responses. The results show that combination immunotherapy consisting of vaccination with a synthetic peptide corresponding to an immunodominant CTL epitope derived from tyrosinase-related protein-2 administered with CpG-ODN adjuvant and followed by systemic injection of anti-CTLA-4 antibodies increased the survival of mice against the poorly immunogenic B16 melanoma. Interestingly, whereas this combination therapy was effective when administered to tumor-bearing mice (therapeutic protocol), it had no significant effect when applied in the prophylactic mode (i.e., before the tumor challenge). Moreover, the antitumor effect of the combination immunotherapy required the participation of CD4+ and CD8+ T lymphocytes and was accompanied by the induction of antitumor CD4+ T-cell responses. The overall results suggest that peptide vaccination of tumor-bearing mice, applied in combination with a strong adjuvant and CTLA-4 blockade, is capable of eliciting durable antitumor T cell responses that provide survival benefit. These findings bear clinical significance for the design of peptide-based therapeutic vaccines for human cancer patients.     

T-cell responses to human papillomavirus type 16 among women with different grades of cervical neoplasia

Infection with high-risk genital human papillomavirus (HPV) types is a major risk factor for the development of cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma. The design of effective immunotherapies requires a greater understanding of how HPV-specific T-cell responses are involved in disease clearance and/or progression. Here, we have investigated T-cell responses to five HPV16 proteins (E6, E7, E4, L1 and L2) in women with CIN or cervical carcinoma directly ex vivo. T-cell responses were observed in the majority (78%) of samples. The frequency of CD4+ responders was far lower among those with progressive disease, indicating that the CD4+ T-cell response might be important in HPV clearance. CD8+ reactivity to E6 peptides was dominant across all disease grades, inferring that E6-specific CD8+ T cells are not vitally involved in disease clearance. T-cell responses were demonstrated in the majority (80%) of cervical cancer patients, but are obviously ineffective. Our study reveals significant differences in HPV16 immunity during progressive CIN. We conclude that the HPV-specific CD4+ T-cell response should be an important consideration in immunotherapy design, which should aim to target preinvasive disease.     

Adenovirus vector‐based prime‐boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV‐infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus‐specific antigens for vaccine‐induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV‐specific CD8⁺ T cells producing IFN‐γ and TNF‐α to the cervicovaginal tract. Importantly, the cervicovaginal CD8⁺ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue‐resident memory CD8⁺ T cells. This prime‐boost strategy targeting heterologous locations also induced circulating HPV‐specific CD8⁺ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.     

Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia

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 = 11) and healthy individuals (n = 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 HPV16+ 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 CD4+ Th1/Th2 and CD8+ 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.     

Arming tumor-reactive T cells with costimulator B7-1 enhances therapeutic efficacy of the T cells

T cells ectopically expressing costimulators are pathogenic and contribute to autoimmunity against self-antigens. Given that tumor antigens are often self-antigen or mutated self-antigens, we hypothesize that neoexpressing a costimulator on tumor-reactive T cells may likewise enhance their reactivity to tumor. To test this hypothesis, we have expressed B7-1 on OT-1 CD8+ T-cell receptor transgenic T cells via protein transfer (or protein "painting"). Na?ve OT-1 T cells, after being painted with B7-1, can self-costimulate themselves, elicit enhanced proliferative and CTL responses to E.G7-ovalbumin tumor cells (expressing a cognate antigen), and become resistant to CD4+CD25+ regulatory T-cell-mediated suppression. Importantly, these T cells, when coimplanted with E.G7-ovalbumin tumor cells into a syngeneic host, are three to nine times more potent than are control T cells (mock painted with human IgG) in inhibiting tumor growth. Further, on transfer into mice bearing established E.G7-ovalbumin tumors, B7-1-painted ex vivo-amplified OT-1 T cells induced complete tumor regression in 65% of treated mice, whereas the control T cells did so in only 28% of treated mice. Finally, on transfer into mice bearing less immunogenic 4T1 breast tumors, B7-1-painted tumor-reactive CD8+ T cells improved the survival of treated mice to a greater extent than did the control T cells. Hence, this study establishes that arming tumor-reactive T cells with a costimulator can enhance their antitumor efficacy.     

