Comparison of media and serum supplementation for generation of monophosphoryl lipid A/interferon-γ–matured type I dendritic cells for immunotherapy

Background aimsEx vivo–generated monocyte-derived dendritic cells (DCs) matured with monophosphoryl lipid A (MPLA) and interferon-γ (IFN-γ) can be used as cancer immunotherapy. MPLA/IFN-γ DCs induce Th1 T cell responses and have migratory capacity. Different culture regimens have been used for generation of immunotherapeutic DCs, with varying results. In the present study, culture conditions for MPLA/IFN-γ–matured type I DCs were optimized for clinical application.MethodsDCs were generated from monocytes in the clinical grade culture media CellGro DC, AIM V or X-VIVO 15 in the absence or presence of 2% human serum (HS) and matured with the use of MPLA/IFN-γ. DC yield and DC functionality were assessed. DC functionality was determined by means of analysis of cytokines in culture supernatant, migratory capacity, expression of co-stimulatory molecules, T cell stimulatory capacity of DCs and T helper cell (Th) polarization by the DCs.ResultsDCs generated in the presence of 2% HS produced low amounts of pro-inflammatory cytokines and could not migrate irrespective of the medium used. In the absence of HS, MPLA/IFN-γ DCs generated in X-VIVO did not migrate either. MPLA/IFN-γ DCs generated in AIM V have slightly lower capacity to induce Th1 cells than do DCs generated in CellGro or X-VIVO.ConclusionsAddition of HS to different GMP culture media is detrimental for pro-inflammatory DC maturation and migration. In the absence of serum, CellGro is the most optimal medium tested for generation of migratory and Th1-inducing MPLA/IFN-γ DCs for cancer immunotherapy.     

Mobilization of dendritic cells from patients with breast cancer into peripheral blood stem cell leukapheresis samples using Flt-3-Ligand and G-CSF or GM-CSF

Treatment with myeloablative chemotherapy and autologous peripheral blood stem cell (PBSC) transplantation followed by vaccination with autologous dendritic cells (DCs) treated with tumor antigens is a promising therapeutic strategy for several types of cancer. Obtaining sufficient numbers of both PBSCs and DCs is central to this approach. Previously, it has been shown that administration of Flt-3-Ligand (FL) combined with either G-CSF or GM-CSF mobilizes large numbers of PBSCs in patients with cancer. In the current study, we sought to determine whether these same cytokines could simultaneously mobilize DCs into the PBSC leukapheresis collection. DCs were analysed in PBSC leukapheresis samples obtained from five patients with high-risk breast cancer who received G-CSF alone as priming prior to leukapheresis, four patients who received FL+G-CSF and five patients who received FL+GM-CSF. DCs were defined as cells with a lin(dim/-) HLA-DR+ CD11c+ phenotype. The proportions of DCs in the FL+G-CSF and FL+GM-CSF samples were significantly higher than in pre-mobilization peripheral blood and G-CSF leukapheresis samples. The mean yield of DCs/kg in the FL+GM-CSF samples was also significantly higher than the mean yield of DCs in the G-CSF samples. The FL+G-CSF and FL+GM-CSF mobilized DCs were immature by morphologic and phenotypic criteria but stimulated allogeneic T-cells at levels similar to DCs generated in culture from PBMCs. Overnight culture?of the immature DCs obtained from patients receiving either FL+G-CSF or FL+GM-CSF in TNF-alpha?resulted in the generation of mature DCs. In summary, administration of FL in combination with GM-CSF and G-CSF to patients with breast cancer can mobilize large numbers of immature DCs into PBSC leukapheresis collections.     

Ex vivo enhancement of antigen-presenting firnction of dendritic cells and its application for DC-based immunotherapy

Dendritic cells (DCs) are potent antigen presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. In particular DCs generated from peripheral blood monocytes (Mo-DCs) have been used with promising results as a new approach for the immunotherapy of cancer. In this study, we have analyzed the changes in the pattern of expression molecules on Mo-DCs during DC maturation using different maturation cytokine combinations and the expansion capacity of an antigen specific CD8+T cells monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. These analyses revealed that the expansion of antigen specific CD8+T cells is the most effective when T cells were activated by fully maturated DCs by culturing monocytes for 5 days in the presence of GM-CSF and IL-4, followed by 2-3 days of maturation with pro-inflammatory mediators including TNFalpha, IL-6, IL-1beta and PGE2. These results pave the way to a more effective immunotherapy using DCs for patients with malignancy, as well as infectious diseases.     

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.     

Immunotherapy of murine leukemia following non-myeloablative conditioning with naïve or G-CSF mobilized blood or bone marrow stem cells

Allogeneic stem cell transplantation (SCT) is the treatment of choice for a large number of hematologic malignancies. Its major advantage over conventional chemotherapy lies in the graft-versus-leukemia (GVL) effects mediated by allo- or tumor-reactive donor lymphocytes given in the course of SCT or post transplantation as donor lymphocyte infusions (DLI). The benefits of cell-mediated immunotherapy over myeloablative radiochemotherapy have also made it possible to reduce the intensity of conditioning regimens. Mobilized peripheral blood has proved preferable to bone marrow (BM) as a source of stem cells for transplantation, since it provides a larger number of stem cells on the one hand and immunologically competent lymphocytes on the other. The use of granulocyte colony stimulating factor (G-CSF), which is necessary to mobilize and increase the number of stem cells, may down-regulate the GVL effect by suppression of donor effector T lymphocytes by inducing Th₁Th₂ cytokine switch. It has previously been shown that GVL effects may be amplified by both in vivo and in vitro activation of donor lymphocytes with human recombinant interleukin-2 (rIL-2). Our studies using a leukemic murine model prepared for transplantation with low intensity conditioning prior to infusion of G-CSF-mobilized peripheral blood stem cells (PBSC) have demonstrated that mobilization of blood cells with G-CSF and in vivo treatment with rIL-2 following low-intensity conditioning enhances the GVL effects and prolongs survival of recipients inoculated with BCL1. Activation of donor lymphocytes with rIL-2 may thus be useful for amplifying GVL effects following mobilization with G-CSF.     

Immunotherapy of solid cancer using dendritic cells pulsed with the HLA-A24-restricted peptide of carcinoembryonic antigen

Carcinoembryonic antigen (CEA), an oncofetal glycoprotein overexpressed in most gastrointestinal and lung cancers, is a candidate molecule for cancer immunotherapy. Recently, a CEA-derived 9-mer peptide, CEA652 (TYACFVSNL), has been identified as the epitope of cytotoxic T lymphocytes restricted with human leukocyte antigen (HLA)-A24, which is present in 60% of the Japanese population and in some Caucasians. The authors performed a clinical study of a vaccine using autologous dendritic cells (DCs) pulsed with CEA652 and adjuvant cytokines, natural human interferon alpha (nhuIFN-alpha), and natural human tumor necrosis factor alpha (nhuTNF-alpha), for the treatment of patients with CEA-expressing advanced metastatic malignancies. Ten HLA-A24 patients with advanced digestive tract or lung cancer were enrolled in the study to assess toxicity, tolerability and immune responses to the vaccine. DCs were generated from plastic adherent monocytes of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMCs) in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Generated DCs showing an immature phenotype were loaded with CEA652 and injected into patients intradermally and subcutaneously with 50% of the dose administered by each route every 2 weeks for a total of ten vaccinations. The total dose of administered DCs ranged from 2.7x10(7)cells to 1.6x10(8)cells. Adjuvant cytokines, i.e., 1x10(6) U/body of nhuIFN-alpha and nhuTNF-alpha, were administered to patients twice a week during the vaccination period. No severe toxicity directly attributable to the treatment was observed, and the vaccine was well tolerated. In the delayed-type hypersensitivity (DTH) skin test, two patients showed a positive skin response to peptide-pulsed DCs after vaccination, although none of the patients tested positive prior to vaccination. In the two patients who showed a positive skin response disease remained stable for 6 and 9 months respectively. These results suggest that active immunization using DCs pulsed with CEA652 peptide in combination with the administration of adjuvant cytokines is a safe and feasible treatment procedure.     

Generation of functional monocyte-derived fast dendritic cells suitable for clinical application in the absence of interleukin-6

To develop dendritic cells (DCs)-based immunotherapy for cancer patients, it is necessary to have a standardized, reproducible, fast, and easy to use protocol for in vitro generation of fully functional DCs. Recently, a new strategy was described for differentiation and maturation of human monocyte (Mo)-derived fast-DCs with full T cell stimulatory capacity within only 48–72 h of in vitro culture. Interleukin (IL)-6 plus tumour necrosis factor (TNF)-α, IL-1β, and prostaglandin (PG)-E₂ were used in this strategy to induce maturation of the generated DCs. The present study further modifies this strategy by excluding IL-6 from the cytokines cocktail used for DCs maturation. The results showed that maturation of fast-DCs without IL-6 did not significantly alter the morphology, phenotype and the yield of mature DCs (P > 0.05, compared with those generated with IL-6). Moreover, fast-DCs generated without IL-6 are functional antigen presenting cells, have the ability to induce tetanus toxoid-specific autologous T cell proliferation, and are suitable for gene delivery through adenoviral vector transduction as those generated with IL-6 (P > 0.05). In conclusion, the present study proves that fully mature and functional Mo-derived fast-DCs can be generated in vitro without adding IL-6, which not only reduces the number of required recombinant cytokines, but may also resemble DCs development in vivo more closely.     

Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway

Dendritic cells (DCs) mediate cross-priming of tumor-specific T cells by acquiring tumor Ags from dead cancer cells. The process of cross-priming would be most economical and efficient if DCs also induce death of cancer cells. In this study, we demonstrate that normal human in vitro generated immature DCs consistently and efficiently induce apoptosis in cancer cell lines, freshly isolated noncultured cancer cells, and normal proliferating endothelial cells, but not in most normal cells. In addition, in vivo generated noncultured peripheral blood immature DCs mediate similar tumoricidal activity as their in vitro counterpart, indicating that this DC activity might be biologically relevant. In contrast to immature DCs, freshly isolated monocytes (myeloid DC precursors) and in vitro generated mature DCs are not cytotoxic or are less cytotoxic, respectively, suggesting that DC-mediated killing of cancer cells is developmentally regulated. Comparable cytotoxic activity is mediated by untreated DCs, paraformaldehyde-fixed DCs, and soluble products of DCs, and is destructible by proteases, indicating that both cell membrane-bound and secreted proteins mediate this DC function. Overall, our data demonstrate that human immature DCs are capable of inducing apoptosis in cancer cells and thus to both directly mediate anticancer activity and initiate processing of cellular tumor Ags.     

Granulocyte colony-stimulating factor modulates alpha-galactosylceramide-responsive human Valpha24+Vbeta11+NKT cells

Despite more than a 10-fold increase in T cell numbers in G-CSF-mobilized peripheral blood stem cell (PBSC) grafts, incidence and severity of acute graft-vs-host disease (GVHD) are comparable to bone marrow transplantation. As CD1d-restricted, Valpha24+Vbeta11+ NKT cells have pivotal immune regulatory functions and may influence GVHD, we aimed to determine whether G-CSF has any effects on human NKT cells. In this study, we examined the frequency and absolute numbers of peripheral blood NKT cells in healthy stem cell donors (n = 8) before and following G-CSF (filgrastim) treatment. Effects of in vivo and in vitro G-CSF on NKT cell cytokine expression profiles and on responsiveness of NKT cell subpopulations to specific stimulation by alpha-galactosylceramide (alpha-GalCer) were assessed. Contrary to the effects on conventional T cells, the absolute number of peripheral blood NKT cells was unaffected by G-CSF administration. Furthermore, responsiveness of NKT cells to alpha-GalCer stimulation was significantly decreased (p < 0.05) following exposure to G-CSF in vivo. This hyporesponsiveness was predominantly due to a direct effect on NKT cells, with a lesser contribution from G-CSF-mediated changes in APC. G-CSF administration resulted in polarization of NKT cells toward a Th2, IL-4-secreting phenotype following alpha-GalCer stimulation and preferential expansion of the CD4+ NKT cell subset. We conclude that G-CSF has previously unrecognized differential effects in vivo on NKT cells and conventional MHC-restricted T cells, and effects on NKT cells may contribute to the lower than expected incidence of GVHD following allogeneic peripheral blood stem cell transplantation.     

A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth

Intravesical bacillus Calmette-Guerin (BCG) therapy is considered the most successful immunotherapy against solid tumors of human bladder carcinoma. To determine the actual effector cells activated by intravesical BCG therapy to inhibit the growth of bladder carcinoma, T24 human bladder tumor cells, expressing very low levels of class I MHC, were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) with live BCG. The proliferation of T24 cells was markedly inhibited when BCG-infected dendritic cells (DCs) were added to the culture although the addition of either BCG or uninfected DCs alone did not result in any inhibition. The inhibitory effect was much stronger when the DCs were infected with live BCG rather than with heat-inactivated BCG. The live BCG-infected DCs secreted TNF-α and IL-12 within a day and this secretion continued for at least a week, while the heat-inactivated BCG-infected DCs secreted no IL-12 and little TNF-α. Such secretion of cytokines may activate innate alert cells, and indeed NKT cells expressing IL-12 receptors apparently proliferated and were activated to produce cytocidal perforin among the PBMCs when live BCG-infected DCs were externally added. Moreover, depletion of γδ T-cells from PBMCs significantly reduced the cytotoxic effect on T24 cells, while depletion of CD8β cells did not affect T24 cell growth. Furthermore, the innate effectors seem to recognize MICA/MICB molecules on T24 via NKG2D receptors. These findings suggest the involvement of innate alert cells activated by the live BCG-infected DCs to inhibit the growth of bladder carcinoma and provide a possible mechanism of intravesical BCG therapy.     

Novel IL-15 dendritic cells have a potent immunomodulatory effect in immunotherapy of multiple myeloma

Dendritic cells (DCs) are the most potent antigen-presenting cells, and have thus been used in clinical cancer vaccines. However, the effects of DC vaccines are still limited, leading researchers to explore novel ways to make them effective. In this study, we investigated whether human monocyte-derived DCs generated via the addition of interleukin 15 (IL-15) had a higher capacity to induce antigen-specific T cells compared to conventional DCs. We isolated CD14⁺ monocytes from peripheral blood from multiple myeloma (MM) patients, and induced immature DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 in the presence or absence of IL-15 for 4–6 days. Then we generated mature DCs (mDCs) with lipopolysaccharide for another 2 days [IL-15 mDCs (6 days), IL-15 mDCs (8 days), and conventional mDCs (8 days)]. IL-15 mDCs (6 days) showed higher expression of MHC I and II, CD40, CD86, and CCR7, and the secretion of IFN-γ was significantly higher compared to conventional mDCs. IL-15 mDCs (6 days) showed superior polarization of naïve T cells toward Th1 cells and a higher proportion of activated T cells, cytokine-induced killer (CIK) cells, and natural killer (NK) cells for inducing strong cytotoxicity against myeloma cells, and lower proportion of regulatory T cells compared to conventional mDCs. These data imply that novel multipotent mDCs generated by the addition of IL-15, which can be cultivated in 6 days, resulted in outstanding activation of T cells, CIK cells and NK cells, and may facilitate cellular immunotherapy for cancer patients.     

Activated human γδ T cells induce peptide-specific CD8+ T-cell responses to tumor-associated self-antigens

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.     

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.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

Production of functional dendritic cells from menstrual blood��a new dendritic cell source for immune therapy

Dendritic cells (DCs) are the most professional antigen-presenting cells of the mammalian immune system. They are able to phagocytize, process antigen materials, and then present them to the surface of other cells including T lymphocytes in the immune system. These capabilities make DC therapy become a novel and promising immune-therapeutic approach for cancer treatment as well as for cancer vaccination. Many trials of DC therapy to treat cancers have been performed and have shown their application value. They involve harvesting monocytes or hematopoietic stem cells from a patient and processing them in the laboratory to produce DCs and then reintroduced into a patient in order to activate the immune system. DCs were successfully produced from peripheral, umbilical cord blood-derived monocytes or hematopoietic stem cells. In this research, we produced DCs from human menstrual blood-derived monocytes. Briefly, monocytes were isolated by FACS based on FSC vs. SSC plot from lysed menstrual blood. Obtained monocytes were induced into DCs by a two-step protocol. In the first step, monocytes were incubated in RPMI medium supplemented with 2% FBS, GM-CSF, and IL-4, followed by incubation in RPMI medium supplemented with α-TNF in the second step. Our data showed that induced monocytes had typical morphology of DCs, expressed HLA-DR, HLA-ABC, CD80 and CD86 markers, exhibited uptake of dextran-FITC, stimulated allogenic T cell proliferation, and released IL-12. These results demonstrated that menstrual blood can not only be a source of stromal stem cell but also DCs, which are a potential candidate for immune therapy.     

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.     

