In vivo microRNA-155 expression influences antigen-specific T cell-mediated immune responses generated by DNA vaccination.

BACKGROUND: MicroRNA (miRNA) molecules are potent mediators of post-transcriptional gene silencing that are emerging to be critical in the regulation of innate and adaptive immunity. RESULTS: Here we report that miR-155--an oncogenic miRNA with important function in the mammalian immune system--is induced in dendritic cells (DCs) upon maturation and potentially attenuates their ability to activate T cells. Biolistic epidermal transfection with DNA encoding miR-155 suppressed the induction of antigen-specific T cell-mediated immunity, whereas reduction of endogenous miR-155 by a partially complementary antisense sequence reversed this effect. Because DCs represent a significant component of epidermal tissue and are among the most potent of antigen-presenting cells, the inhibitory actions of miR-155 could be mediated through this subset of cells. CONCLUSIONS: These results suggest that miR-155 may repress the expression of key molecules involved in lymph node migration, antigen presentation, or T cell activation in DCs, and thus forms part of a negative regulatory pathway that dampens the generation of T cell-mediated immune responses. Modulation of miR-155 expression in epidermis therefore represents a potentially promising form of gene therapy for the control of diseases ranging from autoimmunity to cancer and viral infection.

Intraperitoneal administration of poly(I:C) with polyethylenimine leads to significant antitumor immunity against murine ovarian tumors.

Ovarian cancer is currently the most lethal gynecologic cancer in the United States. There is an urgent need for the development of innovative therapies against ovarian cancer, such as immunotherapy. The toll-like receptor 3 ligand, polyriboinosinic:polyribocytidylic acid (poly(I:C), has emerged as a promising adjuvant for activating the host immune responses for the control of tumors. We reasoned that a strategy to enhance the intracellular uptake of poly(I:C) will likely improve the poly(I:C) adjuvant effect. Since polyethylenimine (PEI) has been shown to increase the transfection efficiency of nucleic acids, we characterized the antitumor effects in mouse ovarian surface epithelial cells (MOSEC) tumor-bearing mice treated intraperitoneally with poly(I:C) and PEI. We observed that tumor-bearing mice treated with poly(I:C) and PEI generated significantly better therapeutic antitumor effects against MOSEC tumors compared with treatment with poly(I:C) alone. Furthermore, we found that NK cells play a significant role in the antitumor effects generated by treatment with poly(I:C) in combination with PEI. Intraperitoneal administration of poly(I:C) with PEI led to the uptake of poly(I:C) mainly by CD11b+ macrophages, resulting in the high expression of MHC class II and IL-12 (M1 phenotype). In addition, adoptive transfer of CD11b+ macrophages from mice treated with poly(I:C) and PEI was found to lead to increased number of activated NK cells in the recipient mice. Taken together, our data indicate that PEI can potentially be used to improve the uptake of poly(I:C) by CD11b+ macrophages, leading to the activation of NK cells and the control of murine ovarian tumors.

Vascular disrupting agent DMXAA enhances the antitumor effects generated by therapeutic HPV DNA vaccines.

Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.

Control of cervicovaginal HPV-16 E7-expressing tumors by the combination of therapeutic HPV vaccination and vascular disrupting agents.

Abstract Antigen-specific immunotherapy and vascular disrupting agents, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have emerged as attractive approaches for the treatment of cancers. In the current study, we tested the combination of DMXAA treatment with therapeutic human papillomavirus type 16 (HPV-16) E7 peptide-based vaccination for their ability to generate E7-specific CD8+ T-cell immune responses, as well as their ability to control E7-expressing tumors in a subcutaneous and a cervicovaginal tumor model. We found that the combination of DMXAA treatment with E7 long peptide (amino acids 43-62) vaccination mixed with polyriboinosinic:polyribocytidylic generated significantly stronger E7-specific CD8+ T-cell immune responses and antitumor effects compared with treatment with DMXAA alone or HPV peptide vaccination alone in the subcutaneous model. Additionally, we found that the DMXAA-mediated enhancement of E7-specific CD8+ T-cell immune responses generated by the therapeutic HPV peptide-based vaccine was dependent on the timing of administration of DMXAA. Treatment with DMXAA in tumor-bearing mice was also shown to lead to increased dendritic cell maturation and increased production of inflammatory cytokines in the tumor. Furthermore, we observed that the combination of DMXAA with HPV-16 E7 peptide vaccination generated a significant enhancement in the antitumor effects in the cervicovaginal TC-1 tumor growth model, which closely resembles the tumor microenvironment of cervical cancer. Taken together, our data demonstrated that administration of the vascular disrupting agent, DMXAA, enhances therapeutic HPV vaccine-induced cytotoxic T-lymphocyte responses and antitumor effects against E7-expressing tumors in two different locations. Our study has significant implications for future clinical translation.