Systemic and local human papillomavirus 16-specific T-cell immunity in patients with head and neck cancer

Squamous cell carcinomas of the head and neck (HNSCC), in particular those of the oropharynx, can be caused by human papilloma virus Type 16 (HPV16). Whereas these HPV-induced oropharyngeal carcinomas may express the HPV16 E6 and E7 oncoproteins and are associated with better survival, the nonvirally induced HNSCC are associated with overexpression of p53. In this study we assessed the presence of systemic and local T cells reactive against these oncoproteins in HNSCC. An exploratory study on the presence, type and function of HPV16- and/or p53-specific T cells in the blood, tumor and/or metastatic lymph node as measured by several immune assays was performed in an unselected group of 50 patients with HNSCC. Tumor tissue was tested for HPV DNA and the overexpression of p53 protein. Almost all HPV16+ tumors were located in the oropharynx. Circulating HPV16- and p53-specific T cells were found in 17/47 and 7/45 tested patients. T cells were isolated from tumor cultures and/or lymph nodes of 20 patients. HPV16-specific T cells were detected in six of eight HPV+ tumors, but in none of the 12 HPV-tumors. Tumor-infiltrating p53-specific T cells were not detected. In depth analysis of the HPV16-specific T-cell response revealed that this response comprised a broad repertoire of CD4+ T-helper Type 1 and 2 cells, CD4+ regulatory T cells and CD8+ T cells reactive to HPV16. The local presence of HPV16-specific T-cell immunity in HPV16-induced HNSCC implicates a role in the antitumor response and support the development of immunotherapy for HNSCC.     

Deoxyribonucleic acid (DNA) encoding a pan-major histocompatibility complex class II peptide analogue augmented antigen-specific cellular immunity and suppressive effects on tumor growth elicited by DNA vaccine immunotherapy

Vaccine immunotherapy must induce helper and cytotoxic cell-mediated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was administered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased in the tumor site. Furthermore, immunofluorescent study of the inoculated tumors revealed increased accumulation of both CD4- and CD8-positive T cells producing IFN-gamma in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the development of therapeutic DNA vaccines against cancer.     

Circulating human papillomavirus type 16 specific T-cells are associated with HLA Class I expression on tumor cells, but not related to the amount of viral oncogene transcripts

Human papillomavirus (HPV) is a necessary factor in the pathogenesis of cervical cancer. Circulating HPV-specific T-cells responding to the E6 and E7 HPV proteins can be detected only in half of cervical cancer patients. Potential explanations for the absence of this response are lack of sufficient amounts of antigen to activate the immune response or local immune escape mechanisms. We studied the relationship between HPV 16 E6/E7 oncogene mRNA expression, human leukocyte antigen (HLA) expression on tumor cells and the presence of circulating E6- and E7-specific T-cell responses in cervical cancer patients. The amount of antigen was assessed by HPV E6/E7 mRNA expression levels measured by quantitative polymerase chain reaction. HLA Class I and Class II expression on tumor cells was analyzed by immunohistochemistry. A proliferative HPV-specific T-cell response was detected in 15/29 patients. The amount of HPV E6/E7 mRNA was not related to the presence of immune response. HLA Class I expression was downregulated in 19 patients and completely lost in 7 patients. HLA Class II expression was upregulated in 18 patients. HLA Class I expression on tumor cells showed a strong correlation with immunity (p = 0.001). Explicitly, all patients with complete HLA loss lacked HPV specific T-cell responses. The presence of circulating HPV-specific T-cells might reflect ongoing antitumor response that is sustained by CD8+ T-cells killing HLA Class I positive cancer cells. We hypothesize that HLA Class I expression status on tumor cells might as well influence the response to HPV E6/E7 directed immunotherapy.     

Immune-mediated tumor regression induced by CpG-containing oligodeoxynucleotides.

T-cell based immunotherapy is an attractive approach for the treatment of multiple tumor types including cervical carcinoma. Immunostimulating DNA containing unmethylated cytosine-guanine (CpG) motifs have been successfully used as adjuvants to enhance immune responses to vaccines designed to trigger antitumor T-cell responses. Using a murine model of cervical carcinoma, we report here that repeated administration of synthetic oligodeoxynucleotides bearing CpG motifs (CpG-ODNs) without the need of vaccination into animals bearing large, established tumors resulted in significant antitumor effects. Both tumor regressions and extended survival resulting from CpG-ODN therapy required the participation of CD8+ T cells. On the other hand, CD4+ T cells were not only not required, but also appeared to inhibit the therapeutic effect of CpG-ODN. Tumor regression correlated with increased infiltration of CD8+ T cells into the tumors and with enhanced expression of MHC class I and II antigens by the tumor cells. Together, these results indicate that CpG therapy could be promising as a single agent for the treatment of some tumors such as cervical carcinoma.