Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity

Dendritic cell (DC)-based vaccines are being developed for treatment of patients with cancer, in part because DC are potent inducers of CD8(+) CTL. DC MHC class I:antigenic peptide complexes that are required for CTL elicitation are most often generated by incubating DC with peptides or by transfecting (or transducing) DC with cDNAs (or viral vectors) that encode protein Ags. The former approach is feasible when MHC class I Ags and relevant peptides are known. The latter approach may be hampered by inefficient DC transfection (transduction) and/or difficulties associated with preparation and use of viral vectors. Herein we demonstrate that a bacterial recombinant model tumor-associated Ag (OVA) that contains the HIV TAT protein transduction domain (PTD) was readily engineered and purified, efficiently transduced murine lymphocytes and DC, and was processed by proteasomes for MHC class I-restricted presentation to CTL. In addition, PTD-containing rOVA was processed and presented by DC to CD4 T cells as efficiently as native OVA or rOVA lacking the PTD. PTD-OVA-transduced DC induced CTL in vivo in a Th cell-independent fashion and vaccinated against OVA-expressing tumors. In contrast, rOVA lacking the PTD did not enter the DC MHC class I presentation pathway and DC treated with this protein did not prime OVA-specific CTL in vivo. Treatment of mice harboring clinically apparent OVA-expressing tumors with PTD-OVA-transduced DC resulted in tumor regression in some animals. This straightforward vaccination strategy may translate into DC-based treatments for patients with cancer and other serious diseases.     

In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self antigens and induces potent antitumor immunity

Most tumor-associated Ags are self proteins that fail to elicit a T cell response as a consequence of immune tolerance. Dendritic cells (DCs) generated ex vivo have been used to break tolerance against such self Ags; however, in vitro manipulation of DCs is cumbersome and difficult to control, resulting in vaccines of variable potency. To address this problem we developed a method for loading and activating DCs, in situ, by first directing sufficient numbers of DCs to peripheral tissues using Flt3 ligand and then delivering a tumor-associated Ag and oligonucleotide containing unmethylated CG motifs to these tissues. In this study, we show in three different tumor models that this method can overcome tolerance and induce effective antitumor immunity. Vaccination resulted in the generation of CD8(+) T and NK cell effectors that mediated durable tumor responses without attacking normal tissues. These findings demonstrate that unmodified tumor-associated self Ags can be targeted to DCs in vivo to induce potent systemic antitumor immunity.     

Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.     

Induction of antitumor immunity by dendritic cells loaded with membrane-translocating mucin 1 Peptide antigen

To investigate the role of enhanced antigen presentation in dendritic cell (DC)-based immunotherapy. Here, we describe the development of a cell-penetrating mucin 1 (MUC1) antigen and its immunotherapeutic potential against tumors. After animal groups received two immunizations of MUC1-MPA(11)P-pulsed DCs, we observed a marked tumor regression compared with the mice treated with DCs alone or DCs pulsed with MUC1 peptide. We confirmed the migration and homing of DCs in the popliteal lymph node using magnetic resonance imaging during the study. In summary, enhanced antigen uptake using an MPA(11)P delivery molecule improves cell therapy.     

Induction of impaired antitumor immunity by fusion of MHC class II-deficient dendritic cells with tumor cells

To dissect the role of Ag presentation through MHC class I and/or II pathways by dendritic cell (DC)-tumor fusion cells, we have created various types of DC-tumor fusion cells by alternating fusion cell partners. Fusions of MC38/MUC1 carcinoma cells with DC from wild-type (WT-DC), MHC class I knockout (IKO-DC), class II knockout (IIKO-DC), or class I and II knockout (I/IIKO-DC) mice created WTDC-fusion cells (FC), IKO-FC, IIKO-FC, and I/IIKO-FC, respectively. MHC class II- and MUC1-positive fusion cells were constructed by fusion of B16/MUC1 melanoma cells with IKO-DC (IKO/B16-FC). Immunization of MUC1 transgenic mice with 5 x 10(5) WTDC-FC, IKO-FC, IIKO-FC, or I/IIKO-FC provided 100, 91.7, 61.5, and 15.4% protection, respectively, against tumor challenge with MC38/MUC1 cells. In contrast, all mice immunized with irradiated MC38/MUC1 tumor cells or WT-DC developed tumors. One group of mice was immunized with 5 x 10(5) IKO/B16-FC and then challenged with B16/Ia(+)/MUC1 on one flank and MC38/MUC1 on the other flank. Immunization of these mice with IKO/B16-FC resulted in 100 and 78.6% protection against B16/Ia(+)/MUC1 and MC38/MUC1 tumor challenge, respectively. The antitumor immunity induced by immunization with IKO/B16-FC was able to inhibit the growth of MHC class II-negative tumor. In addition, in vivo results correlated with the induction of Ag-specific CTL. Collectively, the data indicate that MHC class II Ag presentation targeting activation of CD4 T cells is indispensable for antitumor immunity.     