Enhancement of protein vaccine potency by in vivo electroporation mediated intramuscular injection.

Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for innovative approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the employment of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses. Another approach involves employing a method capable of improving the delivery of protein-based vaccine intramuscularly to lead to the slow release of the protein, resulting in improved vaccine potency. In the current study, we aimed to determine whether intramuscular injection of protein-based vaccines in conjunction with CpG followed by electroporation can lead to increased delivery of the protein-based vaccine into muscle cells, resulting in enhanced protein-based vaccine potency. We found that intramuscular injection followed by electroporation can effectively transduce the protein-based vaccine into the muscle cells. Furthermore, we found that intramuscular vaccination with OVA protein in combination with CpG followed by electroporation generates the best OVA-specific CD8+ T cell immune responses as well as the best protective and therapeutic antitumor effects in vaccinated mice. CD8+ T cells were found to play an important role in the observed protective antitumor effects generated by the vaccination. Similar results were observed using the HPV-16 E7 protein-based vaccination system. Thus, our data indicate that intramuscular administration of protein-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+ T cell immune responses. The clinical implications of the study are discussed.

Improving therapeutic HPV peptide-based vaccine potency by enhancing CD4+ T help and dendritic cell activation.

BACKGROUND: Effective vaccination against human papillomavirus (HPV) represents an opportunity to control cervical cancer. Peptide-based vaccines targeting HPV E6 and/or E7 antigens while safe, will most likely require additional strategies to enhance the vaccine potency. METHODS: We tested the HPV-16 E7 peptide-based vaccine in combination with a strategy to enhance CD4+ T help using a Pan HLA-DR epitope (PADRE) peptide and a strategy to enhance dendritic cell activation using the toll-like receptor 3 ligand, poly(I:C). RESULTS: We observed that mice vaccinated with E7 peptide-based vaccine in combination with PADRE peptide and poly(I:C) generated better E7-specific CD8+ T cell immune responses as well as significantly improved therapeutic anti-tumor effects against TC-1 tumors compared to E7 peptide-based vaccine with either PADRE peptide or poly(I:C) alone. Furthermore, we found that intratumoral vaccination with the E7 peptide in conjunction with PADRE peptide and poly(I:C) generates a significantly higher frequency of E7-specific CD8+ T cells as well as better survival compared to subcutaneous vaccination with the same regimen in treated mice. CONCLUSIONS: The combination of PADRE peptide and poly(I:C) with antigenic peptide is capable of generating potent antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated mice. Our study has significant clinical implications for peptide-based vaccination.

Delivery of chemotherapeutic agents using drug-loaded irradiated tumor cells to treat murine ovarian tumors.

BACKGROUND: Ovarian cancer is the leading cause of death among women with gynecologic malignancies in the United States. Advanced ovarian cancers are difficult to cure with the current available chemotherapy, which has many associated systemic side effects. Doxorubicin is one such chemotherapeutic agent that can cause cardiotoxicity. Novel methods of delivering chemotherapy without significant side effects are therefore of critical need. METHODS: In the current study, we generated an irradiated tumor cell-based drug delivery system which uses irradiated tumor cells loaded with the chemotherapeutic drug, doxorubicin. RESULTS: We showed that incubation of murine ovarian cancer cells (MOSEC) with doxorubicin led to the intracellular uptake of the drug (MOSEC-dox cells) and the eventual death of the tumor cell. We then showed that doxorubicin loaded MOSEC-dox cells were able to deliver doxorubicin to MOSEC cells in vivo. Further characterization of the doxorubicin transfer revealed the involvement of cell contact. The irradiated form of the MOSEC-dox cells were capable of treating luciferase-expressing MOSEC tumor cells (MOSEC/luc) in C57BL/6 mice as well as in athymic nude mice resulting in improved survival compared to the non drug-loaded irradiated MOSEC cells. Furthermore, we showed that irradiated MOSEC-dox cells was more effective compared to an equivalent dose of doxorubicin in treating MOSEC/luc tumor-bearing mice. CONCLUSIONS: Thus, the employment of drug-loaded irradiated tumor cells represents a potentially innovative approach for the delivery of chemotherapeutic drugs for the control of ovarian tumors.

