Generation of dendritic cell–tumor cell hybrids by electrofusion for clinical vaccine application

Vaccination with hybrids comprising fused dendritic cells (DCs) and tumor cells is a novel cancer immunotherapy approach designed to combine tumor antigenicity with the antigen-presenting and immune-stimulatory capacities of DCs. For clinical purposes, we have incorporated a large-scale process for the generation of clinical-grade DCs together with novel electrofusion technology. The electrofusion system provides for ease and standardization of method, efficient DC–tumor cell hybrid formation, and large-quantity production of hybrids in a high-volume (6-ml) electrofusion chamber. In addition, we have evaluated DC electrofusion with a variety of allogeneic human tumor cell lines with the rationale that these tumor cell partners would prove a ready, suitable source for the generation of DC–tumor cell hybrid vaccines. The DC production process can generate 6×10⁸ to 2×10⁹ DCs from a single leukapheresis product (~180 ml). As determined by FACS analysis, electrofusion of 6×10⁷ total cells (1:1 ratio of DC and tumor cells) resulted in a consistent average of 8–10% DC–tumor cell hybrids, irrespective of the tumor type used. Hybrids were retained in the population for 48 h postfusion and following freezing and thawing. Upon pre-irradiation of the tumor cell partner for vaccine purposes, the overall fusion efficiency was not altered at doses up to 200 Gy. Evaluation of DC–tumor cell hybrid populations for their ability to stimulate T-cell responses demonstrated that electrofused populations are superior to mixed populations of DCs and tumor cells in generating a primary T-cell response, as indicated by IFN- release. Moreover, hybrids comprising HLA-A^*0201 DCs and allogeneic melanoma tumor cells (Colo 829 cell line) stimulated IFN- secretion by antigen-specific CD8⁺ T cells, which are restricted for recognition of a melanoma gp100 peptide antigen (gp100_209–217) within the context of the DC HLA haplotype. Maturation of the DC-Colo 829 cell hybrid population served to further improve this T-cell gp100-specific response. Overall, our results are promising for the large-scale generation of electrofused hybrids comprising DCs and allogeneic tumor cells, that may prove useful in human vaccine trials.     

Synergistic induction of antigen-specific CTL by fusions of TLR-stimulated dendritic cells and heat-stressed tumor cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- gamma at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+ IFN-gamma+ CD8+ T cells and CD154+ IFN-gamma+ CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.     

Comparative analysis of DC fused with tumor cells or transfected with tumor total RNA as potential cancer vaccines against hepatocellular carcinoma

BACKGROUND: DC vaccination with the use of tumor cells provides the potential to generate a polyclonal immune response to multiple known and unknown tumor Ag. Our study comparatively analyzed DC fused with tumor cells or transfected with tumor total RNA as potential cancer vaccines against hepatocellular carcinoma (HCC). METHODS: Immature DC generated from PBMC of patients with HCC were fused with HepG2-GFP (HepG2 cell line transfected stably with plasmid pEGFP-C3) cells or transfected with their total RNA. Matured DC were used to stimulate autologous T cells, and the resultant Ag-specific effector T cells were analyzed by IFN-gamma ELISPOT assay. RESULTS: DC were capable of further differentiation into mature DC after fusion with HepG2-GFP cells or transfection with HepG2-GFP cell total RNA, and were able to elicit specific T-cell responses in vitro. Both methods of Ag loading could result in stimulating CD4+ and CD8+ T cells, but with the indication that fusion loading was more efficient than RNA loading in priming the Th1 response, while RNA loading was more effective in CTL priming. DISCUSSION: Our results indicate that DC fused with tumor cells or transfected with tumor total RNA represent promising strategies for the development of cancer vaccines for treatment of HCC. They may have potential as an adjuvant immunotherapy for patients with HCC.     

