Leukotriene C4 induces migration of human monocyte-derived dendritic cells without loss of immunostimulatory function.

Generation of human monocyte-derived dendritic cells (DCs) for cancer vaccination involves ex vivo maturation in the presence of proinflammatory cytokines and prostaglandin E(2) (PGE(2)). Although the inclusion of PGE(2) during maturation is imperative for the induction of DC migration, PGE(2) has unfavorable effects on the immunostimulatory capacity of these cells. Like PGE(2), leukotrienes (LTs) are potent mediators of DC migration. We therefore sought to characterize the migratory and immunologic properties of DCs that matured in the presence of LTB(4), LTC(4), LTD(4), and PGE(2). Here, we demonstrate that DCs matured in the presence of LTC(4), but not LTB(4) or LTD(4), are superior to PGE(2)-matured DCs in stimulating CD4(+) T-cell responses and in inducing antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without concomitant induction or recruitment of regulatory T cells (Tregs). LTC(4)-matured DCs migrate efficiently through layers of extracellular matrix and secrete higher levels of immunostimulatory IL-12p70 while producing reduced levels of immune-inhibitory IL-10, IL12p40, indoleamine-2,3-dioxidase, and TIMP-1 (tissue inhibitor of matrix metalloproteinases). Intracellular calcium mobilization and receptor antagonist studies reveal that, in contrast to LTD(4), LTC(4) did not signal through CysLTR(1) in DCs. Collectively, our data suggest that LTC(4) represents a promising candidate to replace PGE(2) in DC maturation protocols for cancer vaccination.

Impact of anti-CD25 monoclonal antibody on dendritic cell-tumor fusion vaccine efficacy in a murine melanoma model.

BACKGROUND: A promising cancer vaccine involves the fusion of tumor cells with dendritic cells (DCs). As such, a broad spectrum of both known and unidentified tumor antigens is presented to the immune system in the context of the potent immunostimulatory capacity of DCs. Murine studies have demonstrated the efficacy of fusion immunotherapy. However the clinical impact of DC/tumor fusion vaccines has been limited, suggesting that the immunosuppresive milieu found in patients with malignancies may blunt the efficacy of cancer vaccination. Thus, novel strategies to enhance fusion vaccine efficacy are needed. Regulatory T cells (Tregs) are known to suppress anti-tumor immunity, and depletion or functional inactivation of these cells improves immunotherapy in both animal models and clinical trials. In this study, we sought to investigate whether functional inactivation of CD4+CD25+FoxP3+ Treg with anti-CD25 monoclonal antibody (mAb) PC61 prior to DC/tumor vaccination would significantly improve immunotherapy in the murine B16 melanoma model. METHODS: Treg blockade was achieved with systemic PC61 administration. This blockage was done in conjunction with DC/tumor fusion vaccine administration to treat established melanoma pulmonary metastases. Enumeration of these metastases was performed and compared between experimental groups using Wilcoxon Rank Sum Test. IFN-gamma ELISPOT assay was performed on splenocytes from treated mice. RESULTS: We demonstrate that treatment of mice with established disease using mAb PC61 and DC/tumor fusion significantly reduced counts of pulmonary metastases compared to treatment with PC61 alone (p=0.002) or treatment with control antibody plus fusion vaccine (p=0.0397). Furthermore, IFN-gamma ELISPOT analyses reveal that the increase in cancer immunity was mediated by anti-tumor specific CD4+ T-helper cells, without concomitant induction of CD8+ cytotoxic T cells. Lastly, our data provide proof of principle that combination treatment with mAb PC61 and systemic IL-12 can lower the dose of IL-12 necessary to obtain maximal therapeutic efficacy. CONCLUSIONS: To our knowledge, this is the first report investigating the effects of anti-CD25 mAb administration on DC/tumor-fusion vaccine efficacy in a murine melanoma model, and our results may aide the design of future clinical trials with enhanced therapeutic impact.

Local secretion of IL-12 augments the therapeutic impact of dendritic cell-tumor cell fusion vaccination.