Carrageenan as an adjuvant to enhance peptide-based vaccine potency.

New innovative therapies are urgently required in order to combat the high mortality and morbidity associated with advanced cancers. Antigen-specific cancer immunotherapy using peptide-based vaccination has emerged as an attractive approach for the control of cancers due to its simplicity and easy preparation. However, such an approach requires the employment of suitable adjuvants. In the current study, we explored the employment of a sulfated polysaccharide compound from red algae, carrageenan (CGN) as an adjuvant for their ability to generate antigen-specific immune responses and antitumor effects in mice vaccinated with human papillomavirus type 16 (HPV-16) E7 peptide vaccine. We found that carrageenan can significantly enhance the E7-specific immune responses generated by E7 peptide vaccination via the TLR4 activation pathway. In addition, carrageenan could enhance the protective and therapeutic antitumor effects generated by E7 peptide vaccination against E7-expressing tumors. Furthermore, the observed enhancement was not restricted to E7 antigen but was also applicable to other antigenic systems. We also found that other structurally similar compounds to CGN, such as dextran, also generated similar immune enhancement. Thus, our data suggest that CGN and its structurally related compounds may serve as innovative adjuvants for enhancing peptide-based vaccine potency.

Treatment with imiquimod enhances antitumor immunity induced by therapeutic HPV DNA vaccination.

BACKGROUND: There is an urgent need to develop new innovative therapies for the control of advanced cancer. The combination of antigen-specific immunotherapy with the employment of immunomodulatory agents has emerged as a potentially plausible approach for the control of advanced cancer. METHODS: In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) with the TLR7 agonist imiquimod for their ability to generate E7-specific immune responses and antitumor effects in tumor-bearing mice. RESULTS: We observed that treatment with CRT/E7 DNA in combination with imiquimod leads to an enhancement in the E7-specific CD8+ T cell immune responses and a decrease in the number of myeloid-derived suppressor cells in the tumor microenvironment of tumor-bearing mice. Furthermore, treatment with CRT/E7 DNA in combination with imiquimod leads to significantly improved antitumor effects and prolonged survival in treated mice. In addition, treatment with imiquimod led to increased number of NK1.1+ cells and F4/80+ cells in the tumor microenvironment. Macrophages and NK1.1+ cells were found to play an important role in the antitumor effects mediated by treatment with CRT/E7 DNA in combination with imiquimod. CONCLUSIONS: Thus, our data suggests that the combination of therapeutic HPV DNA vaccination with topical treatment with the TLR7 agonist imiquimod enhances the antitumor immunity induced by DNA vaccination. The current study has significant implications for future clinical translation.

Ectopic expression of X-linked lymphocyte-regulated protein pM1 renders tumor cells resistant to antitumor immunity.

Tumor immune escape is a major obstacle in cancer immunotherapy, but the mechanisms involved remain poorly understood. We have previously developed an immune evasion tumor model using an in vivo immune selection strategy and revealed Akt-mediated immune resistance to antitumor immunity induced by various cancer immunotherapeutic agents. In the current study, we used microarray gene analysis to identify an Akt-activating candidate molecule overexpressed in immune-resistant tumors compared with parental tumors. X-linked lymphocyte-regulated protein pM1 (XLR) gene was the most upregulated in immune-resistant tumors compared with parental tumor cells. Furthermore, the retroviral transduction of XLR in parental tumor cells led to activation of Akt, resulting in upregulation of antiapoptotic proteins and the induction of immune resistance phenotype in parental tumor cells. In addition, we found that transduction of parental tumor cells with other homologous genes from the mouse XLR family, such as synaptonemal complex protein 3 (SCP3) and XLR-related, meiosis-regulated protein (XMR) and its human counterpart of SCP3 (hSCP3), also led to activation of Akt, resulting in the upregulation of antiapoptotic proteins and induction of immune resistance phenotype. Importantly, characterization of a panel of human cervical cancers revealed relatively higher expression levels of hSCP3 in human cervical cancer tissue compared with normal cervical tissue. Thus, our data indicate that ectopic expression of XLR and its homologues in tumor cells represents a potentially important mechanism for tumor immune evasion and serves as a promising molecular target for cancer immunotherapy.

