Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E(2) (PGE(2)) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE(2) to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter.     

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.     

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.     

Dendritic cells: limited potential in immunotherapy

Dendritic cells (DC) represent the most potent antigen-presenting cells (APC) of the immune system for their unique capability of presenting antigen to T-cells. Their use as cellular vaccines after charging with antigen ex vivo has been shown to induce protective and therapeutic anti-tumor immunity with regression of tumor manifestations in animal models of experimental cancer therapy. Human monocyte-derived DC (MoDC) generated in vitro in the presence of GM-CSF and IL-4 are regarded equivalent to immature DC. They can be induced to mature under various experimental conditions. MoDC, in their immature as well as mature state have been widely used for experimental as well as for clinical purposes. However, unequivocal proof for the clinical efficiency of MoDC-based anti-tumor vaccinations is still missing. There is now increasing experimental evidence demonstrating that MoDC may be hampered in their ability to migrate in response to inflammatory as well as homeostatic chemataxins. We therefore suggest that MoDC may not represent the equivalent of migratory DC in vivo limiting their use as magic bullets in tumor immunotherapy.     

Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma

We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties.     

Complex evaluation of human monocyte‐derived dendritic cells for cancer immunotherapy

Dendritic cell (DC) immunotherapy is capable of generating tumour‐specific immune responses. Different maturation strategies were previously tested to obtain DC capable of anti‐cancer responses in vitro, usually with limited clinical benefit. Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application. DC were generated from monocytes using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, pulsed with whole tumour cell lysate and then matured with one of five selected maturation strategies or cultured without additional maturation stimulus. DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens. We were able to demonstrate extensive variability among different maturation strategies currently used in DC immunotherapeutic protocols that may at least partially explain limited clinical benefit of some clinical trials with such DC. We identified DC matured with interferon‐γ and lipopolysaccharide as the most attractive candidate for future clinical trials in cancer immunotherapy.     

Low Molecular Weight Hyaluronan-Pulsed Human Dendritic Cells Showed Increased Migration Capacity and Induced Resistance to Tumor Chemoattraction

We have shown that ex vivo pre-conditioning of bone marrow-derived dendritic cells (DC) with low molecular weight hyaluronan (LMW HA) induces antitumor immunity against colorectal carcinoma (CRC) in mice. In the present study we investigated the effects of LMW HA priming on human-tumor-pulsed monocytes-derived dendritic cells (DC/TL) obtained from healthy donors and patients with CRC. LMW HA treatment resulted in an improved maturation state of DC/TL and an enhanced mixed leucocyte reaction activity in vivo. Importantly, pre-conditioning of DC/TL with LMW HA increased their ability to migrate and reduced their attraction to human tumor derived supernatants. These effects were associated with increased CCR7 expression levels in DC. Indeed, a significant increase in migratory response toward CCL21 was observed in LMW HA primed tumor-pulsed monocyte-derived dendritic cells (DC/TL/LMW HA) when compared to LWM HA untreated cells (DC/TL). Moreover, LMW HA priming modulated other mechanisms implicated in DC migration toward lymph nodes such as the metalloproteinase activity. Furthermore, it also resulted in a significant reduction in DC migratory capacity toward tumor supernatant and IL8 in vitro. Consistently, LMW HA dramatically enhanced in vivo DC recruitment to tumor-regional lymph nodes and reduced DC migration toward tumor tissue. This study shows that LMW HA –a poorly immunogenic molecule- represents a promising candidate to improve human DC maturation protocols in the context of DC-based vaccines development, due to its ability to enhance their immunogenic properties as well as their migratory capacity toward lymph nodes instead of tumors.     

Provision of continuous maturation signaling to dendritic cells by RIG-I-stimulating cytosolic RNA synthesis of Sendai virus

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors, but the functional capacity of DC must be assessed in detail, especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant, providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol, resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors, or TLRs, do not show these functional features. Therefore, SeV-infected DC have the potential for DC-directed immunotherapy.     

Uptake of antigens from modified vaccinia Ankara virus-infected leukocytes enhances the immunostimulatory capacity of dendritic cells

Background aimsModified vaccinia Ankara (MVA) is a promising vaccine vector for infectious diseases and malignancies. It is fundamental to ascertain its tropism in human leukocyte populations and immunostimulatory mechanisms for application in immunotherapy.MethodsHuman peripheral blood mononuclear cells (PBMC) and leukocyte subpopulations [monocyte-derived dendritic cells (DC), monocytes and B cells] were infected with MVA in order to evaluate their infection rate, changes in surface markers, cytokine expression and apoptosis.ResultsMonocytes, DC and B cells were most susceptible to MVA infection, followed by natural killer (NK) cells. Monocytes were activated strongly, with upregulation of co-stimulatory molecules, major histocompatibility complex (MHC) molecules and chemokine (C-C motif) receptor (CCR7), while immature DC showed partial activation and B cells were inhibited. Furthermore, expression of chemokine (C-X-C motif) ligand (CXCL10), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-12p70 was enhanced but IL-1β and IL-10 were stable or even downregulated. MVA induced a high apoptosis rate of antigen-presenting cells (APC). Nevertheless, incubation of MVA-infected leukocytes with uninfected immature DC (iDC) led to complete maturation of the DC. Subsequently, the matured DC were able to stimulate cytomegalovirus (CMV)-immediate early protein (IE1)-specific T cells.ConclusionsMVA induces a T-helper (Th)-1-polarizing cytokine expression in APC. Furthermore, incubation of MVA-infected leukocytes with uninfected iDC leads to complete maturation of the DC and may be the basis for cross-presentation of MVA-encoded antigens. Thus this approach seems to be an ideal model for further studies with MVA-encoded viral antigens regarding immunotherapy and vaccination strategies.     

The duration of signaling through CD40 directs biological ability of dendritic cells to induce antitumor immunity

Although it has been demonstrated that the functions of dendritic cells (DCs), including Ag capture, Ag presentation, and migratory activity, change dynamically with their maturation, the most appropriate conditioning of DCs for anticancer immunotherapy is still unclear. The help signal is one of the most potent stimuli for DC maturation and is provided by the interaction of CD40 expressed on DCs with CD40 ligand on CD4(+) T cells. To elucidate the appropriate conditioning of DCs for anticancer immunotherapy, we examined the biological activity of DCs stimulated with immobilized anti-CD40 Ab. DCs stimulated for 3 h (3h-DCs) still showed an immature phenotype, but exhibited augmented migration toward secondary lymphoid tissues. Subcutaneous injection of 3h-DCs facilitated priming of T cells, which could mediate potent antitumor therapeutic efficacy, in draining lymph nodes and successfully induced protective immunity. In contrast, 24h-DCs showed a mature phenotype with good Ag presentation ability to induce cell killing by adoptively transferred CD8(+) T cells when injected at tumor sites; however, they showed no migratory activity and were unable to induce protective immunity when injected s.c. This is the first report that functionally distinct DCs, either for the priming phase or for the effector phase, could be obtained by conditioning with CD40 stimulation and that the duration of stimulation determines the biological outcome. The usage of DCs conditioned for the priming phase might provide significant advantages in anticancer immunotherapy.     

Final maturation of dendritic cells is associated with impaired responsiveness to IFN-gamma and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells

Activation of immature CD83- dendritic cells (DC) in peripheral tissues induces their maturation and migration to lymph nodes. Activated DC become potent stimulators of Th cells and efficient inducers of Th1- and Th2-type cytokine production. This study analyzes the ability of human monocyte-derived CD1a+ DC at different stages of IL-1 beta and TNF-alpha-induced maturation to produce the major Th1-driving factor IL-12. DC at the early stages of maturation (2 and 4 h) produced elevated amounts of IL-12 p70 during interaction with CD40 ligand-bearing Th cells or, after stimulation with the T cell-replacing factors, soluble CD40 ligand and IFN-gamma. The ability to produce IL-12 was strongly down-regulated at later time points, 12 h after the induction of DC maturation, and in fully mature CD83+ cells, at 48 h. In contrast, the ability of mature DC to produce IL-6 was preserved or even enhanced, indicating their intact responsiveness to CD40 triggering. A reduced IL-12-producing capacity of mature DC resulted mainly from their impaired responsiveness to IFN-gamma, a cofactor in CD40-induced IL-12 p70 production. This correlated with reduced expression of IFN-gamma R (CD119) by mature DC. In addition, while immature DC produced IL-12 and IL-6 after stimulation with LPS or Staphylococcus aureus Cowan I strain, mature DC became unresponsive to these bacterial stimuli. Together with the previously described ability of IL-10 and PGE2 to stably down-regulate the ability to produce IL-12 in maturing, but not in fully mature, DC, the current data indicate a general resistance of mature DC to IL-12-modulating factors.     

Cutting edge: peripheral neuropeptides attract immature and arrest mature blood-derived dendritic cells

Dendritic cells (DC) are highly motile and play a key role in mediating immune responses in various tissues and lymphatic organs. We investigated locomotion of mononuclear cell-derived DC at different maturation stages toward gradients of sensory neuropeptides in vitro. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, secretin, and secretoneurin induced immature DC chemotaxis comparable to the potency of RANTES, whereas substance P and macrophage-inflammatory protein-3beta stimulated immature cell migration only slightly. Checkerboard analyses revealed a true chemotactic response induced by neuropeptides. Upon maturation of DC, neuropeptides inhibited spontaneous, macrophage-inflammatory protein-3beta- and 6Ckine-induced cell migration. Maturation-dependent changes in migratory behavior coincided with distinct neuropeptide-induced signal transduction in DC. Peripheral neuropeptides might guide immature DC to peripheral nerve fibers where high concentrations of these peptides can arrest the meanwhile matured cells. It seems that one function of sensory nerves is to fasten DC at sites of inflammation.     

Transduction of human dendritic cells with a recombinant modified vaccinia Ankara virus encoding MUC1 and IL-2

The epithelial mucin MUC1 is considered an opportune target antigen for cancer immunotherapy, as it is over-expressed and exhibits aberrant glycosylation in malignant cells. Because dendritic cells (DC) are powerful initiators of immune responses, efforts have focused on tumor antigen-bearing DC as potent cancer vaccines. In this study we have characterized the transduction of monocyte-derived DC with a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] encoding human MUC1 and the immunostimulatory cytokine IL-2. Analysis of transduced DC cultures generated from a number of donors revealed MUC1 expression in the range of 27-54% of the cells and a co-regulated secretion of bioactive IL-2. As shown by FACS analysis with MUCI-specific antibodies, the MVA-MUC1/IL-2-transduced DC predominantly expressed the fully processed glycoform of MUC1, typical of that displayed by normal epithelia. Over a 3-day period after transduction, transgene expression declined concurrent with an increase in MVA-induced cytopathic effects. The transduced DC stimulated allogeneic lymphocyte proliferation, indicating that DC immunostimulatory function is not impaired by vector transduction. In the presence of IL-2, MVA-transduced DC were able to enhance autologous lymphocyte proliferation. Also, vector expression was analyzed in DC cultures treated with TNF-alpha, a known DC maturation factor. As indicated by the up-regulation of several DC maturation markers, neither virus infection nor transgene expression influenced the maturation capacity of the cells. The MVA-MUC1/IL-2 vector effectively transduced both immature and TNF-alpha-matured DC. Overall, our results are encouraging for the clinical application of MVA-MUC1/IL-2-transduced DC.     

Increased extracellular pressure provides a novel adjuvant stimulus for enhancement of conventional dendritic cell maturation strategies

Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40 mmHg increased pressure for 12 h, then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.     