BACKGROUND: The development of dendritic cell (DC)-tumor fusion vaccines is a promising approach in cancer immunotherapy. Using fusion vaccines allows a broad spectrum of known and unidentified tumor-associated antigens to be presented in the context of MHC class I and class II molecules, with potent co-stimulation provided by the DCs. Although DC-tumor fusion cells are immunogenic, murine studies have shown that effective immunotherapy requires a third signal, which can be provided by exogenous interleukin 12 (IL-12). Unfortunately, systemic administration of IL-12 induces severe toxicity in cancer patients, potentially precluding clinical use of this cytokine to augment fusion vaccine efficacy. To overcome this limitation, we developed a novel approach in which DC-tumor fusion cells locally secrete IL-12, then evaluated the effectiveness of this approach in a murine B16 melanoma model. MATERIALS AND METHODS: Tumor cells were stably transduced to secrete murine IL-12p70. These tumor cells were then electrofused to DC to form DC-tumor heterokaryons. These cells were used to treat established B16 pulmonary metastases. Enumeration of these metastases was performed and compared between experimental groups using Wilcoxon rank sum test. Interferon γ enzyme-linked immunosorbent spot assay was performed on splenocytes from treated mice. RESULTS: We show that vaccination with DCs fused to syngeneic melanoma cells that stably express murine IL-12p70 significantly reduces counts of established lung metastases in treated animals when compared with DC-tumor alone (P = 0.029). Interferon γ enzyme-linked immunosorbent spot assays suggest that this antitumor response is mediated by CD4(+) T cells, in the absence of a tumor-specific CD8(+) T cell response, and that the concomitant induction of antitumor CD4(+) and CD8(+) T cell responses required exogenous IL-12. CONCLUSIONS: This study is, to the best of our knowledge, the first report that investigates the impact of local secretion of IL-12 on antitumor immunity induced by a DC-tumor fusion cell vaccine in a melanoma model and may aid the rational design of future clinical trials.

Enhancement of anti-tumor immunity through local modulation of CTLA-4 and GITR by dendritic cells.

Cancer vaccines have now demonstrated clinical efficacy, but immune modulatory mechanisms that prevent autoimmunity limit their effectiveness. Systemic administration of mAbs targeting the immune modulatory receptors CTLA-4 and glucocorticoid-induced TNFR-related protein (GITR) on Treg and effector T cells augments anti-tumor immunity both experimentally and clinically, but can induce life-threatening autoimmunity. We hypothesized that local delivery of anti-CTLA-4 and anti-GITR mAbs to the sites where T cells and tumor antigen-loaded DC vaccines interact would enhance the induction of anti-tumor immunity while avoiding autoimmunity. To achieve this goal, DCs transfected with mRNA encoding the H and L chains of anti-mouse CTLA-4 and GITR mAbs were co-administered with tumor antigen mRNA-transfected DCs. We observed enhanced induction of anti-tumor immunity and significantly improved survival in melanoma-bearing mice, without signs of autoimmunity. Using in vitro assays with human DCs, we demonstrated that DCs transfected with mRNA encoding a humanized anti-CTLA-4 mAb and mRNA encoding a soluble human GITR-L fusion protein enhance the induction of anti-tumor CTLs in response to DCs transfected with mRNAs encoding either melanoma or breast cancer antigens. Based on these results, this approach of using local delivery of immune modulators to enhance vaccine-induced immunity is currently being evaluated in a phase I clinical cancer immunotherapy trial.

Reversal of myeloid cell-mediated immunosuppression in patients with metastatic renal cell carcinoma.

PURPOSE: Tumor-induced immunosuppression remains a significant obstacle that limits the efficacy of biological therapy for renal cell carcinoma. Here we evaluate the role of CD33 myeloid-derived suppressor cells (MDSC) in the regulation of T-cell responses in renal cell carcinoma patients. We also examine effect of all-trans-retinoic acid (ATRA) on MDSC-mediated immune suppression. EXPERIMENTAL DESIGN: CD33-positive myeloid cells were isolated from the peripheral blood of renal cell carcinoma patients with magnetic beads and tested in vitro for their ability to inhibit T-cell responses. T-cell function was evaluated using ELISPOT and CTL assays. RESULTS: MDSC isolated from renal cell carcinoma patients, but not from healthy donors, were capable of suppressing antigen-specific T-cell responses in vitro through the secretion of reactive oxygen species and nitric oxide upon interaction with CTL. MDSC-mediated immune suppression and IFN-gamma down-regulation was reversible in vitro by exposing cells to the reactive oxygen species inhibitors. Moreover, ATRA was capable of abrogating MDSC-mediated immunosuppression and improving T-cell function by direct differentiation into antigen-presenting cell precursors. CONCLUSIONS: These results may have significant implications regarding the future design of active immunotherapy protocols that may include differentiation agents as part of a multimodal approach to renal cell carcinoma immunotherapy.

Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells.