Immunotherapy for cervical cancer: Research status and clinical potential.

The high-risk types of human papillomavirus (HPV) have been found to be associated with most cervical cancers and play an essential role in the pathogenesis of the disease. Despite recent advances in preventive HPV vaccine development, such preventive vaccines are unlikely to reduce the prevalence of HPV infections within the next few years, due to their cost and limited availability in developing countries. Furthermore, preventive HPV vaccines may not be capable of treating established HPV infections and HPV-associated lesions, which account for high morbidity and mortality worldwide. Thus, it is important to develop therapeutic HPV vaccines for the control of existing HPV infection and associated malignancies. Therapeutic vaccines are quite different from preventive vaccines in that they require the generation of cell-mediated immunity, particularly T cell-mediated immunity, instead of the generation of neutralizing antibodies. The HPV-encoded early proteins, the E6 and E7 oncoproteins, form ideal targets for therapeutic HPV vaccines, since they are consistently expressed in HPV-associated cervical cancer and its precursor lesions and thus play crucial roles in the generation and maintenance of HPV-associated disease. Our review covers the various therapeutic HPV vaccines for cervical cancer, including live vector-based, peptide or protein-based, nucleic acid-based, and cell-based vaccines targeting the HPV E6 and/or E7 antigens. Furthermore, we review the studies using therapeutic HPV vaccines in combination with other therapeutic modalities and review the latest clinical trials on therapeutic HPV vaccines.

DNA vaccines for cervical cancer.

Human papillomavirus (HPV), particularly type 16, has been associated with more than 99% of cervical cancers. There are two HPV oncogenic proteins, E6 and E7, which play a major role in the induction and maintenance of cellular transformation. Thus, immunotherapy targeting these proteins may be employed for the control of HPV-associated cervical lesions. Although the commercially available preventive HPV vaccines are highly efficient in preventing new HPV infection, they do not have therapeutic effects against established HPV infection or HPV-associated lesions. Since T cell-mediated immunity is important for treating established HPV infections and HPV-associated lesions, therapeutic HPV vaccine should aim at generating potent E6 and E7-specific T cell-mediated immune responses. DNA vaccines have now developed into a promising approach for antigen-specific T cell-mediated immunotherapy to combat infection and cancer. Because dendritic cells are the most potent professional antigen-presenting cells, and are highly effective in priming antigen-specific T cells, several DNA vaccines have employed innovative strategies to modify the properties of dendritic cells (DCs) for the enhancement of the DNA vaccine potency. These studies have revealed impressive pre-clinical data that has led to several ongoing HPV DNA vaccine clinical trials.

Enhancing the therapeutic effect against ovarian cancer through a combination of viral oncolysis and antigen-specific immunotherapy.

Cancer therapy using oncolytic viruses represents a promising new approach for controlling ovarian cancer. In this study, we have circumvented the limitation of repeated vaccination by employing different virus vectors, Semliki Forest Virus (SFV) and vaccinia virus (VV) for boosting the immune response. We found that infection of tumor-bearing mice with VV followed by infection with SFV or vice versa leads to enhanced antitumor effects against murine ovarian surface epithelial carcinoma (MOSEC) tumors. Furthermore, infection with VV-ovalbumin (OVA) followed by infection with SFV-OVA or vice versa was found to lead to enhanced OVA-specific CD8(+) T-cell immune responses. In addition, we found that infection with SFV-OVA followed by infection with VV-OVA leads to enhanced antitumor effects in vivo and enhanced tumor killing in vitro through a combination of viral oncolysis and antigen-specific immunity. The clinical implications of this study are discussed.