Generation of mature dendritic cells with unique phenotype and function by in vitro short-term culture of human monocytes in the presence of interleukin-4 and interferon-beta

Dendritic cell (DC)-based immunotherapy has been utilized for the treatment of not only a number of human malignancies but also a select group of infectious diseases. Conventional techniques for the generation and maturation of DCs require 7 days of in vitro culture, which prompted us to seek alternative methods that would hasten the generation of functional human myeloid DCs in vitro. Following the use of a number of cytokines/growth factors, we found that in vitro culture of purified human monocytes, in media containing interleukin (IL)-4, together with interferon (IFN)-beta for 24 hrs, followed by the addition of non-specific antigenic stimuli, such as keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), or inactivated human immunodeficiency virus (HIV)-1 induced the monocytes to differentiated by 3 days into mature DCs (4B-DCs). These 4B-DCs expressed high levels of CD83 and CD11c, as well as markers of immune activation, including CD80 and CD86, human leukocyte antigen (HLA) class I and II, and CD14, but not CD1a. Anti-CD14 blocking antibody interfered with generation of 4B-DCs by LPS, but not by KLH or HIV-1. Interestingly, 4B-DCs, but not conventional DCs generated using macrophage-colony stimulating factor and IL-4 (G4-DCs), expressed OX40 and OX40L. 4B-DCs showed phagocytic activity, and spontaneously produced IL-12 and tumor necrosis factor (TNF)-alpha, but not IL-10. 4B-DCs promoted proliferation of allogeneic na?ve CD4(+) T cells, producing IFN-(lambda) at lower levels than those stimulated with G4-DCs. 4B-DCs were more potent stimulators of allogeneic bulk CD8(+) T cells producing IFN-(lambda) than G4-DCs. These data indicate that 4B-DCs are unique and may provide a relatively more rapid alternative tool for potential clinical use, as compared with conventional G4-DCs.     

International Meeting “Immunotherapy of Cancer: Challenges and Needs”

The main aims of the international meeting “Immunotherapy of Cancer: Challenges and Needs” were to review the state of the art of cancer immunotherapy and to identify critical issues which deserve special attention for promoting progress of research in this field, with a particular focus on the perspectives of clinical research. Novel concepts and strategies for identifying, monitoring and predicting effective responses to cancer immunotherapy protocols were presented, focused on the use of adjuvants (CpG oligonucleotides) or cytokines (IFN-alpha) to enhance the efficacy of cancer vaccines. Moreover, the possible advantages of using different types of dendritic cells (for active immunization strategies) or T cells (for adoptive immunotherapy protocols) were debated. A consensus was achieved on the need for enhancing the efficacy of cancer vaccines or adoptive cell immunotherapy by combining these strategies with other anti-cancer treatments, including chemotherapy. Finally, initiatives for promoting clinical research by establishing a strategic cooperation in the field of cancer immunotherapy based on the active participation of all the relevant actors, including public institutions responsible of Public Health, National Cancer Institutes, industry, representatives of regulatory bodies, and patients’ organizations were proposed.     

Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans

We used multiparameter flow cytometry to characterize leukocyte immunophenotypes and cytokines in skin and peripheral blood of patients with erythema migrans (EM). Dermal leukocytes and cytokines were assessed in fluids aspirated from epidermal suction blisters raised over EM lesions and skin of uninfected controls. Compared with corresponding peripheral blood, EM infiltrates were enriched for T cells, monocytes/macrophages, and dendritic cells (DCs), contained lower proportions of neutrophils, and were virtually devoid of B cells. Enhanced expression of CD14 and HLA-DR by lesional neutrophils and macrophages indicated that these innate effector cells were highly activated. Staining for CD45RO and CD27 revealed that lesional T lymphocytes were predominantly Ag-experienced cells; furthermore, a subset of circulating T cells also appeared to be neosensitized. Lesional DC subsets, CD11c(+) (monocytoid) and CD11c(-) (plasmacytoid), expressed activation/maturation surface markers. Patients with multiple EM lesions had greater symptom scores and higher serum levels of IFN-alpha, TNF-alpha, and IL-2 than patients with solitary EM. IL-6 and IFN-gamma were the predominant cytokines in EM lesions; however, greater levels of both mediators were detected in blister fluids from patients with isolated EM. Circulating monocytes displayed significant increases in surface expression of Toll-like receptor (TLR)1 and TLR2, while CD11c(+) DCs showed increased expression of TLR2 and TLR4; lesional macrophages and CD11c(+) and CD11c(-) DCs exhibited increases in expression of all three TLRs. These results demonstrate that Borrelia burgdorferi triggers innate and adaptive responses during early Lyme disease and emphasize the interdependence of these two arms of the immune response in the efforts of the host to contain spirochetal infection.