In this study, we investigated whether elimination of CD4+/CD25+ Tregs using the recombinant IL-2 diphtheria toxin conjugate DAB(389)IL-2 (also known as denileukin diftitox and ONTAK) is capable of enhancing the immunostimulatory efficacy of tumor RNA-transfected DC vaccines. We show that DAB(389)IL-2 is capable of selectively eliminating CD25-expressing Tregs from the PBMCs of cancer patients without inducing toxicity on other cellular subsets with intermediate or low expression of CD25. DAB(389)IL-2-mediated Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB(389)IL-2 was omitted during T cell priming. DAB(389)IL-2 significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma (RCC) patients and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, DAB(389)IL-2-mediated elimination of Tregs followed by vaccination with RNA-transfected DCs significantly improved the stimulation of tumor-specific T cell responses in RCC patients when compared with vaccination alone. Our findings may have implications in the design of immune-based strategies that may incorporate the Treg depletion strategy to achieve potent antitumor immunity with therapeutic impact.

Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors.

Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell-mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA-transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer.

Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product.

Dendritic cells (DCs) transfected with mRNA encoding human telomerase reverse transcriptase (hTERT) have been shown to represent potent inducers of CTLs and antitumor immunity. However, it has become widely accepted that not only CTLs but also CD4(+) T helper cells are critical to the generation, as well as to the maintenance, of potent antitumor responses in vivo. In this study, we sought to determine whether human DCs transfected with mRNA encoding a chimeric hTERT/lysosome-associated membrane protein (LAMP-1) protein, carrying the endosomal/lysosomal sorting signal of the LAMP-1, are capable of stimulating concomitant hTERT-specific CD8(+) and CD4(+) T-cell responses in vitro. We show that processing of hTERT/LAMP-1 transcripts leads to enhanced stimulation of hTERT-specific CD4(+) T cells and does not negatively affect intracellular generation and subsequent presentation of MHC class I epitopes, hence, generating a CTL response. These findings provide a preclinical rationale of using DCs transfected with the chimeric hTERT/LAMP-1 RNA in vaccine trials to facilitate generation of antigen-specific CD4(+) T-cell responses that may be required to stimulate and maintain an optimal CD8(+) CTL response in vivo.

Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells.

Autologous dendritic cells transfected with total renal tumor RNA have been shown to be potent stimulators of CTLs and antitumor immunity in vitro. A Phase I trial was conducted to evaluate this strategy for feasibility, safety, and efficacy to induce tumor-specific T-cell responses in subjects with metastatic renal cell carcinoma. Renal tumor RNA-transfected dendritic cells were administered to 10 evaluable study patients with no evidence of dose-limiting toxicity or vaccine-related adverse effects including autoimmunity. In six of seven evaluable subjects, expansion of tumor-specific T cells was detected after immunization. The vaccine-induced T-cell reactivities were directed against a broad set of renal tumor-associated antigens, including telomerase reverse transcriptase, G250, and oncofetal antigen, but not against self-antigens expressed by normal renal tissues. Although most patients underwent secondary therapies after vaccination, tumor-related mortality of the study subjects was unexpectedly low with only 3 of 10 patients dying from disease after a mean follow-up of 19.8 months. These data provide a scientific rationale for continued clinical investigation of this polyvalent vaccine strategy in the treatment of metastatic renal cell carcinoma and, potentially, other cancers.

Transfecting Human Monocytes with RNA.

Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

Gene Expression Profile of Dendritic Cell-Tumor Cell Hybrids Determined by Microarrays and Its Implications for Cancer Immunotherapy.

BACKGROUND: Dendritic cell- (DC-) tumor fusion cells stimulate effective in vivo antitumor responses. However, therapeutic approaches are dependent upon the coadministration of exogenous 3rd signals. The purpose of this study was to determine the mechanisms for inadequate 3rd signaling by electrofused DC-tumor cell hybrids. METHODS: Murine melanoma cells were fused with DCs derived from C57BL/6 mice. Quantitative real-time PCR (qPCR) was used to determine relative changes in Th (T helper) 1 and Th2 cytokine gene expression. In addition, changes in gene expression of fusion cells were determined by microarray. Last, cytokine secretion by fusion cells upon inhibition of signaling pathways was analyzed by ELISA. RESULTS: qPCR analyses revealed that fusion cells exhibited a downregulation of Th1 associated cytokines IL-12 and IL-15 and an upregulation of the Th2 cytokine IL-4. Microarray studies further showed that the expression of chemokines, costimulatory molecules, and matrix-metalloproteinases was deregulated in fusion cells. Lastly, inhibitor studies demonstrate that inhibition of the PI3K/Akt/mTOR signaling pathway could restore the secretion of bioactive IL-12p70 by fusion cells. CONCLUSION: Our results suggest that combining fusion cell-based vaccination with administration of inhibitors of the PI3K/Akt/mTOR signaling pathway may enhance antitumor responses in patients.