Therapeutic HPV DNA vaccines.

Human papillomavirus (HPV) has been associated with several human cancers, including cervical cancer, vulvar cancer, vaginal and anal cancer, and a subset of head and neck cancers. The identification of HPV as an etiological factor for HPV-associated malignancies creates the opportunity for the control of these cancers through vaccination. Currently, the preventive HPV vaccine using HPV virus-like particles has been proven to be safe and highly effective. However, this preventive vaccine does not have therapeutic effects, and a significant number of people have established HPV infection and HPV-associated lesions. Therefore, it is necessary to develop therapeutic HPV vaccines to facilitate the control of HPV-associated malignancies and their precursor lesions. Among the various forms of therapeutic HPV vaccines, DNA vaccines have emerged as a potentially promising approach for vaccine development due to their safety profile, ease of preparation and stability. However, since DNA does not have the intrinsic ability to amplify or spread in transfected cells like viral vectors, DNA vaccines can have limited immunogenicity. Therefore, it is important to develop innovative strategies to improve DNA vaccine potency. Since dendritic cells (DCs) are key players in the generation of antigen-specific immune responses, it is important to develop innovative strategies to modify the properties of the DNA-transfected DCs. These strategies include increasing the number of antigen-expressing/antigen-loaded DCs, improving antigen processing and presentation in DCs, and enhancing the interaction between DCs and T cells. Many of the studies on DNA vaccines have been performed on preclinical models. Encouraging results from impressive preclinical studies have led to several clinical trials.

Combination of viral oncolysis and tumor-specific immunity to control established tumors.

PURPOSE: Advanced-stage cancers are extremely difficult to treat and rarely result in a cure. The application of oncolytic viruses is a potential strategy for controlling advanced-stage cancer because intratumoral (i.t.) injection of an oncolytic virus, such as vaccinia virus, results in tumor cell lysis and subsequent release of tumor antigens into the microenvironment. Furthermore, the viruses can serve as a vehicle for delivering genes of interest to cancer cells. EXPERIMENTAL DESIGN: In the current study, we hypothesize that in tumor-bearing mice primed with DNA encoding an immunogenic foreign antigen, ovalbumin (OVA) followed by a boost with i.t. administration of vaccinia virus encoding the same foreign antigen, OVA, can generate enhanced antitumor effects through the combination of viral oncolysis and tumor-specific immunity. RESULTS: We observed that tumor-bearing mice primed with OVA DNA and boosted with vaccinia encoding OVA (Vac-OVA) generated significant therapeutic antitumor effects as well as induced significant levels of OVA-specific CD8+ T cells in two different tumor models. Furthermore, treatment with Vac-OVA not only kills the tumor and stromal cells directly but also renders the tumor cells and surrounding stromal cells susceptible to OVA-specific CD8+ T-cell killing, resulting in enhanced antitumor therapeutic effects. CONCLUSIONS: Thus, the current study may provide a novel therapeutic strategy for the control of advanced-stage cancers.

Treatment with demethylating agent, 5-aza-2'-deoxycytidine enhances therapeutic HPV DNA vaccine potency.

DNA vaccines have emerged as a potential alternative to current strategies to control cancer for their safety, stability and ease of preparation. We have previously demonstrated that a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) can generate significant E7-specific immune responses and antitumor effects in vaccinated mice, thus enhancing DNA vaccine potency. Another strategy to improve DNA vaccine potency is by enhancing the level of expression of the antigen encoded in the vaccine. DNA methylation has been shown to lead to silencing of the genes that would affect the expression of the encoded antigen of the DNA vaccines. In the current study, we reasoned that CRT/E7 DNA vaccination combined with demethylating agent, 5-aza-2'-deoxycytidine (DAC) would lead to upregulation of CRT/E7 expression, resulting in improved DNA vaccine potency. We found that pre-treatment with DAC led to increased CRT/E7 DNA expression, leading to enhanced E7-specific CD8+ T cell immune responses as well as the antitumor effects generated by the CRT/E7 DNA vaccine. Thus, our data suggest that combination of CRT/E7 DNA vaccination with DAC treatment may represent a potentially promising approach to control HPV-associated malignancies. The clinical implications of this study are discussed.