A critical role for prostaglandin E2 in podosome dissolution and induction of high-speed migration during dendritic cell maturation

Dendritic cells (DCs) are professional APCs of the immune system that play a key role in regulating T cell-based immunity. The capacity of DCs to activate T cells depends on their maturation state as well as their ability to migrate to the T cell areas of draining lymph nodes. In this study, we investigated the effects of DC maturation stimuli on the actin cytoskeleton and beta(1) integrin-dependent adhesion and migration. Podosomes, specialized adhesion structures found in immature monocyte-derived DCs as well as myeloid DCs, rapidly dissolve in response to maturation stimuli such as TNF-alpha and PGE(2), whereas the TLR agonist LPS induces podosome dissolution only after a long lag time. We demonstrate that LPS-mediated podosome disassembly as well as the onset of high-speed DC migration are dependent on the production of PGs by the DCs. Moreover, both of these processes are inhibited by Ab-induced activation of beta(1) integrins. Together, these results show that maturation-induced podosome dissolution and loss of alpha(5)beta(1) integrin activity allow human DCs to undergo the transition from an adhesive to a highly migratory phenotype.     

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.     

Monocyte-derived CD1a+ and CD1a- dendritic cell subsets differ in their cytokine production profiles, susceptibilities to transfection, and capacities to direct Th cell differentiation

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-gamma, but they remained CD1a- and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.     

MSC-DC interactions: MSC inhibit maturation and migration of BM-derived DC

BACKGROUND: Mesenchymal stromal cells (MSC) comprise one of the BM stromal cells that are known to support hematopoiesis. It has also been suggested recently that MSC display immunosuppressive capacities through inhibiting the differentiation of monocyte-derived DC. DC travel to the lymph nodes (LN) to present Ag to T cells, and CCL21 is the chemokine that plays an important role in DC migration into the T-cell area of LN. We addressed the effect of MSC on this chemotactic activity of DC, one of the typical characteristics upon maturation. METHODS: BM cells were isolated and then cultured for generation of myeloid DC in the presence of GM-CSF and/or lipopolysaccharide with or without MSC. MSC were identified by flow cytometry of the immunologic markers and by performing colony-forming unit fibroblast assay. Migration of DC was observed with a newly developed time-lapse video microscopic technique. RESULTS: MSC co-culture inhibited the initial differentiation of DC, as well as their maturation. The matured DC actively migrated directionally in response to CCL21, a powerful DC-attracting chemokine, whereas the MSC co-cultured DC did not. DISCUSSION: Collectively, the findings of these experiments raise the possibility that MSC suppress the migratory function of DC and so they may serve immunoregulatory activities through the modulation of the Ag-presenting function of DC.     

A novel approach to specific allergy treatment: the recombinant allergen-S-layer fusion protein rSbsC-Bet v 1 matures dendritic cells that prime Th0/Th1 and IL-10-producing regulatory T cells

An ideal vaccine for allergen-specific immunotherapy of type I allergies should display reduced mediator-releasing capacity, induce maturation of APC, and modify the disease-eliciting Th2-dominated allergen-specific response to a more physiological response. We have previously shown that rSbsC-Bet v 1, the recombinant fusion protein of a bacterial surface (S-layer) protein of Geobacillus stearothermophilus ATCC 12980 and the major birch pollen allergen Bet v 1, exhibited reduced allergenicity and induced IFN-gamma and IL-10 synthesis in Bet v 1-specific Th2 clones. In this study, we characterized the effects of rSbsC-Bet v 1 on immature monocyte-derived dendritic cells (mdDC) and the consequences for the polarization of naive CD4(+) T lymphocytes isolated from the blood of birch pollen-allergic patients. mdDC responded to rSbsC-Bet v 1 with a significant up-regulation of costimulatory molecules, functional maturation, and the synthesis of IL-10 and IL-12. mdDC matured with rSbsC-Bet v 1 induced the differentiation of naive T cells into IFN-gamma-producing cells. This effect was IL-12 dependent. In parallel, a substantial number of naive T cells developed into IL-10-producing CD25(+)Foxp3(+)CLTA-4(+) cells capable of active suppression. Thus, rSbsC-Bet v 1 showed immune stimulatory capacity on DC, which then promoted the simultaneous differentiation of Th0/Th1 cells and regulatory T cells. These data further support that the concept of conjugating allergens to bacterial agents is a promising approach to improve vaccines for specific immunotherapy of atopic allergies.     

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8⁺ T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.