Tumor Antigen-pulsed Dendritic Cells Research Articles

A CTLA-4 blocking strategy based on Nanobody in dendritic cell-stimulated cytokine-induced killer cells enhances their anti-tumor effects

BackgroundCytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, it sefficacy is restricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells, we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, we tested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity, or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy.ResultsAfter stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice.ConclusionsOur findings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential to treat malignant tumors through in vivo adoptive therapy.

Contribution of pre-existing neoantigen-specific T cells to a durable complete response after tumor-pulsed dendritic cell vaccine plus nivolumab therapy in a patient with metastatic salivary duct carcinoma

ABSTRACT Although immune checkpoint inhibitors (ICIs) have emerged as new therapeutic options for refractory cancer, they are only effective in select patients. Tumor antigen-pulsed dendritic cell (DC) vaccine therapy activates tumor-specific cytotoxic T lymphocytes, making it an important immunotherapeutic strategy. Salivary ductal carcinoma (SDC) carries a poor prognosis, including poor long-term survival after metastasis or recurrence. In this study, we reported a case of refractory metastatic SDC that was treated with a tumor lysate-pulsed DC vaccine followed by a single injection of low-dose nivolumab, and a durable complete response was achieved. We retrospectively analyzed the immunological factors that contributed to these long-lasting clinical effects. First, we performed neoantigen analysis using resected metastatic tumor specimens obtained before treatment. We found that the tumor had 256 non-synonymous mutations and 669 class I high-affinity binding neoantigen peptides. Using synthetic neoantigen peptides and ELISpot analysis, we found that peripheral blood mononuclear leukocytes cryopreserved before treatment contained pre-existing neoantigen-specific T cells, and the cells obtained after treatment exhibited greater reactivity to neoantigens than those obtained before treatment. Our results collectively suggest that the rapid and long-lasting effect of this combination therapy in our patient may have resulted from the presence of pre-existing neoantigen-specific T cells and stimulation and expansion of those cells following tumor lysate-pulsed DC vaccine and ICI therapy.

Peptidoglycan-treated tumor antigen-pulsed dendritic cells impart complete resistance against tumor rechallenge.

Solid tumors elicit suppressive T cell responses which impair antigen-presenting cell (APC) functions. Such immune suppression results in uncontrolled tumor growth and mortality. Addressing APC dysfunction, dendritic cell (DC)-mediated anti-tumor vaccination was extensively investigated in both mice and humans. These studies never achieved full resistance to tumor relapse. Herein, we describe a repetitive RM-1 murine tumor rechallenge model for recurrence in humans. Using this newly developed model, we show that priming with tumor antigen-pulsed, Toll-like receptor (TLR)2 ligand-activated DCs elicits a host-protective anti-tumor immune response in C57BL/6 mice. Upon stimulation with the TLR2 ligand peptidoglycan (PGN), the tumor antigen-pulsed DCs induce complete resistance to repetitive tumor challenges. Intra-tumoral injection of PGN reduces tumor growth. The tumor resistance is accompanied by increased expression of interleukin (IL)-27, T-box transcription factor TBX21 (T-bet), IL-12, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with heightened cytotoxic T lymphocyte (CTL) functions. Mice primed four times with PGN-stimulated tumor antigen-pulsed DCs remain entirely resistant to repeat challenges with RM-1 tumor cells, suggesting complete prevention of relapse and recurrence of tumor. Adoptive transfer of T cells from these mice, which were fully protected from RM-1 rechallenge, confers anti-tumor immunity to syngeneic naive recipient mice upon RM-1 challenge. These observations indicate that PGN-activated DCs induce robust host-protective anti-tumor T cells that completely resist tumor growth and recurrence.

Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation.

Dendritic cell (DC) vaccination involves administration of multiple doses. Cryopreservation of tumor antigen-pulsed DCs can provide a ready to use vaccine source and eliminate the need of frequent withdrawal of the patient's blood for vaccine preparation. The aim of this study was to assess the effect of addition of trehalose in the freezing medium on the recovery of DCs after cryopreservation. DCs were generated from mononuclear cells from apheresis samples of healthy donors. For long-term storage of 6 months, cells were frozen with a rate-controlled programmable freezer and stored in liquid nitrogen. For short-term storage of 1 month, cells were frozen and stored at -80°C. DCs frozen with Iscove's Modified Dulbecco's Medium + 10% dimethyl sulfoxide + 20% fetal bovine serum served as the control group, while the test group was additionally supplemented with 50 μg/mL of trehalose. After revival of control and test DCs, they were assessed for viability, morphology, phenotype, and functions. The addition of trehalose to the conventional freezing medium helped to preserve the viability and functionality of DCs better than dimethyl sulfoxide alone in both long- and short-term cryopreservation. Trehalose also protected the mitochondrial membrane potential and cytoskeleton integrity of DCs, which are necessary for their functionality. Mediators of the intrinsic apoptotic pathway like Caspase-9 and Bim-1 were found to be low in the test. Supplementation of conventional freezing medium with trehalose results in better quality of DCs revived after cryopreservation. This finding could help improve DC vaccine preparation for cancer immunotherapy.

Tumor exosomes block dendritic cells maturation to decrease the T cell immune response

Tumors can induce the generation and accumulation of immunosuppression in a tumor microenvironment, contributing to the tumor’s escape from immunological surveillance. Although tumor antigen-pulsed dendritic cell can improve anti-tumor immune responses, tumor associated regulatory dendritic cells are involved in the induction of immune tolerance. The current study sought to investigate whether exosomes produced by tumor cells had any effect on DCs in immune suppression. In this study, we examined the effect of tumor exosomes on DCs and found that exosomes from LLC Lewis lung carcinoma or 4T1 breast cancer cell blocked the differentiation of myeloid precursor cells into CD11c+ DCs and induced cell apoptosis. Tumor exosome treatment inhibited the maturation and migration of DCs and promoted the immune suppression of DCs. The treatment of tumor exosomes drastically decreased CD4+IFN-γ+ Th1 differentiation but increased the rates of regulatory T (Treg) cells. The immunosuppressive ability of tumor exosome-treated DCs were partially restored with PD-L1 blockage. These data suggested that PD-L1 played a role in tumor exosome-induced DC-associated immune suppression.

Immunotherapy of patient with hepatocellular carcinoma using cytotoxic T lymphocytes ex vivo activated with tumor antigen-pulsed dendritic cells.

Purpose The aim of this study was to evaluate the clinical response of immunotherapy with dendritic cell-cytotoxic T lymphocytes (DC-CTLs) in patients with hepatocellular carcinoma (HCC).Method Sixty-eight patients with a confirmed diagnosis of HCC and who received follow-up until December 2015 were enrolled. We measured immune phenotypes of DCs and activated T cells using flow cytometry and clinical indexes using an electrochemiluminescence method.Results DCs exhibited up-regulation of the maturation markers CD83, CD80, CD11c, and CD86 on day8. Levels of IFN-γ and TNF-α were higher in the DCs pulsed with tumor-associated antigens (TAAs) than in DCs with a non-proliferative recombinant adenovirus. The percentage of regulatory T cells (Tregs) decreased in patients after DC-CTLs therapy. In addition, serum levels of AFP, AFP-L3, ALT, and CA19-9 were significantly reduced in these patients. Quality of life was improved, especially on physical functioning scales. Median overall survival (OS) and progression-free survival (PFS) were 8.2 months and 4.3 months, respectively, for the control group and 12.8 months and 9 months, respectively, for the DC-CTL group. Patients treated with DC-CTLs therapy showed a statistically significant PFS and OS curve (OS: p=0.016; PFS: p<0.0001). In addition, no serious adverse reactions were observed.Conclusion This study indicated that Tregs, as well as serum levels of AFP, AFP-L3, ALT, and CA19-9, which were correlated with a poor prognosis, decreased after DC-CTL treatments. The OS, PFS and the quality of life of HCC patients partially improved.