Combination of apigenin treatment with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects.

BACKGROUND: It is important to develop innovative therapies for advanced stage cancers in addition to the conventional therapies including chemotherapy, radiation and surgery. Antigen-specific immunotherapy has emerged as a novel alternate therapy for advanced stage cancers, which may be employed in conjunction with conventional therapies. METHODS: In the current study, we tested the effect of treatment with the chemotherapeutic agent, apigenin in combination with DNA vaccines encoding the HPV-16 E7 antigen linked to heat shock protein 70 (HSP70) in the control of the E7-expressing tumor, TC-1. RESULTS: We observed that treatment with apigenin rendered the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic CD8+ T cells. Furthermore, treatment of TC-1 tumor cells with apigenin was found to enhance apoptotic tumor cell death in vitro in a dose-dependant manner. We showed that TC-1 tumor-bearing mice treated with apigenin combined with E7-HSP70 DNA generate highest frequency of primary and memory E7-specific CD8+ T cells, leading to potent therapeutic anti-tumor effects against E7-expressing tumors. CONCLUSION: Thus, apigenin represents a promising chemotherapeutic agent, which may be used in combination with immunotherapy for the treatment of advanced stage cancers. The clinical implications of the current strategy are discussed.

Treatment with LL-37 peptide enhances antitumor effects induced by CpG oligodeoxynucleotides against ovarian cancer.

There is an urgent need for innovative therapies against ovarian cancer, one of the leading causes of death from gynecological cancers in the United States. Immunotherapy employing Toll-like receptor (TLR) ligands, such as CpG oligodeoxynucleotides (CpG-ODN), may serve as a potentially promising approach in the control of ovarian tumors. The CpG-ODN requires intracellular delivery into the endosomal compartment, where it can bind to TLR9 in order to activate the immune system. In the current study, we aim to investigate whether the antimicrobial polypeptide from the cathelicidin family, LL-37, could enhance the immunostimulatory effects of CpG-ODN by increasing the uptake of CpG-ODN into the immune cells, thus enhancing the antitumor effects against ovarian cancer. We found that treatment with the combination of CpG-ODN and LL-37 generated significantly better therapeutic antitumor effects and enhanced survival in murine ovarian tumor-bearing mice compared with treatment with CpG-ODN or LL-37 alone. We also observed that treatment with the combination of CpG-ODN and LL-37 enhanced proliferation and activation of natural killer (NK) cells, but not CD4(+) or CD8(+) T cells, in the peritoneal cavity. Furthermore, in vivo antibody depletion experiments indicated that peritoneal NK cells played a critical role in the observed antitumor effects. Thus, our data suggest that the combination of CpG-ODN with LL-37 peptide may lead to the control of ovarian tumors through the activation of innate immunity.

Expression of IL-15RA or an IL-15/IL-15RA fusion on CD8+ T cells modifies adoptively transferred T-cell function in cis.

IL-15 and IL-15 receptor alpha (IL-15RA) play a significant role in multiple aspects of T-cell biology. However, given the evidence that IL-15RA can present IL-15 in trans, the functional capacity of IL-15RA expressed on CD8(+) T cells to modify IL-15 functions in cis is currently unclear. In the current study, we explore the functional consequences of IL-15RA, expression on T cells using a novel method to transfect naive CD8(+) T cells. We observed that RNA nucleofection led to highly efficient, non-toxic, and rapid manipulation of protein expression levels in unstimulated CD8(+) T cells. We found that transfection of unstimulated CD8(+) T cells with IL-15RA RNA led to enhanced viability of CD8(+) T cells in response to IL-15. Transfection with IL-15RA enhanced IL-15-mediated phosphorylation of STAT5 and also promoted IL-15-mediated proliferation in vivo of adoptively transferred naïve CD8(+) T cells. We demonstrated that IL-15RA can present IL-15 via cis-presentation on CD8(+) T cells. Finally, we showed that transfection with a chimeric construct linking IL-15 to IL-15RA cell autonomously enhances the viability and proliferation of primary CD8(+) T cells and cytotoxic potential of antigen-specific CD8(+) T cells. The clinical implications of the current study are discussed.

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts.

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed.