The immunization site of cytokine-secreting tumor cell vaccines influences the trafficking of tumor-specific T lymphocytes and antitumor efficacy against regional tumors

Tumor cells engineered to secrete cytokines, referred to as tumor cell vaccines, can often generate systemic antitumor immunity and, in many cases, cause tumor regression. We compared the efficacy of s.c. immunization or intrahepatic immunization of GM-CSF-expressing tumor cell vaccines on the growth of s.c. or orthotopic liver tumors. A chemically transformed hepatic epithelial cell line, GP7TB, derived from Fischer 344 rats, was used to generate tumor models and tumor cell vaccines. Our results demonstrated that two s.c. injections of an irradiated tumor cell vaccine significantly controlled the growth of s.c. tumors, but was completely ineffective against orthotopic liver tumors. Effector cell infiltration in liver tumors was markedly reduced compared with s.c. tumors. Enhanced apoptosis of some effector cells was observed in the liver tumors compared with the s.c. tumors. Furthermore, the T cells induced by s.c. immunization preferentially migrated to s.c. tumor sites, as demonstrated by adoptive transfer experiments. In contrast, intrahepatic immunization, using parental tumor cells admixed with adenoviruses carrying the GM-CSF gene, yielded significantly better therapeutic effects on the liver tumors than on the s.c. tumors. Adoptive transfer experiments further confirmed that the T cells induced by liver immunization preferentially migrated to the liver tumor sites. Our results demonstrate that distinct T cell populations are induced by different immunization routes. Thus, the homing behavior of T cells depends on the route of immunization and is an important factor determining the efficacy of immunotherapy for regional tumors.     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

Active immunization combined with cisplatin confers enhanced therapeutic protection and prevents relapses of HPV-induced tumors at different anatomical sites

The active immunotherapy concept relies on the use of vaccines that are capable of inducing antitumor immunity, reversion of the suppressive immunological environment, and long-term memory responses. Previously, antitumor vaccines based on a recombinant plasmid (pgDE7h) or a purified protein (gDE7) led to regression of early-established human papillomavirus (HPV)-associated tumors in a preclinical model. In this work, the anticancer vaccines were combined with cisplatin to treat HPV-induced tumors at advanced growth stages. The antitumor effects were evaluated in terms of tumor regression, induction of specific CD8⁺ T cells, and immune modulation of the tumor microenvironment. Acute toxicity induced by the treatment was measured by weight loss and histological alterations in the liver and kidneys. Our results revealed that the combination of cisplatin with either one of the tested immunotherapies (pgDE7h or gDE7) led to complete tumor regression in mice. Also, the combined treatment resulted in synergistic effects, particularly among mice immunized with gDE7, including activation of systemic and tumor-infiltrating E7-specific CD8⁺ T cells, tumor infiltration of macrophages and dendritic cells, and prevention of tumor relapses at different anatomical sites. Furthermore, the protocol allowed the reduction of cisplatin dosage and its intrinsic toxic effects, without reducing antitumor outcomes. These results expand our knowledge of active immunotherapy protocols and open perspectives for alternative treatments of HPV-associated tumors.     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.     

Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor

Immunotherapy of cancer is attractive because of its potential for specificity and limited side effects. The efficacy of this approach may be improved by providing adjuvant signals and an inflammatory environment for immune cell activation. We evaluated antitumor immune responses in mice after treatment of OVA-expressing B16-F0 tumors with single (15 Gy) or fractionated (5 x 3 Gy) doses of localized ionizing radiation. Irradiated mice had cells with greater capability to present tumor Ags and specific T cells that secreted IFN-gamma upon peptide stimulation within tumor-draining lymph nodes than nonirradiated mice. Immune activation in tumor-draining lymph nodes correlated with an increase in the number of CD45(+) cells infiltrating single dose irradiated tumors compared with nonirradiated mice. Similarly, irradiated mice had increased numbers of tumor-infiltrating lymphocytes that secreted IFN-gamma and lysed tumor cell targets. Peptide-specific IFN-gamma responses were directed against both the class I and class II MHC-restricted OVA peptides OVA(257-264) and OVA(323-339), respectively, as well as the endogenous class I MHC-restricted B16 tumor peptide tyrosinase-related protein 2(180-188). Adoptive transfer studies indicated that the increased numbers of tumor Ag-specific immune cells within irradiated tumors were most likely due to enhanced trafficking of these cells to the tumor site. Together these results suggest that localized radiation can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site.     

Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8⁺ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8⁺ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8⁺ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8⁺ CD44^hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-α or IFN-γ or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8⁺ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8⁺ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8⁺ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8⁺ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8⁺ T cells.     

