Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Streptococcal preparation OK-432: a new maturation factor of monocyte-derived dendritic cells for clinical use

For vaccinations based on dendritic cells (DCs), maturation of DCs is critical to the induction of T-cell responses. We tested the efficacy of streptococcal preparation OK-432 as a Good Manufacturing Practice (GMP)-grade maturation agent. OK-432 is currently used in Japan as a cancer immunotherapy drug. Immature monocyte-derived dendritic cells (imMo-DCs) isolated from human peripheral blood monocytes stimulated with granulocyte-macrophage colony stimulating factor and interleukin-4 were exposed to maturation factors, i.e., lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 (PGE2), and OK-432 for 2 days. OK-432 increased expression of activation- and maturation-related molecules such as HLA-DR, CD80, CD83, and CD86 in imMo-DCs at levels similar to that of TNF-alpha plus PGE2, and higher than that of LPS. All agents examined induced allogeneic T-cell proliferation at a similar level. Only OK-432 caused significant production of interleukin-12 (IL-12) p70 and interferon gamma (IFN-gamma) at both the mRNA and protein levels in imMo-DCs. Neutralizing antibody against IL-12 p70 blocked IFN-gamma secretion from OK-432-stimulated Mo-DCs. IL-12 p70 produced by OK-432-stimulated imMo-DCs induced secretion of IFN-gamma by CD4+ T cells. OK-432 and LPS activated nuclear factor kappa B (NF-kappaB) in imMo-DCs. Both secretion of IL-12 p70 and IFN-gamma and activation of NF-kappaB induced by OK-432 were suppressed when imMo-DCs were pretreated with cytochalasin B. These results indicate that uptake of OK-432 by imMo-DCs is an early critical event for IL-12 p70 production and that NF-kappaB activation induced by OK-432 also contributes partially to IL-12 p70 production. In conclusion, OK-432 is a GMP-grade maturation agent and may be a potential tool for DC-based vaccine therapies.     

The Pseudomonas aeruginosa Mannose Sensitive Hamemagglutination Strain (PA-MSHA) Induces a Th1-Polarizing Phenotype by Promoting Human Dendritic Cells Maturation

Pseudomonas aeruginosa mannose sensitive hamemagglutination strain (PA-MSHA) is a kind of peritrichous P. aeruginosa strain with MSHA fimbriae and has been shown to activate kinds of immunocytes. Dendritic cells (DCs) are specialized antigen-presenting cells required for the stimulating and priming CD4⁺ T cells toward the T helper cell type 1 (Th1), Th2 and other different phenotypes. PA-MSHA effecting on Th1 remains an important missing link. Here we demonstrated that PA-MSHA augmented monocytes derived-dendritic cells (Mo-DCs) expression of HLA-DR, co-stimulatory and adhesion molecules, and induced Th1-promoting interleukin-12 and tumor necrosis factor α secretion, in addition, PA-MSHA treated Mo-DCs displayed lesser endocytic capacity. Furthermore, in mixed lymphocyte reactions, allostimulatory capacity of Mo-DCs was enhanced by PA-MSHA, CD4⁺ T cells stimulated by PA-MSHA -activated Mo-DCs showed a Th1-polarized cytokine production, increasing secretion of IFN-γ and decreasing secretion of IL-10 and IL-4. Our findings identified PA-MSHA as an important exogenous factor that induced DCs maturation toward a Th1-promoting phenotype.     

Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.     

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.     

Induction of dendritic cell maturation by IL-18

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.     

The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs’ antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and Dcbased immunotherapy may be adopted in the treatment of RB.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

Monophosphoryl lipid A plus IFNγ maturation of dendritic cells induces antigen-specific CD8+ cytotoxic T cells with high cytolytic potential

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNγ generates mature DCs that produce IL-12 and polarize CD4⁺ T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNγ induce superior tumour antigen-specific CD8⁺ CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNγ DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNγ DCs. The high amounts of induced CTLs are functional as they produce IFNγ and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNγ DCs are very promising for the use in future cancer immunotherapy.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

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.     

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.     

Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram-positive bacteria to CD4+ T lymphocytes.

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.     

An IFN-gamma-IL-18 signaling loop accelerates memory CD8+ T cell proliferation

Rapid proliferation is one of the important features of memory CD8(+) T cells, ensuring rapid clearance of reinfection. Although several cytokines such as IL-15 and IL-7 regulate relatively slow homeostatic proliferation of memory T cells during the maintenance phase, it is unknown how memory T cells can proliferate more quickly than na?ve T cells upon antigen stimulation. To examine antigen-specific CD8(+) T cell proliferation in recall responses in vivo, we targeted a model antigen, ovalbumin(OVA), to DEC-205(+) dendritic cells (DCs) with a CD40 maturation stimulus. This led to the induction of functional memory CD8(+) T cells, which showed rapid proliferation and multiple cytokine production (IFN-gamma, IL-2, TNF-alpha) during the secondary challenge to DC-targeted antigen. Upon antigen-presentation, IL-18, an IFN-gamma-inducing factor, accumulated at the DC:T cell synapse. Surprisingly, IFN-gamma receptors were required to augment IL-18 production from DCs. Mice genetically deficient for IL-18 or IFN-gamma-receptor 1 also showed delayed expansion of memory CD8(+) T cells in vivo. These results indicate that a positive regulatory loop involving IFN-gamma and IL-18 signaling contributes to the accelerated memory CD8(+) T cell proliferation during a recall response to antigen presented by DCs.     

A role for IL-16 in the cross-talk between dendritic cells and T cells.

Dendritic cells (DCs) in the periphery capture and process Ags, migrate to lymphoid organs, and initiate immune responses in T cells. IL-16, the soluble ligand for CD4, is a potent chemoattractant for CD4+ T cells, eosinophils, and monocytes and is mainly derived from activated T cells. Because migration is a fundamental property of DCs, we asked whether IL-16 induces chemotaxis in DCs and whether DCs are a source of IL-16. DCs were generated by culture of monocytes in IL-4 and GM-CSF for 6 days and subsequently highly purified employing magnetic beads. Migration was assayed by nitrocellulose and polycarbonate filter-based assays, and distinction of chemotaxis and chemokinesis was performed by a checkerboard analysis. Messenger RNA and protein data revealed constitutive expression and release of IL-16 by day-6 DCs. Gradients of rIL-16 induced a chemotactic response of DCs. Furthermore, the chemotactic activity of DC supernatant toward DCs themselves and T cells was mainly due to IL-16, because the addition of neutralizing Abs completely abrogated the migratory response. However, after induction of maturation by the addition of TNF-alpha and PGE2 DCs, neither expressed IL-16 mRNA nor produced IL-16 protein. We conclude that IL-16 may play a role in the trafficking of DCs and may be a major chemotactic signal from DCs toward themselves and toward T cells.     

Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.     

Human dendritic cells sequentially matured with CD4(+) T cells as a secondary signal favor CTL and long-term T memory cell responses

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.