Cancer vaccines based on dendritic cells loaded with tumor-associated antigens

Dendritic cells (DCs) can be used as an antigen presentation platform for vaccination against cancer. In this approach, DCs are expanded in vitro from monocyte-derived progenitors, and subsequently loaded with well-characterized tumor-associated antigens (TAAs). TAAs can be incubated with DCs in various forms, including peptides, recombinant proteins, plasmid DNA, formulated RNA, or recombinant viruses. Advantages and limitations of DC-based cellular vaccines against cancers, as well as preliminary results of clinical studies already performed in humans, are discussed. Importantly, significant advances in our understanding of the biology of DCs can be used to support the design of new vaccines or adjuvants in order to elicit T_H1 cellular immune responses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

Mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens.

Chemokine receptors are differentially expressed on immature and mature dendritic cells (DC). Herein, we demonstrate for the first time that murine antimicrobial peptides beta-defensins 2 and 3 bind murine CCR6, similarly to inflammatory chemokine macrophage-inflammatory protein 3alpha, and they chemoattract bone marrow-derived immature, but not mature DC. Using various chemokines or defensins fused with nonimmunogenic tumor Ags, we studied their capacity to delivery Ags to subsets of immune cells to elicit antitumor immunity. We demonstrate that DNA immunizations with fusion constructs with beta-defensin 2 or inflammatory chemokines that target immature DC, but not homeostatic chemokines secondary lymphoid tissue chemokine, CCL21, or stromal cell-derived factor 1, CXCL12, which chemoattract mature DC, elicit humoral, protective, and therapeutic immunity against two different syngeneic lymphomas.     

Reovirus activates human dendritic cells to promote innate antitumor immunity

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-alpha, TNF-alpha, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-kappaB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-gamma production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-gamma) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus's direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.     

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.     

Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells

Vaccination with dendritic cells (DCs) pulsed with antigenic peptides derived from various tumor antigens has great, but as yet significantly unrealized, potential in cancer treatment. Here, we describe a strategy for prolonged presentation of an MHC class I-restricted self-peptide on DCs through linkage of it to a cell penetrating peptide (CPP). DCs loaded with a peptide derived from tyrosinase-related protein 2 (TRP2) covalently linked to a CPP1 sequence retained full capacity to stimulate T cells for at least 24 h, completely protected immunized mice from subsequent tumor challenge, and significantly inhibited lung metastases in a 3-day tumor model. DCs pulsed with TRP2 alone failed to provide any of these protections. In addition, we demonstrate that both CD4+ and CD8+ T cells were required for potent antitumor immunity. This CPP-based approach may be generally applicable to enhance the efficacy of DC-based peptide vaccines against cancer and other diseases.     

Effect of thymosin alpha 1 on the antitumor activity of tumor-associated macrophage-derived dendritic cells

We have previously suggested that thymosin ₁ (thy1), an immunomodulating thymic hormone, can activate tumor-associated macrophages to a tumoricidal state in a murine model bearing a transplantable T-cell lymphoma of spontaneous origin designated as Dalton's lymphoma (DL). Since tumor-infiltrating dendritic cells (DC) also play an important role in the host's antitumor response and are as such in an immunocompromised state in a tumor-bearing host, in the present investigation we studied if thy1 is able to influence the differentiation of tumor-associated macrophages (TAM) into DC with granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor (TNF) and whether these TAM-derived DC show enhanced antitumor activity. It was observed that DC generated from thy1-administered tumor-bearing mice showed augmented antitumor activity in vitro. Adoptive immunotherapy using TAM-derived DC showed a significant delay in the tumor growth and a prolongation of the survival time in tumor-bearing mice. DC obtained from TAM of thy1-administered mice also produced an enhanced amount of cytokines like IL-1 and TNF-. This is the first study of its kind regarding the effect of thy1 on the differentiation of DC from TAM and the role of TAM-derived DC in tumor progression.     

Induction of antitumor immunity by indomethacin

Irradiated tumor cells given, together with indomethacin, to syngeneic mice induced an antitumor response and conferred protection against a challenge of a lethal dose of murine mammary (4T1) and lung (3LL) carcinoma cells. Continuous administration of indomethacin was crucial throughout the entire period of immunization and challenge, as no protection was achieved when the drug was given during only one of these procedures. Antitumor immunity was long-lasting and, when tested in the 4T1 model, 48% of mice were resistant to a second challenge of lethal tumor cells. Tumor-free immune mice that were given indomethacin for more than 300 days remained healthy with normal white blood cell counts and normal spleen size. Cells isolated from immune mice were able to kill tumor cells in culture after in vitro activation by interleukin-2, in a manner similar to cells from naive normal control mice. In addition, the mitogenic response of their T cells was as high as that of the control naive mice. While indomethacin was able to induce antitumor immunity to 4T1 and 3LL murine carcinoma cells, both of which contain a high concentration of endogenic prostaglandin E₂ (PGE2), no such immunity was achieved to murine tumor cells with a low concentration of endogenic PGE2. These results suggest a correlation between PGE2 concentration and the ability of indomethacin to induce antitumor immunity. We therefore suggest that an immunotherapy protocol with long-term dispensation of a tolerable dose of an immunomodulator, given together with irradiated autologous tumor cells, may stimulate antitumor responses to tumors containing high concentrations of endogenic PGE2. Received: 12 August 1999 / Accepted: 21 September 1999     

Release of tumor-associated antigens by murine melanoma cells.

Synthesis and release of radiolabeled macromolecules and tumor-associated antigens (MAA) by murine B16 melanoma was studied by pulse labeling cells in culture with 3H-leucine. Approximately 36% of newly synthesized macromolecules and 44% of newly synthesized MAA were released in 48 hr. MAA release was slightly, but consistently, more rapid than the average release of other macromolecules. Release of MAA did not result solely from cell death since it was greater than that of 51Cr-labeled molecules and cell viability was over 98%. The rate of release of newly synthesized MAA was not significantly influenced by cell replication. However, synthesis of MAA was much greater during the logarithmic than the stationary phase of cell growth, suggesting a concomitant increase in the amount of MAA available for release. These findings indicate that antigens and other macromolecules can be rapidly released by viable tumor cells.     

Recombinant cholera toxin B subunit activates dendritic cells and enhances antitumor immunity

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non-toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7-2 on DC was upregulated and the secretion of IL-12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB-treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB-treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB-treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell-depleted mice, although substantial antitumor effect was observed in CD4+ T cell-depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.     

Induction of efficient antitumor immunity using dendritic cells activated by recombinant Sendai virus and its modulation by exogenous IFN-beta gene

Dendritic cell (DC)-based cancer immunotherapy has been paid much attention as a new and cancer cell-specific therapeutic in the last decade; however, little clinical outcome has been reported. Current limitations of DC-based cancer immunotherapy include sparse information about which DC phenotype should be administered. We here report a unique, representative, and powerful method to activate DCs, namely recombinant Sendai virus-modified DCs (SeV/DC), for cancer immunotherapy. In vitro treatment of SeV without any bioactive gene solely led DCs to a mature phenotype. Even though the expression of surface markers for DC activation ex vivo did not always reach the level attained by an optimized amount of LPS, superior antitumor effects to B16F1 melanoma, namely tumor elimination and survival, were obtained with use of SeV-GFP/DC as compared with those seen with LPS/DC in vivo, and the effect was enhanced by SeV/DC-expressing IFN-beta (SeV-murine IFN-beta (mIFN-beta)/DC). In case of the treatment of an established tumor of B16F10 (7-9 mm in diameter), a highly malignant subline of B16 melanoma, SeV-modified DCs (both SeV-GFP/DC and SeV-mIFN-beta/DC), but not immature DC and LPS/DC, dramatically improved the survival of animals. Furthermore, SeV-mIFN-beta/DC but not other DCs could lead B16F10 tumor to the dormancy, associated with strongly enhanced CD8+ CTL responses. These results indicate that rSeV is a new and powerful tool as an immune booster for DC-based cancer immunotherapy that can be significantly modified by IFN-beta, and SeV/DC, therefore, warrants further investigation as a promising alternative for cancer immunotherapy.     

Induction of anti-tumor immunity by vaccination with dendritic cells pulsed with anti-CD44 IgG opsonized tumor cells

Due to the pivotal role that dendritic cells (DC) play in eliciting and maintaining functional anti-tumor T cell responses, these APC have been exploited against tumors. DC express several receptors for the Fc portion of IgG (Fcγ receptors) that mediate the internalization of antigen-IgG complexes and promote efficient MHC class I and II restricted antigen presentation. In this study, the efficacy of vaccination with DC pulsed with apoptotic B16 melanoma cells opsonized with an anti-CD44 IgG (B16-CD44) was explored. Immature bone marrow derived DC grown in vitro with IL-4 and GM-CSF were pulsed with B16-CD44. After 48 h of pulsing, maturation of DC was demonstrated by production of IL-12 and upregulation of CD80 and CD40 expression. To test the efficacy of vaccination with DC+B16-CD44, mice were vaccinated subcutaneously Lymphocytes from mice vaccinated with DC+B16-CD44 produced IFN-γ in response to B16 melanoma lysates as well as an MHC class I restricted B16 melanoma-associated peptide, indicating B16 specific CD8 T cell activation. Upon challenge with viable B16 cells, all mice vaccinated with DC alone developed tumor compared to 40% of mice vaccinated with DC+B16-CD44; 60% of the latter mice remained tumor free for at least 8 months. In addition, established lung tumors and distant metastases were significantly reduced in mice treated with DC+B16-CD44. Lastly, delayed growth of established subcutaneous tumors was induced by combination therapy with anti-CD44 antibodies followed by DC injection. This study demonstrates the efficacy of targeting tumor antigens to DC via Fcγ receptors.     

The potential of DNA vaccination against tumor-associated antigens for antitumor therapy

Conventional treatment approaches for malignant tumors are highly invasive and sometimes have only a palliative effect. Therefore, there is an increasing demand to develop novel, more efficient treatment options. Increased efforts have been made to apply immunomodulatory strategies in antitumor treatment. In recent years, immunizations with naked plasmid DNA encoding tumor-associated antigens have revealed a number of advantages. By DNA vaccination, antigen-specific cellular as well as humoral immune responses can be generated. The induction of specific immune responses directed against antigens expressed in tumor cells and displayed e.g., by MHC class I complexes can inhibit tumor growth and lead to tumor rejection. The improvement of vaccine efficacy has become a critical goal in the development of DNA vaccination as antitumor therapy. The use of different DNA delivery techniques and coadministration of adjuvants including cytokine genes may influence the pattern of specific immune responses induced. This brief review describes recent developments to optimize DNA vaccination against tumor-associated antigens. The prerequisite for a successful antitumor vaccination is breaking tolerance to tumor-associated antigens, which represent "self-antigens." Currently, immunization with xenogeneic DNA to induce immune responses against self-molecules is under intensive investigation. Tumor cells can develop immune escape mechanisms by generation of antigen loss variants, therefore, it may be necessary that DNA vaccines contain more than one tumor antigen. Polyimmunization with a mixture of tumor-associated antigen genes may have a synergistic effect in tumor treatment. The identification of tumor antigens that may serve as targets for DNA immunization has proceeded rapidly. Preclinical studies in animal models are promising that DNA immunization is a potent strategy for mediating antitumor effects in vivo. Thus, DNA vaccines may offer a novel treatment for tumor patients. DNA vaccines may also be useful in the prevention of tumors with genetic predisposition. By DNA vaccination preventing infections, the development of viral-induced tumors may be avoided.