Successful tumor eradication was achieved by collaboration of augmented cytotoxic activity and anti-angiogenic effects following therapeutic vaccines containing helper-activating analog-loaded dendritic cells and tumor antigen DNA

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4⁺ and CD8⁺ T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.     

Targeting molecular and cellular inhibitory mechanisms for improvement of antitumor memory responses reactivated by tumor cell vaccine

Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor Ags via restimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms, which function to limit the recall responses of tumor-specific immunity, remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional antitumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survival. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting in which tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

Vaccine-stimulated, adoptively transferred CD8+ T cells traffic indiscriminately and ubiquitously while mediating specific tumor destruction

It has been suggested that antitumor T cells specifically traffic to the tumor site, where they effect tumor destruction. To test whether tumor-reactive CD8(+) T cells specifically home to tumor, we assessed the trafficking of gp100-specific pmel-1 cells to large, vascularized tumors that express or do not express the target Ag. Activation of tumor-specific CD8(+) pmel-1 T cells with IL-2 and vaccination with an altered peptide ligand caused regression of gp100-positive tumors (B16), but not gp100-negative tumors (methylcholanthrene 205), implanted on opposing flanks of the same mouse. Surprisingly, we found approximately equal and very large numbers of pmel-1 T cells (>25% of all lymphocytes) infiltrating both Ag-positive and Ag-negative tumors. We also found evidence of massive infiltration and proliferation of activated antitumor pmel-1 cells in a variety of peripheral tissues, including lymph nodes, liver, spleen, and lungs, but not peripheral blood. Most importantly, evidence for T cell function, as measured by production of IFN-gamma, release of perforin, and activation of caspase-3 in target cells, was confined to Ag-expressing tumor. We thus conclude that CD8(+) T cell-mediated destruction of tumor is the result of specific T cell triggering at the tumor site. The ability to induce ubiquitous homing and specific tumor destruction may be important in the case of noninflammatory metastatic tumor foci.     

Role of tumor cell apoptosis in tumor antigen migration to the draining lymph nodes

Establishment of an immune response against cancer may depend on the capacity of dendritic cells to transfer tumor Ags into T cell-rich areas. To check this possibility, we used a colon cancer cell variant that yields tumors undergoing complete T cell-dependent rejection when injected into syngeneic rats. We previously demonstrated that immunogenicity of these tumors depended on the early apoptosis of a part of these tumor cells. In this paper we show that fluorescent tumor cell proteins are released from FITC-labeled tumor cells and undergo engulfment by tumor-infiltrating monocytes without a phenotype of mature dendritic cells or macrophages. Fluorescence-labeled mononuclear cells with a phenotype of MHC class II+ dendritic cells are also found in the T cell areas of the draining lymph nodes. Interestingly, no fluorescent cell can be found in lymph nodes after a s.c. injection of Bcl2-transfected apoptosis-resistant tumor cells that yielded progressive tumors. Proliferation of tumor-immune T lymphocytes was induced by dendritic cells isolated from the draining lymph nodes recovered after a s.c. injection of apoptosis-sensitive, but not apoptosis-resistant, tumor cells. These results show that tumor cell apoptosis releases proteins that are engulfed by inflammatory cells in the tumor, then transported to lymph node T cell areas where they can induce a specific immune response leading to tumor rejection.     

Endogenous dendritic cells are required for amplification of T cell responses induced by dendritic cell vaccines in vivo

Dendritic cells (DCs) loaded in vitro with Ag are used as cellular vaccines to induce Ag-specific immunity. These cells are thought to be responsible for direct stimulation of Ag-specific T cells, which may subsequently mediate immunity. In this study, in transgenic mouse models with targeted MHC class II expression specifically on DCs, we show that the DC vaccine is responsible only for partial CD4(+) T cell activation, but to obtain optimal expansion of T cells in vivo, participation of endogenous (resident) DCs, but not endogenous B cells, is crucial. Transfer of Ag to endogenous DCs seems not to be mediated by simple peptide diffusion, but rather by DC-DC interaction in lymph nodes as demonstrated by histological analysis. In contrast, injection of apoptotic or necrotic DC vaccines does not induce T cell responses, but rather represents an immunological null event, which argues that viability of DC vaccines can be crucial for initial triggering of T cells. We propose that viable DCs from the DC vaccine must migrate to the draining lymph nodes and initiate a T cell response, which thereafter requires endogenous DCs that present transferred Ag in order induce optimal T cell expansion. These results are of specific importance with regard to the applicability of DC vaccinations in tumor patients, where the function of endogenous DCs is suppressed by either tumors or chemotherapy.     

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

Dendritic cell-based vaccines suppress metastatic liver tumor via activation of local innate and acquired immunity

Background  Dendritic cell (DC)-based vaccines have been applied clinically in the setting of cancer, but tumor-associated antigens (TAAs) have not yet been enough identified in various cancers. In this study, we investigated whether preventive vaccination with unpulsed DCs or peptide-pulsed DCs could offer anti-tumor effects against MC38 or BL6 liver tumors. Methods  Mice were subcutaneously (s.c.) immunized with unpulsed DCs or the recently defined TAA EphA2 derived peptide-pulsed dendritic cells (Eph-DCs) to treat EphA2-positive MC38 and EphA2-negative BL6 liver tumors. Liver mononuclear cells (LMNCs) from treated mice were subjected to ⁵¹Cr release assays against YAC-1 target cells. In some experiments, mice were injected with anti-CD8, anti-CD4 or anti-asialo GM1 antibody to deplete each lymphocyte subsets. Results  Immunization with unpulsed DCs displayed comparable efficacy against both MC38 and BL6 liver tumors when compared with Eph-DCs. Both DC-based vaccines significantly augmented the cytotoxicity of LMNCs against YAC-1 cells. In vivo antibody depletion studies revealed that NK cells, as well as, CD4+ and CD8+ T cells play critical roles in the anti-tumor efficacy associated with either DC-based modality. Tumor-specific cytotoxic T lymphocyte (CTL) activity was generally higher if mice had received Eph-DCs versus unpulsed DCs. Importantly, the mice that had been protected from MC38 liver tumor by either unpulsed DCs or Eph-DCs became resistant to s.c. MC38 rechallenge, but not to BL6 rechallenge. Conclusions  These results demonstrate that unpulsed DC vaccines might serve as an effective therapy for treating metastatic liver tumor, for which TAA has not yet been identified. Shinjiro Yamaguchi and Tomohide Tatsumi contributed equally to this work.     

Cytokine-gene-modified tumor vaccination intensified by a streptococcal preparation OK-432

Vaccinations with tumor cells engineered to express certain cytokines have been demonstrated to induce potent and specific antitumor immunity. In our previous report, we carried out a comparative study on the ability of cytokine-gene-modified tumor vaccines to induce host immune responses, and found that irradiated tumor cells, genetically modified to secrete granulocyte/macrophagecolony-stimulating factor (GM-CSF tumor vaccine), were the most potent stimulators of systemic antitumor immunity. In this report, using the experimental tumor models in which the GM-CSF tumor vaccine was less effective in immunopotentiation, we found that the combined use of a biological response modifier (BRM) OK-432 remarkably enhanced the antitumor activity induced by the GM-CSF tumor vaccine. These data indicate the possible role of a BRM such as OK-432 to intensify further the specific tumor vaccination therapy.     

Tumor-derived TGF-beta reduces the efficacy of dendritic cell/tumor fusion vaccine

Dendritic cell (DC)-based antitumor vaccine is a novel cancer immunotherapy that is promising for reducing cancer-related mortality. However, results from early clinical trials were suboptimal. A possible explanation is that many tumors secrete immunosuppressive factors such as TGF-beta, which may hamper host immune response to DC vaccine. In this study, we demonstrated that TGF-beta produced by tumors significantly reduced the potency of DC/tumor fusion vaccines. TGF-beta-secreting (CT26-TGF-beta) stable mouse colon cancer cell lines were generated using a retroviral vector expressing TGF-beta. A non-TGF-beta-secreting (CT26-neo) cell line was generated using an empty retroviral vector. The efficacies of DC/tumor fusion vaccines were assessed in vitro and in vivo. DC/CT26-TGF-beta fusion cells failed to induce a strong T cell proliferative response in vitro, mainly due to the effect of TGF-beta on T cell responsiveness rather than DC stimulatory capability. Animals vaccinated with DC/CT26-TGF-beta fusion vaccine had lower tumor-specific CTL activity and had significantly lower survival after tumor challenge as compared with animals immunized with DC/CT26-neo hybrids (45 vs 77%, p < 0.05). Ex vivo exposure of DCs to TGF-beta did not appear to lessen the efficacy of DC vaccine. These data suggest that tumor-derived TGF-beta reduces the efficacy of DC/tumor fusion vaccine via an in vivo mechanism. Neutralization of TGF-beta produced by the fusion cells may enhance the effectiveness of DC-based immunotherapy.     

Photodynamic therapy-generated vaccine for cancer therapy

A target tumor-derived whole cancer cell therapeutic vaccine was developed based on an in vitro pre-treatment by photodynamic therapy (PDT) and was investigated using a poorly immunogenic tumor model. The vaccine was produced by incubating in vitro expanded mouse squamous cell carcinoma SCCVII cells for 1 h with photosensitizer benzoporphyrin derivative (BPD), then exposing to light (690 nm, 1 J/cm²) and finally to a lethal X-ray dose. Treatment of established subcutaneous SCCVII tumors growing in syngeneic C3H/HeN mice with 2x10⁷ PDT-vaccine cells per mouse by a peritumoral injection produced a significant therapeutic effect, including growth retardation, regression and cures. Tumor specificity of this PDT-generated vaccine was demonstrated by its ineffectiveness when prepared from a mismatched tumor cell line. Vaccine cells retrieved from the treatment site at 1 h postinjection were intermixed with dendritic cells (DC), exhibited heat shock protein 70 on their surface, and were opsonized by complement C3. Tumor-draining lymph nodes treated by the PDT-vaccine contained dramatically increased numbers of DC as well as B and T lymphocytes (with enlarged memory phenotype fraction in the latter), while high levels of surface-bound C3 were detectable on DC and to a lesser extent on B cells. The PDT-vaccine produced no therapeutic benefit against tumors growing in C3-deficient hosts. It is suggested that surface expression of heat shock proteins and complement opsonization are the two unique features of PDT-treated cells securing avid immune recognition of vaccinated tumor and the development of a strong and effective antitumor adaptive immune response.     

TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines

A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.     

Potent in vivo anti-tumor activity of isolated CD62L(low) lymph node cells sensitized in vivo with tumor lysate-pulsed DC-based vaccines

BACKGROUND: DC are potent APC that can activate both CD4 and CD8 T cells in vitro and in vivo. Although the efficacy of DC-based cancer vaccines is currently being evaluated in clinical trials, the systemic immune suppression in cancer patients negatively impacts the clinical benefit of this therapeutic approach. Therefore, in this study we tested the feasibility and anti-tumor effect of adoptive immunotherapy using in vitro-activated CD62L(low) lymph node cells that were isolated from DC-vaccinated draining lymph nodes (VDLN). METHODS: DC were prepared from BM cells and loaded with tumor lysate for inoculating into naive mice. Subsequently, the VDLN were removed and CD62L(low) cells in the VDLN population isolated, expanded in vitro by 5-day culture with IL-2 and immobilized anti-CD3 stimulation, then injected into mice with established pulmonary tumors. Eighteen days after treatment, mice were killed in order to enumerate pulmonary tumor nodes. RESULTS: DC phagocytosed the tumor lysate efficiently and induced detectable T-cell responses and significant cell expansion in the draining lymph nodes. After induction of maturation by LPS treatment, DC expressed higher levels of CD40, CD86 and MHC class II molecules. When CD62L(low) VDLN cells that had been isolated and expanded in vitro were transferred into tumor-bearing mice, as few as 3 x 10(6) cells were able to cure metastatic pulmonary tumors in vivo. DISCUSSION: DC-based VDLN T cells are an important source of anti-tumor effector for adoptive immunotherapy. This study provides a novel and an effective protocol using T-cell adoptive immunotherapy for application in cancer patients; therefore, clinical trials based on this protocol may be warranted.     

Provision of granulocyte-macrophage colony-stimulating factor converts an autoimmune response to a self-antigen into an antitumor response

Many tumor Ags recognized by T cells are self-Ags. Because high avidity, self-reactive T cells are deleted in the thymus, any residual self-reactive T cells existing in the periphery are likely to be low avidity and nonresponsive due to peripheral tolerance mechanisms. Activation of these residual T cells is critical for targeting tumors for immunotherapy. In this study, we studied immune responses against the murine B16 melanoma using a tyrosinase-related protein 2 (TRP-2) peptide as a model tumor/self-Ag. Our results showed that TRP-2 peptide vaccination alone elicited a weak T cell response and modestly decreased B16 lung tumor nodules. The combination of peptide vaccination and treatment with an Ab directed against the inhibitory receptor CTLA-4 enhanced the immune response against TRP-2 peptide, inducing autoimmune depigmentation and further decreasing lung tumor nodules. However, both vaccination methods failed to protect against orthotopic (s.c.) B16 tumor challenge. The addition of an irradiated GM-CSF-expressing, amelanotic tumor cell vaccine significantly delayed s.c. B16 tumor growth. Subsequent studies revealed that provision of GM-CSF increased dendritic cell numbers in lymph nodes and spleen. Furthermore, addition of CTLA-4 blockade increased the frequency of TRP-2-specific, IFN-secreting T cells in spleen and lymph nodes. Overall, our results indicate that combining enhancement of Ag presentation with removal of CTLA-4-mediated inhibition can convert a "weaker" autoimmune response into a more potent antitumor immune response.