Superior anti-tumor protection and therapeutic efficacy of vaccination with allogeneic and semiallogeneic dendritic cell/tumor cell fusion hybrids for murine colon adenocarcinoma

Cancer immunotherapy by dendritic cell (DC)/tumor cell fusion hybrids (DC/TC hybrids) has been shown to elicit potent anti-tumor effects via the induction of immune responses against multiple tumor-associated antigens. In the present study, we compared the anti-tumor effects of vaccinating Balb/c mice (H-2^d) with CT26CL25 colon carcinoma cells that had been fused with either syngeneic DCs from Balb/c mice, allogeneic DCs from C57BL/6 mice (H-2^b) or semiallogeneic DCs from B6D2F1 mice (H-2^b/d). Preimmunization with either semiallogeneic or allogeneic DC/TC hybrids induced complete protection from tumor challenge, whereas mice preimmunized with syngeneic DC/TC hybrids were only partially protected (75% tumor rejection). The average number of pulmonary metastases after intravenous tumor injection decreased significantly following immunization with semiallogeneic or allogeneic DC/TC hybrids (8.3 ± 7.9 or 16.3 ± 3.5, mean ± SD) relative to syngeneic DC/TC hybrids (67.8 ± 6.3). These data demonstrate that vaccination with semiallogeneic DC/TC hybrids resulted in the greatest anti-tumor efficacy. Anti-tumor effects showed by in vivo studies were virtually accomplished by the frequency of induced CTLs specific to both gp70 and β-galactosidase assessed by using pentameric assay. Among the fusion vaccines tested, semiallogeneic DC/TC hybrids induced the highest ratio of Th1 cytokine IFN-γ to Th2 cytokine IL-10. In addition, allogeneic or semiallogeneic DC/TC hybrids elicited a significantly stronger NK activity than syngeneic DC/TC hybrids. These findings suggest that in clinical settings, DCs derived from a healthy donor (which are generally characterized as more semiallogeneic than allogeneic) may be more capable than autologous DCs of inducing promising anti-tumor effects in vaccinations with DC/TC hybrids.     

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4⁺ and CD8⁺ T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

An ex vivo readout for evaluation of dendritic cell-induced autologous cytotoxic T lymphocyte responses against esophageal cancer

Esophageal cancer is a highly malignant disease that despite surgery and adjuvant therapies has an extremely poor outcome. Dendritic cell (DC) immunotherapy as a novel promising strategy could be an alternative for treating this malignancy. Effective DC-mediated immune responses can be achieved by raising cytotoxic T lymphocyte (CTL) response against multiple antigens through loading DCs with total tumor RNA. However, the efficacy of this strategy first needs to be evaluated in a pre-clinical setting. The aim of the study was to set up an ex vivo autologous human readout assay for assessing the effects of DC-mediated cytotoxic responses, using total tumor RNA as an antigen load. Biopsy specimens of seven esophageal cancer patients were used to establish primary cultures of normal and cancer cells and to obtain autologous RNA for loading DCs. Mature DCs loaded with either normal or tumor RNA were obtained and subsequently used to raise various lymphocytes populations. Apoptosis levels of the autologous cultures were measured before and after incubating the cultures with the different lymphocytes populations. The mean apoptosis levels in the tumor cell cultures, induced by lymphocytes instructed by DCs loaded with tumor RNA, significantly increased with 15.6% ±2.9 SEM (range 3.4–24.5%, t-test, P < 0.05). Incubation of the normal cultures with the lymphocytes populations showed a mean non-significant increase in apoptosis of 0.4% ±3.4 SEM (range −13.9 to 9.8%, t-test, P = 0.7). Here, we introduce a practical, patient-specific autologous readout assay for pre-clinical testing of DC-mediated cytotoxic responses. Additionally, we demonstrated that the use of autologous tumor RNA as a strategy for raising cytotoxic responses against multiple tumor antigens could be effective for treating esophageal cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antigen loading of DCs with irradiated apoptotic tumor cells induces improved anti-tumor immunity compared to other approaches

Dendritic cells (DCs) serve as central regulators of adaptive immunity by presenting antigens and providing necessary co-signals. Environmental information received by the DCs determines the co-signals delivered to the responding adaptive cells and, ultimately, the outcome of the interaction. DCs loaded with relevant antigens have been used as therapeutic cellular vaccines, but the optimal antigen loading method has not been determined. We compared different methods to load class I and class II epitopes from the male antigenic complex, HY, onto DCs for the potency of the immune response induced in vivo. Co-incubation of female DCs with HY peptides, RNA or cell lysate from HY expressing tumor induced immune responses equivalent to male DCs. In contrast, female DCs incubated with irradiated, apoptotic HY expressing tumor cells (or male B cells) generated a stronger immune response than male DCs or female DCs loaded using any of the other methods. DC loading with apoptotic tumor resulted in complete protection against high dose HY-expressing tumor challenge whereas 100% lethality was observed in groups receiving DCs that were loaded with peptides, RNA, or lysate. We conclude that signals provided to the DCs by apoptotic cells substantially augment the potency of DC vaccines.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses

Due to their capacity to induce primary immune responses, dendritic cells (DC) are attractive vectors for immunotherapy of cancer. Yet the targeting of tumor Ags to DC remains a challenge. Here we show that immature human monocyte-derived DC capture various killed tumor cells, including Jurkat T cell lymphoma, malignant melanoma, and prostate carcinoma. DC loaded with killed tumor cells induce MHC class I- and class II-restricted proliferation of autologous CD8+ and CD4+ T cells, demonstrating cross-presentation of tumor cell-derived Ags. Furthermore, tumor-loaded DC elicit expansion of CTL with cytotoxic activity against the tumor cells used for immunization. CTL elicited by DC loaded with the PC3 prostate carcinoma cell bodies kill another prostate carcinoma cell line, DU145, suggesting recognition of shared Ags. Finally, CTL elicited by DC loaded with killed LNCap prostate carcinoma cells, which express prostate specific Ag (PSA), are able to kill PSA peptide-pulsed T2 cells. This demonstrates that induced CTL activity is not only due to alloantigens, and that alloantigens do not prevent the activation of T cells specific for tumor-associated Ags. This approach opens the possibility of using allogeneic tumor cells as a source of tumor Ag for antitumor therapies.     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Purified Dendritic Cell-Tumor Fusion Hybrids Supplemented with Non-Adherent Dendritic Cells Fraction Are Superior Activators of Antitumor Immunity

Background

Strong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown.

Methods

Patient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared.

Results

Except for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction.

Conclusions

The non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy.     

Third generation dendritic cell vaccines for tumor immunotherapy

This review summarizes our studies of the past several years on the development of third generation dendritic cell (DC) vaccines. These developments have implemented two major innovations in DC preparation: first, young DCs are prepared within 3 days and, second, the DCs are matured with the help of Toll-like receptor agonists, imbuing them with the capacity to produce bioactive IL-12 (p70). Based on phenotype, chemokine-directed migration, facility to process and present antigens, and stimulatory capacity to polarize Th1 responses in CD4⁺ T cells, induce antigen-specific CD8⁺ CTL and activate natural killer cells, these young mDCs display all the important properties needed for initiating good antitumor responses in a vaccine setting.     

Patient-derived renal cell carcinoma cells fused with allogeneic dendritic cells elicit anti-tumor activity: in vitro results and clinical responses

Renal cell carcinoma (RCC) has been shown to be susceptible to immunotherapeutic treatment strategies. In the present study, patient-derived tumor cells were fused with allogeneic dendritic cells (DC) to elicit anti-tumor activity against RCC. DC from HLA-A2+ healthy donors were fused with primary RCC cells from ten patients. Phenotype of fusion cells were characterized by flow cytometer and confocal microscopy. In vitro, T cell proliferation, IFN-γ secretion and cytotocic T lymphocytes (CTL) activity elicited by allogeneic DC/RCC fusion cells were assessed. Clinically, ten patients were vaccinated with allogeneic DC/RCC fusion vaccine. The adverse effects and toxicity were observed. The clinical response was evaluated by CT scans. After fusion, the created hybrids expressed both tumor associated antigen and DC-derived molecules and could stimulate the proliferation and IFN-γ secretion of T cells as well as elicit strong CTL activity against RCC cells in vitro. In vivo, no serious adverse effects, toxicity, or signs of autoimmune disease were observed after vaccination therapy. Percentage of T lymphocyte subsets in peripheral blood of patients was increased significantly. One of ten patients exhibited a partial response with regression of lung metastases. Six patients showed stable disease with stabilization of previously progressive disease (follow up 1.5 years). The PR and SD responses, exhibited by 7/10 patients who received the allogeneic DC/RCC fusion vaccine treatment, suggest that this approach is safe and can elicit immunological responses in a significant portion of patients with RCC. J. Zhou and D. Weng contributed equally.     