Tumor vaccines: from gene therapy to dendritic cells--the emerging frontier.

Gene-modified tumor cells have been employed in a vaccination setting to trigger therapeutic antitumor immunity against metastatic renal cell carcinoma. Recent studies suggest that dendritic cells may be even more potent, because these cells can efficiently present tumor antigens to effector T cells, thereby circumventing the poor antigen-presenting properties of tumor cells. Proof of concept studies using antigen-loaded dendritic cells have been performed, establishing clear evidence of vaccine safety and bioactivity by stimulating immunologic and even clinical responses in cancer patients. Nevertheless, key aspects of such vaccination remain undefined. The critical challenge remains to understand fully the mechanisms of action and to further optimize dendritic cell vaccines to produce effective, durable, and, ultimately, therapeutic antitumor responses.

Whole blood cells loaded with messenger RNA as an anti-tumor vaccine.

The use of a cell-based vaccine composed of autologous whole blood cells loaded with mRNA is described. Mice immunized with whole blood cells loaded with mRNA encoding antigen develop anti-tumor immunity comparable to DC-RNA immunization. This approach offers a simple and affordable alternative to RNA-based cellular therapy by circumventing complex, laborious and expensive ex vivo manipulations required for DC-based immunizations.

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes.

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19.

Melanoma immunotherapy using mature DCs expressing the constitutive proteasome.

BACKGROUND: Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. METHODS: Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1, MAGE-3, gp100, and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4), transfected with control siRNA (Arm B; n = 3), or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). RESULTS: Vaccination stimulated antigen-specific T cell responses in all subjects, which peaked after 3-4 vaccinations, but remained elevated in Arm C subjects. Also in Arm C, circulating melanoma cell levels (as detected by quantitative PCR) fell, and T cell lytic activity against autologous melanoma was induced. In HLA-A2⁺ subjects, CD8⁺ T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C), one had a partial clinical response, while the other, who exhibited diffuse dermal and soft tissue metastases, had a complete response. CONCLUSION: These results suggest that the efficacy of melanoma DC-based immunotherapy is enhanced when tumor antigen-loaded DCs used for vaccination express cPs. TRIAL REGISTRATION: Clinicaltrials.gov NCT00672542. FUNDING: Duke Clinical Research Institute/Duke Translational Medicine Institute, Duke Melanoma Consortium, and Duke University Department of Surgery.

Immunoproteasome down-modulation enhances the ability of dendritic cells to stimulate antitumor immunity.

The process of dendritic cell (DC) maturation, critical for effective DC-based immunotherapy, also alters the proteasome such that peptides presented in the context of HLA class I are generated not by the constitutive proteasome, but by the immunoproteasome. Cytotoxic T lymphocytes (CTLs) induced by such DCs might not optimally recognize tumor cells normally expressing the constitutive proteasome. Using small interfering RNA (siRNA) transfection of DCs to inhibit expression of the 3 inducible immunoproteasome subunits in mature DCs, we found that such DCs expressed increased intracellular levels of constitutive proteasomes and presented an altered repertoire of tumor-antigenic peptides. When DCs generated from the monocytes of 3 patients with melanoma were transfected with immunoproteasome siRNA, induced to mature, and then trans-fected with RNA encoding defined melanoma antigens, these DCs were superior inducers of antigen-specific CTLs against autologous melanoma cells. This alteration of DC proteasome composition, which enhances the ability of mature antigen-loaded DCs to stimulate anti-tumor immune responses, may lead to more effective DC-based tumor immunotherapy.

Technology Insight: vaccine therapy for prostate cancer.

The lack of effective therapies for advanced prostate cancer mandates continued development of alternative treatment strategies. Insights into the regulation of immune responses and the malignant process have facilitated the emergence of new immune-based strategies, currently under investigation in clinical trials. Like other forms of targeted therapy, cancer vaccines hold the promise of achieving cancer control without inducing overt toxicity. Many prostate cancer vaccines at different phases of development have been tested in clinical trials. Vaccination strategies under consideration include: immunization with defined antigenic preparations such as synthetic peptides, proteins or plasmid DNA; antigen-loaded dendritic cells; manipulated tumor cells; or with viral vectors engineered to express immunogenic genes. Although the underlying mechanisms of immunization may vary, all strategies share the common goal of eliciting immune responses against prostate tumor-associated antigens or of enhancing an otherwise weak antitumor response in the cancer patient. Unlocking the therapeutic potential of cancer vaccines will require a thorough understanding of cellular and molecular mechanisms that modulate the immune response. In this review, we provide an overview of vaccine-based strategies for prostate cancer therapy, discuss their mechanisms of action, and provide relevant clinical trial data.

Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand.

The objective of this study was to investigate whether the immunostimulatory properties of human monocyte-derived dendritic cells (DCs) could be enhanced by triggering OX40/OX40L signaling. Since monocyte-derived DCs possess only low-cell surface levels of OX40L in the absence of CD40 signaling, OX40L was expressed by transfection of DCs with the corresponding mRNA. We show that OX40L mRNA transfection effectively enhanced the immunostimulatory function of DCs at multiple levels: OX40L mRNA transfection augmented allogeneic and HLA class II epitope-specific CD4+ T-cell responses, improved the stimulation of antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without interfering with the prostaglandin E2 (PGE2)-mediated migratory function of the DCs, and facilitated interleukin 12 p70 (IL-12p70)-independent T helper type 1 (Th1) polarization of naive CD4+ T-helper cells. Furthermore, vaccination of tumor-bearing mice using OX40L mRNA-cotransfected DCs resulted in significant enhancement of therapeutic antitumor immunity due to in vivo priming of Th1-type T-cell responses. Our data suggest that transfection of DCs with OX40L mRNA may represent a promising strategy that could be applied in clinical immunotherapy protocols, while circumventing the current unavailability of reagents facilitating OX40 ligation.

Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer.

Telomerase reverse transcriptase (hTERT) represents an attractive target for cancer immunotherapy because hTERT is reactivated in most human tumors. A clinical trial was initiated in which hTERT mRNA-transfected dendritic cells (DC) were administered to 20 patients with metastatic prostate cancer. Nine of these subjects received DC transfected with mRNA encoding a chimeric lysosome-associated membrane protein-1 (LAMP) hTERT protein, allowing for concomitant induction of hTERT-specific CD8+ and CD4+ T cell responses. Treatment was well tolerated. Intense infiltrates of hTERT-specific T cells were noted at intradermal injection sites after repeated vaccination. In 19 of 20 subjects, expansion of hTERT-specific CD8+ T cells was measured in the peripheral blood of study subjects, with 0.9-1.8% of CD8+ T cells exhibiting Ag specificity. Patients immunized with the chimeric LAMP hTERT vaccine developed significantly higher frequencies of hTERT-specific CD4+ T cells than subjects receiving DC transfected with the unmodified hTERT template. Moreover, CTL-mediated killing of hTERT targets was enhanced in the LAMP hTERT group, suggesting that an improved CD4+ response could augment a CTL response. Vaccination was further associated with a reduction of prostate-specific Ag velocity and molecular clearance of circulating micrometastases. Our findings provide a rationale for further development of hTERT-transfected DC vaccines in the treatment of prostate and other cancers.

Cotransfection of DC with TLR4 and MART-1 RNA induces MART-1-specific responses.

BACKGROUND: Cotransfection of dendritic cells (DC) with MART-1 and constitutively active TLR4 (caTLR4) RNA enhances the maturation of DC. MATERIALS AND METHODS: Immature DC were cotransfected with RNA constructs encoding MART-1 and caTLR4, and CTL responses were analyzed. RESULTS: Cotransfection of DC with MART-1 + caTLR4 enhanced the expression of CD80 and CD83 surface markers and increased the secretion of cytokines IL-6, IL-12, and TNFalpha. Neither the native nor the A27L-modified MART-1 RNA could induce significant DC maturation or cytokine secretion. More importantly, DC cotransfected with caTLR4 + MART-1 RNA induced MART-1-specific CTL responses of a higher magnitude than DC transfected with either the native or A27L MART-1 RNA. When the MART-1 RNA-transfected DC were treated with DC-maturing cytokines, the induced CTL were less frequent and less lytic than those induced with MART-1 + caTLR4. A 2- to 100-fold increase in MART-1 tetramer+ cells and 2- to 10-fold increases in IFNgamma secretion and cytotoxicity were seen in CTL induced with MART-1 + caTLR4 compared to CTL induced with either MART-1 or A27L RNA. CTL induced with the mixed RNA displayed high percentages of CD8+ cells coexpressing CD45RA, CD56, and 2B4 antigens. Transfection with caTLR4 alone induced DC maturation, but did not induce lytic CTL, suggesting that CTL responses were induced solely by MART-1 epitopes. CONCLUSIONS: caTLR4 increases the CTL-inducing capacity of DC generating a lytic response specific for the accompanying antigen. These results demonstrate the possibility of enhancing the immunogenicity of the native MART-1 and other RNA derived from weakly immunogenic tumors in DC-based immunotherapy.