Dendritic Cell Maturation is a Critical Step in Dendritic Cell Vaccine Preparation for Cancer Therapy

Background: Dendritic cell (DC) vaccine is a hopeful approach for cancer treatment. In clinical trials, DC vaccines have produced clinical responses in some cancer patients. However, DC vaccines efficacy is not satisfactory in most types of cancer and more efforts must be done to improve their effectiveness in advanced cancers. Understanding the influence of tumor cells and tumor stromal cells on DCs and the antitumor activity of ex vivo generated DCs in the tumor microenvironment can help to augment antitumor efficiency of ex vivo generated DCs. In a fibrosarcoma tumor model, we explored effects of the tumor microenvironment on the antitumor efficacy of ex vivo generated DCs. Methods: DCs were generated from mouse bone marrow precursor cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were pulsed with tumor antigens and matured in the presence of tumor necrosis factor-alpha (TNF-α), lipopolysaccharide (LPS), or TNF-α plus LPS. Mature or immature DCs were injected subcutaneously before tumor inoculation or were directly injected into the tumor tissue. Results: Tumor antigen-pulsed DCs matured in the presence of TNF-α plus LPS showed appropriate functionality in vitro, including IL-12 secretion and induction of lymphocyte proliferation. Tumor lysate-loaded DCs matured in the presence of TNF-α did not show appropriate antitumor function in vivo. Injection of antigen-unpulsed mature DCs two days before tumor inoculation resulted in antitumor effects. In contrast, injection of immature DCs directly into the tumor tissue enhanced the tumor growth. Conclusion: These results suggest that tumor cells, tumor stromal cells, or tumor derived factors can influence DCs to have tumor-promoting function. Appropriate maturation induction in ex vivo generated DCs and manipulating the tumor microenvironment before DC vaccination may improve antitumor activity of DC vaccines in cancer patients.

Tumor antigen–specific T cells for immune monitoring of dendritic cell–treated glioblastoma patients

Background aimsCD8+ T cells are part of the adaptive immune system and, as such, are responsible for the elimination of tumor cells. Dendritic cells (DC) are professional antigen-presenting cells (APC) that activate CD8+ T cells. Effector CD8+ T cells in turn mediate the active immunotherapeutic response of DC vaccination against the aggressive glioblastoma (GBM). The lack of tumor response assays complicates the assessment of treatment success in GBM patients. MethodsA novel assay to identify specific cytotoxicity of activated T cells by APC was evaluated. Tumor antigen-pulsed DCs from HLA-A*02-positive GBM patients were cultivated to stimulate autologous cytotoxic T lymphocytes (CTL) over a 12-day culture period. To directly correlate antigen specificity and cytotoxic capacity, intracellular interferon (IFN)-γ fluorescence flow cytometry-based measurements were combined with anti-GBM tumor peptide dextramer staining. IFN-γ response was quantified by real-time polymerase chain reaction (PCR), and selected GBM genes were compared with healthy human brain cDNA by single specific primer PCR characterization. ResultsUsing CTL of GBM patients stimulated with GBM lysate-pulsed DCs increased IFN-γ messenger RNA levels, and intracellular IFN-γ protein expression was positively correlated with specificity against GBM antigens. Moreover, the GBM peptide-specific CD8+ T-cell response correlated with specific GBM gene expression. Following DC vaccination, GBM patients showed 10-fold higher tumor-specific signals compared with unvaccinated GBM patients. DiscussionThese data indicate that GBM tumor peptide-dextramer staining of CTL in combination with intracellular IFN-γ staining may be a useful tool to acquire information on whether a specific tumor antigen has the potential to induce an immune response in vivo.

Endostatin enhances antitumor effect of tumor antigen-pulsed dendritic cell therapy in mouse xenograft model of lung carcinoma.

Objective To investigate the antitumor effect of endostatin combined with tumor antigen-pulsed dendritic cell (DC)-T cell therapy on lung cancer. Methods Transplanted Lewis lung cancer (LLC) models of C57BL/6 mice were established by subcutaneous injection of LLC cells in left extremity axillary. Tumor antigen-pulsed DC-T cells from spleen cells and bone of mice were cultured in vitro. Tumor-bearing mice were randomly divided into three groups, including DC-T+endostatin group, DC-T group, and phosphate-buffered saline (PBS) control group. Microvessel density (MVD) of tumor tissue in tumor-bearing mice was determined by immunohistochemistry (IHC). The expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by Western blotting and IHC staining. The proportions of CD8+ T cells, mature dendritic cells (mDC), tumor-associated macrophages [TAM (M1/M2)], and myeloid-derived suppressor cells (MDSC) in suspended cells of tumor tissue were determined by flow cytometry. The expressions of interleukin (IL)-6, IL-10, IL-17, transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) in suspended cells of tumor tissue were detected by enzyme-linked immune sorbent assay (ELISA). Results DC-T cells combined with endostatin remarkably suppressed tumor growth. MVD of mice in DC-T+endostatin group was significantly lower than that of the control group and DC-T monotherapy group. The expressions of VEGF, IL-6 and IL-17 in tumors were markedly decreased, but IFN-γ and HIF-1α increased after treating with DC-T cells combined with endostatin, compared to control group and DC-T group. In the DC-T+endostatin group, the proportions of MDSC and TAM (M2 type) were significantly decreased, mDC and TAM (M1 type) were up-regulated, and CD8+ T cells were recruited to infiltrate tumors, in contrast to PBS control and DC-T monotherapy. DC-T cells combined with endostatin potently reduced the expressions of IL-6, IL-10, TGF-β and IL-17 in tumor tissue, and enhanced the expression of IFN-γ. Conclusions The study indicated the synergic antitumor effects between endostatin and tumor antigen-pulsed DC-T cells, which may be a prospective therapy strategy to achieve potent antitumor effects on lung cancer.

Abstract 2539: Clinical prospective study of PepTivator® WT1-pulsed DC vaccines with assessment of immunological responses in solid tumors

Abstract Wilms' tumor gene 1 (WT1) is reported to be expressed in various types of malignancies and be one of promising target antigens for cancer immunotherapy. However, the synthetic short peptide vaccines based on the WT1 sequences limit a candidate patient by its HLA restriction. In this study, we performed the clinical study for dendritic cell (DC) vaccines using peptide pools consisting mainly of 15-mer sequences with 11 amino acids overlaps of full-length WT1 protein (PepTivator® WT1). We previously reported that copulsing tumor antigen-pulsed DC with zoledronate (Zol) could efficiently enhance expansion of tumor antigen specific CD8+ T cells by activation of gamma-delta T cells. In the present study, we studied the immunological response induced by PepTivator® WT1-pulsed Zol-DC vaccines in solid tumors, and also investigated the feasibility and safety of PepTivator® WT1-pulsed Zol-DC vaccines. Five patients with WT1-positive solid tumor (ovary: 4, lung: 1) were enrolled. PBMCs were collected by leukapheresis from the patients and the adherent cell fraction was differentiated into DCs by conventional method. In this vaccine therapy, PepTivator® WT1-pulsed Zol-DC (more than 5x106 cells per injection) was administered to patients subcutaneously for six times every two weeks. In order to examine the specific immune responses to WT1 antigen, PBMCs before therapy (pre-PBMC) and PBMCs after therapy (post-PBMC) were in vitro restimulated with PepTivator® WT1-pulsed Zol-DCs for 14 days and the expanded cells were separated into CD8+ cells and CD4+ cells by MACS, and the antigen specific IFN-gamma- producing cells were detected by ELISpot assay. As a result, no serious adverse events were seen in three patients who had completed all DC vaccines (Patient ID: #1, #2 and #5). DTH reaction was observed in all three patients. During in vitro restimulation and expansion, there was more than three-fold higher expansion of T cells responding to PepTivator® WT1-pulsed Zol-DC in post-PBMC compared to pre-PBMC among two of three patients (#1, #2). In these two patients, the antigen specific IFN-gamma-producing cells were detected in CD8+ T cells from post-PBMC, but not from pre-PBMC. As for CD4+ T cells, however, the antigen specific IFN-gamma-producing cells were not detected, neither in pre-PBMC, nor in post-PBMC. We found that PepTivator® WT1-pulsed Zol-DC vaccines are feasible and safe, and can induce antigen specific CD8+ T cells. We need more extensive investigation for the immunological monitoring and clinical outcome. Citation Format: Shoko Saiwaki, Shigenori Goto, Masashi Takahara, Haruka Matsushita, Takashige Kondo, Hermann Bohnenkamp, Ryuji Maekawa, Takashi Kamigaki. Clinical prospective study of PepTivator® WT1-pulsed DC vaccines with assessment of immunological responses in solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2539. doi:10.1158/1538-7445.AM2014-2539

Clinical efficacy of tumor antigen-pulsed DC treatment for high-grade glioma patients: evidence from a meta-analysis.

BackgroundThe effectiveness of immunotherapy for high-grade glioma (HGG) patients remains controversial. To evaluate the therapeutic efficacy of dendritic cells (DCs) alone in the treatment of HGG, we performed a systematic review and meta-analysis in terms of patient survival with relevant published clinical studies.Materials and methodsA total of 409 patients, including historical cohorts, nonrandomized and randomized controls with HGG, were selected for the meta-analysis.ResultsThe treatment of HGG with DCs was associated with a significantly improved one-year survival (OS) (p<0.001) and 1.5-, 2-, 3-, 4-, and 5-year OS (p<0.001) compared with the non-DC group. A meta-analysis of the patient outcome data revealed that DC immunotherapy has a significant influence on progression-free survival (PFS) in HGG patients, who showed significantly improved 1-,1.5-, 2-, 3- and 4-year PFS (p<0.001). The analysis of Karnofsky performance status (KPS) demonstrated no favorable results for DC cell therapy arm (p = 0.23).The percentages of CD3+CD8+ and CD3+CD4+ T cells and CD16+ lymphocyte subset were not significantly increased in the DC group compared with the baseline levels observed before treatment (p>0.05), whereas CD56+ lymphocyte subset were significantly increased after DC treatment (p = 0.0001). Furthermore, the levels of IFN-γ in the peripheral blood of HGG patients, which reflect the immune function of the patients, were significantly increased after DC immunotherapy (p<0.001).ConclusionsThus, our meta-analysis showed that DC immunotherapy markedly prolongs survival rates and progression-free time, enhances immune function, and improves the efficacy of the treatment of HGG patients.

Autologous cytokine-induced killer and dentritic cells in treatment of advanced and unresectable lung cancer.

e13069 Background: Both experimental and clinical studies showed cytokine-induced killer (CIK) cells have been shown capable of efficiently lysing target tumor cells, which activity can be enhanced by dendritic cell (DC) stimulation. This study demonstrated that co-cultured autologous CIK and tumor antigen-pulsed DC are effective in in treatment of advanced and unresectable lung cancer Methods: Sixteen patients having stage III-IV lung cancer with a malignant pleural effusion, 9 females and 7 males with an average age of 54.3 years old, were included in this study. Autologous CIK and DC were prepared from isolated peripheral blood mononuclear cells and were induced by multiple cytokines. Generated DCs were pulsed by the antigen generated from tumor tissues and co-cultured with CIK. Those patients received 4-6 times of CIK-DC intravenous infusion, 2-3 ×109 cells each time. Results: Eleven (68.8%) patients achieved a partial remission. The pleural effusion decreased in all those patients, and in 9 of them the effusion decreased over 50%. The clinical symptoms were relieved in all the patients. There were no significant adverse effects were found. Those patients are still followed for long-term survival outcome. Conclusions: CIK-DCs immunotherapy provides a new and effective treatment for the late stage lung cancer.

A High-Affinity T-Helper Epitope Enhances Peptide-Pulsed Dendritic Cell–Based Vaccine

The NV epitope, a dominant helper determinant from the circumsporozoite antigen of Plasmodium falciparum, is strongly immunogenic and can provide help for cytotoxic T-lymphocyte (CTL) activation. In this study, we evaluated whether the addition of NV peptide can augment the efficacy of peptide-pulsed dendritic cell (DC) immunization in vivo. Using B16 melanoma as tumor model, we demonstrated that DCs pulsed with both NV and gp100 (a melanoma-specific antigen) peptide enhanced immune priming and protection from tumor challenge in vivo. Further, we showed the mechanisms of the NV epitope that help CTL activation; MHC-II-restricted NV peptide induced dramatically more effective helper cells, with a higher level of CD40L expression and IFN-γ production, which, in turn, more effectively conditioned DCs for CTL activation. The improved helper cells also induced greater IL-12 production by DCs, accounting for the reciprocal T-helper polarization to Th1, and increased the expression of costimulatory molecules. Collectively, these findings demonstrate that NV peptide in addition to tumor antigen-pulsed DC immunizations augment helper cell activation, which in turn promotes maturation of DC, and enhance in vivo antitumor activity.

Dendritic cells stimulated with outer membrane protein A (OmpA) of Salmonella typhimurium generate effective anti-tumor immunity

Gram-negative bacterial outer membrane proteins (Omps) have an important role in pathogenesis and signal reception. We previously reported that Acinetobacter OmpA ( AbOmpA) induced maturation of bone marrow-derived dendritic cells (BMDCs) and that AbOmpA-primed DCs produced IL-12 which generated Th1 CD4 + T-cells. We analyzed the effects of Salmonella typhimurium OmpA (OmpA-Sal) on dendritic cell (DC) maturation in the present study, and determined that tumor antigen-pulsed DCs stimulated with OmpA-Sal induced anti-tumor responses in a mouse model. OmpA-Sal activated BMDCs by augmenting expression of MHC class II and of the co-stimulatory molecules CD80 and CD86. RT-PCR revealed that IL-12(p40) gene expression is highly augmented in OmpA-Sal-stimulated BMDCs. DNA (CRT/E7) vaccination combined with OmpA-Sal stimulation generated more antigen-specific CD8 + T-cells in the present study. Certain antigen-pulsed BMDCs stimulated with OmpA-Sal induced strong PADRE-specific CD4 + and E7-specific CD8 + T-cell responses. In addition, BMDCs stimulated with OmpA-Sal (OmpA-Sal-BMDCs) and pulsed with both E7 and PADRE peptide generated greater numbers of E7-specific CD8 + effector and memory T-cells than those pulsed with E7 peptide alone. E7- and PADRE-expressing OmpA-Sal-BMDC vaccines resulted in significant long-term protective anti-tumor effects in vaccinated mice. Our data suggested that E7- and PADRE-expressing BMDCs that were matured in the presence of OmpA-Sal might enhance anti-tumor immunity and support the therapeutic use of OmpA-Sal in DC-based immunotherapy.

Reduction of major histocompatibility complex class I expression on bladder carcinoma following tumor antigen‐pulsed dendritic cell vaccine: Implications for immunoresistance in therapy

To clarify the relationship between a decreased major histocompatibility complex class I (MHC-I) expression on bladder tumors and decreased immunological efficacy of tumor antigen-pulsed dendritic cell vaccine in a rat bladder carcinoma model induced by N-methyl-N-nitrosourea irrigation. Enzyme-linked immunosorbent assay was used to evaluate interferon-gamma concentration in the serum and colorimetric lactate dehydrogenase release assay in vitro was used to test the cytotoxicity capability of T lymphocytes. MHC-I expression on tumor cells was detected by flow cytometry and analyzed with CellQuest software. The tumor antigen sensitized dendritic cell vaccine group showed decreased hyperplastic formations, lower pathological stages in rat bladders and more potent cytotoxicity activity (P < 0.001) than the dendritic cell vaccine group. Additionally, immunization with pulsed dendritic cell vaccine induced higher specific cytokine production of interferon-gamma. Nevertheless, a decreased MHC-I expression on bladder tumors was tested after immunotherapy by pulsed dendritic cell vaccine on week 15. As expected, the cytotoxic activity of T lymphocytes from rats on tumor cells with low MHC-I expression was also decreased to 19.70 +/- 4.82% as compared with tumor cells with high MHC-I (52.10 +/- 8.66%, P = 0.005). Tumor antigen sensitized dendritic cell vaccine has beneficial activity on N-methyl-N-nitrosourea-induced bladder cancer in situ in rats, but therapeutic responses are accompanied by decreased MHC-I expression on tumors, possibly suggesting poor long-term therapeutic outcomes.

Vaccination with immature dendritic cells combined with CD40 mAb induces protective immunity against B lymphoma in hu-SCID mice

Dendritic cells (DCs) pulsed by tumor antigens have been widely used as tumor vaccines to specifically trigger the cytotoxicity of CD8+ T cells. But the tumor microenviroment with enriched immunosuppressants hampered DC maturation and co-stimulation. CD40/CD40L signaling, one of the most important co-stimulatory molecules is capable of effectively skewing the immune response by promoting DCs maturation and co-stimulation. To establish a novel specific immunotherapeutic approach for the use of DC vaccine in the treatment of B lymphoma, hu-SCID mice bearing B lymphoma were vaccinated by different combination of tumor antigen pulsed DC or imDC vaccines and immune-enhancing agencies such as agonist CD40 mAb and T cells. The results of immature DCs combined with agonistic CD40 mAb were encouraging with achievement of tumor regression and induction of antigen-specific immune responses. These findings demonstrated the potential utility of imDC-based tumor vaccination combining with agonistic CD40 mAb in the treatment of malignant lymphoma.

Immunological Factors Relating to the Antitumor Effect of Temozolomide Chemoimmunotherapy in a Murine Glioma Model

In this study, we investigated the potential of combined treatment with temozolomide (TMZ) chemotherapy and tumor antigen-pulsed dendritic cells (DCs) and the underlying immunological factors of TMZ chemoimmunotherapy with an intracranial GL26 glioma animal model. The combined treatment enhanced the tumor-specific immune responses and prolonged the survival more effectively than either single therapy in GL26 tumor-bearing animals. Apoptosis was induced in the tumors of the animals by the treatment with TMZ. Calreticulin (CRT) surface exposure was detected by immunofluorescence staining of TMZ-treated GL26 cells. TMZ chemotherapy increased tumor antigen cross-priming from tumor cells, leading to cross-priming of tumor antigen-specific CD4(+) T cells and CD8(+) T cells. This chemotherapy appeared to suppress the frequency of CD4(+) CD25(+) regulatory T cells (Treg). Moreover, this combined therapy resulted in an increase in the tumor infiltration of CD4(+) and CD8(+) T cells. Collectively, the findings of this study provide evidence that the combination of TMZ chemotherapy and treatment with DC-based vaccines leads to the enhancement of antitumor immunity through increased tumor-specific immune responses via the cross-priming of apoptotic tumor cell death mediated by CRT exposure and, in part, the suppression of Treg. Therefore, CRT exposure, regulatory T cells, and cross-priming by TMZ chemotherapy may be immunological factors related to the enhancement of the antitumor effects of chemoimmunotherapy in an experimental brain tumor model.

Improvement of a Dendritic Cell-Based Therapeutic Cancer Vaccine with Components ofToxoplasma gondii

The use of dendritic cells (DCs) as a cellular adjuvant is a promising approach to the immunotherapy of cancer. It has previously been demonstrated that DCs pulsed ex vivo with Toxoplasma gondii antigens trigger a systemic Th1-biased specific immune response and induce protective and specific antitoxoplasma immunity. In the present study, we demonstrate that tumor antigen-pulsed DCs matured in the presence of Toxoplasma gondii components induce a potent antitumor response in a mouse model of fibrosarcoma. Bone-marrow derived DCs (BMDCs) were cultured in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. After 5 days, tumor lysates with or without the T. gondii lysate were added to the culture for another 2 days. The cytokine production in the BMDC culture and the coculture supernatants of DCs and splenic cells was evaluated. For immunization, 7 days after tumor challenge, different groups of BALB/c mice received different kinds of DCs subcutaneously around the tumor site. Tumor growth was monitored, and 2 weeks after DC immunotherapy, the cytotoxic activity and the infiltration of CD8(+) T cells were monitored in different groups. According to the findings, immunotherapy with T. gondii-matured DCs led to a significant increase in the activity of cytotoxic T cells and decreased the rate of growth of the tumor in immunized animals. Immature DCs did not cause any change in cytotoxic activity or the tumor growth rate compared to that in the healthy controls. The current study suggests that a specific antitumor immune response can be induced by DCs matured with T. gondii components and provide the basis for the use of T. gondii in DC-targeted clinical therapies.

Stimulatory effects of CpG oligodeoxynucleotide on dendritic cell‐based immunotherapy of colon cancer in CEA/HLA‐A2 transgenic mice

Immunostimulatory DNA containing unmethylated cytosine-guanine (CpG) motifs have been successfully used as adjuvants to enhance the immunity of vaccines designed to trigger antitumor T-cell responses. We examined the effect of a CpG oligodeoxynucleotide (CpG ODN) for its ability to potentiate the activity of tumor antigen-pulsed dendritic cells (DC) in a clinically relevant mouse model, which is transgenic for both carcinoembryonic antigen (CEA) and HLA-A2 for the treatment of colon carcinoma in a therapeutic setting. The systemic administration of CpG ODN 1826 alone had modest effect on tumor growth when tumors were palpable and had no effect with larger tumor burden. However, coadministration of CpG ODN 1826 with the vaccine provided significant increase in tumor-free survival compared with mice immunized with DC-based vaccines alone. The DC/CpG combined vaccination strategy resulted in increased secretion of Th1 cytokines and HLA-A2-restricted CEA-specific CTL responses were also enhanced. Both tumor regression and extended tumor-free survival resulting from DC/CpG combination therapy required the participation of T cells. Tumor-free mice were resistant to tumor rechallenge and immunity conferred by the vaccine was transferable in athymic nude mice. These results provide evidence that vaccination with antigen-pulsed DC with CpG ODN as adjuvant can lead to effective tumor regression and long-term survival in a murine model of colon carcinoma.

Listeria monocytogenes activated dendritic cell based vaccine for prevention of experimental tumor in mice.

The use of dendritic cells (DCs) as a cellular adjuvant provides a promising approach in immunotherapy of cancer. It has been demonstrated that Listeria monocytogenes activated DCs pulsed ex vivo with tumor antigens trigger a systemic Th1-biased specific immune response and a single dose of this vaccine will cause a considerable anti tumor immunity. The present study was designed to evaluate the ability of multiple doses of tumor antigen-pulsed DCs, matured in the presence of Listeria monocytogenes components in induction of a potent anti-tumor response and the prevention of tumor formation in an experimental model. Bone-marrow derived DCs (BMDCs) were cultured in the presence of GM-CSF and IL-4. After 5 days, tumor lysates with/without Listeria monocytogenes lysate were added to the culture media for another 2 days. Mice received mature and tumor antigen pulsed dendritic cells subcutaneously in 3 groups. Tumor growth was monitored and two weeks after immunotherapy, cytotoxic activity of CD8+ T cells was evaluated in different groups. According to the findings, repeated doses of vaccine did not lead to a significant increase in the activity of cytotoxic T cells and decreased tumor growth of immunized animals. The current study suggests that increased doses of vaccine do not have sufficient efficiency for prevention of tumor induction. Generation of T regulatory responses upon repeated doses of such vaccines should be considered in future investigations.