Preparation of Triple-Negative Breast Cancer Vaccine through Electrofusion with Day-3 Dendritic Cells

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) in human immune system. DC-based tumor vaccine has met with some success in specific malignancies, inclusive of breast cancer. In this study, we electrofused MDA-MB-231 breast cancer cell line with day-3 DCs derived from peripheral blood monocytes, and explored the biological characteristics of fusion vaccine and its anti-tumor effects in vitro. Day-3 mature DCs were generated from day-2 immature DCs by adding cocktails composed of TNF-α, IL-1β, IL-6 and PEG₂. Day-3 mature DCs were identified and electofused with breast cancer cells to generate fusion vaccine. Phenotype of fusion cells were identified by fluorescence microscope and flow cytometer. The fusion vaccine was evaluated for T cell proliferation, secretion of IL-12 and IFN-γ, and induction of tumor-specific CTL response. Despite differences in morphology, day-3 and day-7 DC expressed similar surface markers. The secretion of IL-12 and IFN-γ in fusion vaccine group was much higher than that in the control group. Compared with control group, DC-tumor fusion vaccine could better stimulate the proliferation of allogeneic T lymphocytes and kill more breast cancer cells (MDA-MB-231) in vitro. Day-3 DCs had the same function as the day-7 DCs, but with a shorter culture period. Our findings suggested that day-3 DCs fused with whole apoptotic breast cancer cells could elicit effective specific antitumor T cell responses in vitro and may be developed into a prospective candidate for adoptivet immunotherapy.     

Specific antihepatocellular carcinoma T cells generated by dendritic cells pulsed with hepatocellular carcinoma cell line HepG2 total RNA

Dendritic cell (DC) vaccination with the use of total tumor RNA provides the potential to generate a polyclonal immune response to multiple known and unknown tumor antigens without HLA restriction. Our study evaluated this approach as potential immunotherapy for patients with hepatocellular carcinoma (HCC). Immature DCs generated from peripheral blood mononuclear cells of patients with HCC were transfected with HepG2-GFP (HepG2 cells transfected stably with plasmid pEGFP-C3) cells total RNA. Transfected, matured DCs were used to stimulate autologous T cells. Results revealed that DCs transfected with HepG2-GFP cells total RNA expressed EGFP when observed by flow cytometry. Compared with those before transfection, the expressions of membrane molecules were increased dramatically, and interleukin-12p70 release in the supernatant was elevated significantly. Specific T cells generated by DCs transfected with HepG2-GFP total RNA recognized HLA-matched HepG2 cell lines specifically. These findings indicate that these RNA-transfected DCs successfully generate specific T cells that specifically recognize HCC cells. Total tumor RNA-pulsed DCs may have potential as an adjuvant immunotherapy for patients with HCC.     

Dendritic cells derived from metastatic cancer patients vaccinated with allogeneic dendritic cell–autologous tumor cell hybrids express more CD86 and induce higher levels of interferon-gamma in mixed lymphocyte reactions

Dendritic cells (DCs) are highly effective antigen-presenting cells that, when derived from cancer patients, seem to be functionally deficient. Herein, we show that vaccination with allogeneic DC–autologous tumor cell hybrids affects the phenotype and improves the function of monocyte-derived DCs (Mo-DCs) from cancer patients. Mononuclear cells were isolated from patients peripheral blood by density gradient centrifugation, and adherent cells were cultured in medium containing GM-CSF plus IL-4 and, after 5 days, TNF-. After 2 more days, Mo-DCs were harvested and their CD80, CD86, and CD83 expression was assessed by flow cytometry. They were also used as stimulators in mixed lymphocyte reactions (MLR), where IFN- production was measured by ELISA. Mo-DCs from unvaccinated patients expressed significantly lower levels of CD86, and tended to express lower levels of CD83 than Mo-DCs from healthy donors. However, Mo-DCs generated after hybrid cell vaccination presented increased expression of the same markers and induced significantly higher levels of IFN- in MLR. These results indicate that the use of allogeneic DC–based cancer vaccines induces recovery of DC function in metastatic cancer patients and, therefore, could precede the use of autologous DCs for vaccine preparation. Such an approach could be relevant and should be investigated in clinical trials.     

Antibody response after vaccination with antigen-pulsed dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system capable of initiating immune responses to antigens. It is also well documented that cancer patients often experience anergy against tumor antigens. In this study we selected the best protocol for inducing the production of antibodies against the HER2 oncoprotein using DCs to overcome anergy. Murine DCs were pulsed in vitro, using different protocols, with recombinant HER2 fused to a human Fc (in order to improve DC antigen uptake) and were used to vaccinate mice. The obtained results indicate that antigen-pulsed DCs can induce an antibody response and that adding CpG after antigen pulsing greatly increases anti-HER2 antibody production.     

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust