The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles

Biodegradable microparticles (MP) represent a promising and efficient delivery system for parenteral vaccination. Recently, MP have also been explored as tool for the ex vivo antigen loading of professional antigen-presenting cells such as dendritic cells (DC) to be used as cellular vaccines. The purpose of this study was to investigate various polycationic coatings on poly(lactide-co-glycolide) (PLGA) MP, with regard to their effect on phenotypic and functional maturation of monocyte-derived DC (MoDC) that had previously been loaded with the MP in vitro. The preparation and concomitant coating of the PLGA was performed by means of a solvent extraction/evaporation method using a recently developed microextrusion-based technique. The polyelectrolytes tested for MP coating encompassed aminodextran, chitosan, poly(ethylene imine) (PEI), poly(L-lysine) and protamine. Uncoated and differently coated PLGA MP were fed to immature MoDC, which ingested efficiently the different MP types irrespective of their surface coating. The MP-loaded immature MoDC were then matured with the help of a cytokine/PGE-2 maturation cocktail. Here, the presence of the ingested MP did not affect the MoDC maturation in terms of expression of the surface markers CD80, CD83, CD86, HLA-DR and MMR, irrespective of the MP surface coating. Importantly, none of the PLGA MP types alone induced significant maturation of MoDC in the absence of the maturation cocktail. MP-loaded and subsequently matured MoDC expressed high levels of the chemokine receptor CCR7, whose functional activity was evidenced by the migration of MoDC towards CCL21, irrespective of the presence of ingested MP. Further, MP-loaded and subsequently matured MoDC also secreted comparable amounts of IL-10 and IL-12p70, irrespective of the presence of ingested MP except for PEI-coated PLGA MP, which enhanced significantly the secretion of IL-12p70 in mature MoDC. In conclusion, phenotypic and functional maturation of MoDC by means of a maturation cocktail remained unchanged irrespective of the presence of previously ingested differently coated PLGA MP. This offers interesting perspectives for using these particulate systems together with entrapped antigens for ex vivo loading of MoDC in view of cellular immunotherapy.     

IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity

Dendritic cells (DC) of myeloid origin can be generated from mouse bone marrow (BM) using granulocyte macrophage-colony stimulating factor (GM-CSF). Immature major histocompatibility complex (MHC) II(low) DC are known to bear a high endocytosis capacity, in contrast to DC precursors and mature DC. Now we found that a subset of MHC II(low) DC in BM-DC cultures is unable to exert mannose receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (DX) and resembles immature Langerhans cells (LC). The FITC-DX endocytosis activity of LC-like cells occurs at an earlier stage of development, where the surface MHC II expression is absent or very weak. This LC-like subset expresses higher levels of E-cadherin but lower amounts of the markers Gr-1, scavenger receptor 2F8, and CD11b, when compared with the highly endocytic DC subset. The latter myeloid DC resemble monocyte-derived DC (MoDC). The sorted LC-like population develops completely and exclusively into mature MHC IIhigh DC, and the MoDC-like cells remain immature MHC II(low) DC or develop into adherent MHC IIneg macrophages or mature into MHC IIhigh DC. The development of LC-like cells is promoted by interleukin-4. Thus, we show here that the simultaneous development of LC-like and MoDC-like DC subsets occurs in standard bulk cultures with GM-CSF, suggesting the existence of two different precursors for LC and MoDC in BM.     

IFN-alpha enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells

Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte-derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)-induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up-regulated the expression of HLA-DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen-uptake function, increased the levels of IL-12p35 mRNA, and prolonged surface expression of peptide-MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co-factors for CD40L-mediated function. Here, they enhanced CD40L-mediated IL-6, IL-10 and IL-12p70 secretion. Furthermore, when combined with IL-1beta and/or IL-4, IFN-alpha2a type I IFN increased CD40L-mediated IL-12p70 production by 2- to 3-fold, and biased the IL-12 p40/p70 ratio towards the IFN-gamma inducing p70 heterodimer, this correlating with higher levels of IFN-gamma secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL-1beta at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L-mediated DC activation.     

Differentiation and activation of equine monocyte‐derived dendritic cells are not correlated with CD206 or CD83 expression

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte–macrophage colony‐stimulating factor and interleukin‐4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon‐γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system.     

Dendritic cells lose ability to present protein antigen after stimulating antigen-specific T cell responses, despite upregulation of MHC class II expression

Immature dendritic cells (DC) take up, process and present protein antigens; mature DC are specialized for stimulating primary T cell responses with increased expression of MHC class II and co-stimulatory molecules, but are incapable of processing and presenting soluble protein. The current study examined whether maturation of DC is triggered by T cell recognition of antigens presented by immature DC. Human DC derived from CD34+ progenitor cells by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) in serum-free medium could prime naive CD4+ T cells to keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). The cultured DC retained the ability to prime T cells to native protein for at least 15 days. To test for changes in DC function after participation in an immune response, DC were co-cultured with either allogeneic or autologous CD4+ T cells. DC co-cultured with autologous T cells retained the ability to prime T cells to intact protein antigens. By contrast, DC which had previously stimulated an allogeneic T cell response lost ability to prime T cells to soluble proteins. However, such induced a MLR and stimulated peptide-specific primary CD4+ T cell responses. This indicated that did not die or lose the ability to prime, but lost the ability to process and present subsequent antigens. Following participation in T cell activation, DC increased surface expression of MHC class II, co-stimulatory molecules CD40 and B7.2, and the intercellular adhesion molecule-1 (ICAM-1). In addition, our data suggest that interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) are involved in this T cell-mediated DC maturation.     

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “nature's adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS^®-fractionated MHC class II^low (termed immature DC) or MHC class II^high populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-α. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.     

The polysaccharide capsule of Cryptococcus neoformans interferes with human dendritic cell maturation and activation

The ability of encapsulated and acapsular strains of Cryptococcus neoformans to activate dendritic cells (DC) derived from monocytes stimulated with granulocyte macrophage-colony stimulating factor and interleukin-4 was evaluated. Profound differences in DC response to encapsulated and acapsular C. neoformans strains were observed. In particular, (i) the acapsular strain was easily phagocytosed by immature DC, and the process induced several molecular markers, such as major histocompatibility complex (MHC) class I and class II, CD40, and CD83, which are characteristic of mature DC; (ii) the encapsulated strain did not up-regulate MHC class I and class II and CD83 molecules; (iii) the soluble capsular polysaccharide glucuronoxylomannan (GXM) is unable to regulate MHC class I and class II molecules; (iv) the addition of monoclonal antibody to GXM (anti-GXM) to the encapsulated strain facilitated antigen-presenting cell maturation by promoting ingestion of C. neoformans via Fc receptor for immunoglobulin G (FcgammaR)II (CD32) and FcgammaRIII (CD16); (v) pertubation of FcRgammaII or FcgammaRIII was insufficient to promote DC maturation; and (vi) optimal DC maturation permitted efficient T cell activation and differentiation, as documented by the enhancement of lymphoproliferation and interferon-gamma production. These results indicate that the C. neoformans capsule interferes with DC activation and maturation, indicating a new pathway by which the fungus may avoid an efficient T cell response.     

Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro

Dendritic cells (DC) can be generated from mouse bone marrow (BM) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bacterial stimuli such as endotoxin / lipopolysaccharide (LPS) can induce their final maturation. When BM-DC cultures were treated at day 6 or later with LPS, this final maturation was induced in vitro within 24 h. Such mature DC exhibited high levels of surface MHC II molecules and potent T cell sensitizing, but reduced endocytosis capacity. In contrast, immature DC express only few MHC II molecules and are weak T cell stimulators but highly endocytic. When BM-DC cultures in GM-CSF were treated with 1 microg / ml LPS at day 0 of the culture or throughout the culture, only immature DC developed as defined by phenotype (MHC II low) and function (high endocytosis, weak primary mixed lymphocyte reaction). Those early LPS-treated immature DC induced alloantigen-specific anergy of CD4(+) T cells in vitro. These findings might contribute to the understanding of reduced T cell immunity in the course of septic shock and find application in DC-mediated tolerogenic immunotherapy strategies.     

Monocytes from Wiskott-Aldrich patients differentiate in functional mature dendritic cells with a defect in CD83 expression.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by congenital thrombocytopenia and progressive deterioration of the immune function. Dendritic cells (DC) are key effectors in the induction of specific immunity and are highly specialized in antigen uptake and subsequent migration to draining lymph nodes. DC were generated in vitro from circulating monocytes from ten WAS patients characterized by a different disease score. Immature DC showed similar morphology and membrane phenotype, as compared to normal DC. In chemotaxis assay, immature DC had a reduced migration in response to MIP-1alpha/CCL3, but efficiently endocytosed the macromolecules FITC-dextran and FITC-albumin. Upon terminal differentiation with LPS or CD40 ligand, the acquisition of a mature surface phenotype was variably achieved among WAS patients, with increased expression of CD80, CD86 and DC-LAMP. In contrast, the expression of CD83 was usually low. A defective up-regulation of CD83 was also observed in the lymph node from one WAS patient, whose DC stained positively for DC-LAMP. Mature DC from all the patients tested, but one, significantly migrated in vitro in response to MIP-3beta, a finding confirmed in vivo by the detection of HLA-DR/DC LAMP-positive cells in secondary lymphoid organs. When tested in MLR assays, both immature and mature WAS DC induced allogenic T cell proliferation in a manner comparable to control DC. Collectively these results suggest that, although many functional activities of WAS DC are essentially similar to normal DC, subtle and selective alterations of DC differentiation were also observed, with reduced migratory activity of immature DC and defective CD83 expression upon maturation.     

Maturation of Peyer's patch dendritic cells in vitro upon stimulation via cytokines or CD40 triggering

In mouse Peyer's patches (PP), dendritic cells (DC) are localized in T cell areas as NLDC145⁺ CD11c+ cells, and in the dome and corona region of the follicle as NLDC145? CD11c+ cells, respectively, suggesting the presence of two different DC populations with distinct roles in antigen uptake, processing, and presentation. However, it is not clear how this relates to DC maturation. In this report, we demonstrate that freshly-isolated CD11c+ DC have the properties of immature DC since they endocytose soluble antigens, phagocytose particulate material such as latex beads, synthetize major histocompatibility complex (MHC) class II and invariant chain, but, at the same time, display low stimulatory activity for resting T cells, as shown in mixed-lymphocyte reaction and oxidative mitogenesis assays. When cultured for 24 h in the presence of the cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor or anti-CD40, the cells undergo dramatic phenotypic and functional changes characteristic of DC maturation. After 24 h stimulation in vitro, CD11c+ cells lose the ability to take up proteins such as ovalbumin, and in parallel with this decline, the biosynthesis of MHC class II and invariant chain is dramatically down-regulated or eliminated. On the other hand cells treated in vitro exhibit on the cell surface higher levels of MHC class II, of co-stimulatory molecules (CD80, CD86), of adhesion molecules (CD44, intercellular adhesion molecule-1), and acquire expression of the interdigitating DC surface marker NLDC145. Concomitantly, the ability to stimulate naive T cells drastically increased after in vitro treatment with both stimuli. Taken together, our results indicate that the majority of DC in the PP are immature in terms of their antigen-uptake capacity. These sentinel antigen presenting cells are strategically positioned at the dome region of PP, where antigens are transcytosed via the M cells from the gut lumen. A second population of mature interdigitating NLDC145⁺ CD11c+ DC stimulates naive unprimed T cells in interfollicular areas by up-regulation of surface ligands and accessory signals.     

Both maturation and survival of human dendritic cells are impaired in the presence of anergic/suppressor T cells

T cell suppression is a well established phenomenon, but the mechanisms involved are still a matter of debate. Mouse anergic T cells were shown to suppress responder T cell activation by inhibiting the antigen presenting function of DC. In the present work we studied the effects of co-culturing human anergic CD4+ T cells with autologous dendritic cells (DC) at different stages of maturation. Either DC maturation or survival, depending on whether immature or mature DC where used as APC, was impaired in the presence of anergic cells. Indeed, MHC and costimulatory molecule up-regulation was inhibited in immature DC, whereas apoptotic phenomena were favored in mature DC and consequently in responder T cells. Defective ligation of CD40 by CD40L (CD154) was responsible for CD95-mediated and spontaneous apoptosis of DC as well as for a failure of their maturation process. These findings indicate that lack of activation of CD40 on DC by CD40L-defective anergic cells might be the primary event involved in T cell suppression and support the role of CD40 signaling in regulating both activation and survival of DC.     

Differential regulation of maturation and apoptosis of human monocyte-derived dendritic cells mediated by MHC class II

Antigen-driven interaction of dendritic cells (DC) with CD4(+) T(h) cells results in the exchange of bidirectional activating signals. Cross-linking of TCR by MHC class II-bound antigen activates T(h) cells, resulting in their up-regulation of CD40 ligand. Here we show that MHC class II molecules, in addition to their passive role in DC-T(h) cell interaction, can also actively induce DC maturation. Cross-linking of MHC class II molecules on human monocyte-derived DC results in the up-regulation of the surface expression of CD83, CD80, CD86, CD54, CD1a and CD40 molecules, the typical DC maturation-associated markers. It also promotes a rapid homotypic aggregation of DC paralleled by the up-regulation of such adhesion molecules as VLA-4, tissue transglutaminase, CD54 and CD11c. The impact of MHC class II cross-linking upon DC was context dependent. The outcome of MHC class II signaling depends on the maturation status of DC. While the cross-linking of MHC class II on immature DC promoted their maturation, the dominant effect upon the DC that were previously matured was the induction of DC apoptosis. Our current observations indicate that, in addition to the previously reported negative impact of MHC class II-mediated signaling on DC function, it also promotes DC maturation, participating in the enhancement of DC stimulatory function. Importantly, MHC class II-induced DC maturation and apoptosis are mediated by different signaling pathways, sensitive to different sets of inhibitors. This opens the possibility of differential regulation of each of these events in immunotherapy.     

Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses

Vaccination with in vitro-generated dendritic cells (DC) that present tumor-associated antigens is a promising approach for immunotherapy of malignant tumors. For optimization of DC-based vaccination protocols, preclinical tumor models that mimic the clinical situation closely are highly desirable. Strong non-specific T cell activation was observed in experimental immunization of mice with syngeneic DC generated in standard FCS-supplemented culture medium. To avoid deviation of the immune response to FCS-derived antigens, a serum-free culture protocol for in vitro generation of murine DC from bone marrow progenitor cells was developed. In comparison to DC differentiated with FCS supplementation, DC generated under serum-free conditions (sfDC) have a more homogeneous phenotype with higher expression of IL-12 and the differentiation and activation markers CD11c, CD40, CD80, CD83, CD86, DEC-205, and MHC class II. Demonstration of strong uptake of protein and carbohydrate antigens and analysis of the in vivo migration behaviour of sfDC also indicated excellent APC function. Vaccination of mice with peptide-pulsed sfDC efficiently induced an antigen-specific T cell response as assessed by MHC tetramer staining, IFN-γ ELISPOT and in vivo cytotoxicity assay. sfDC may therefore represent a valuable tool to improve active tumor immunotherapy in animal models.     

Interleukin-10-induced T cell unresponsiveness can be reversed by dendritic cell stimulation

The secretion of immunosuppressive factors like interleukin-10 (IL-10), either by tumor cells or by tumor-infiltrating leukocytes, has been recognized as one of the mechanisms involved in tumor immunological escape and a serious obstacle for successful immunotherapy. Therefore, any therapeutic attempts aimed at inducing antitumor immunity in tumor-bearing hosts must overcome this immunosuppressive state. This study aimed to determine whether dendritic cell (DC) immunization, a promising approach to induce antitumor immunity, could break IL-10-induced anergic state in CD4+ T cells, essential cells in generating tumor-specific immunity. We found that the ability of DC to reverse IL-10-induced anergic state in human CD4+ T cells is dependent on the IL-10 concentration that T cells have been exposed to and the degree of DC maturation. The efficacy of mature DC in reversing T cell anergy can be mimicked by higher cell numbers of immature DC. In addition, activated T cells induced by DC stimulation are sensitive to IL-10 treatment. Collectively, our results suggest the use of mature DC and the necessity of antagonizing the action of tumor-derived IL-10 in immunotherapy of cancer with DC immunization.     

天花粉蛋白联合CD40信号激发的人MoDC介导Th2细胞分化的研究

探讨天花粉蛋白(Tk)联合CD40信号诱导人单核细胞来源DC(MoDC)的成熟活化及其介导Th2细胞分化的作用和机制。采用GM-CSF和IL-4联合方案体外诱导人MoDC,并利用鼠抗人CD40激发型单抗(5C11)刺激负载了Tk的DC制备成熟DC。采用免疫荧光标记和流式细胞术分析DC表型(CD80、CD83、CD86、CD14、PDL1)及其摄取FITC-Dextran的能力;ELISA法测定DC上清中IL-12p70的含量;胞内染色和流式细胞术检测经成熟DC活化的T细胞中CD4~+IFN-γ~+T和CD4~+IL-4~+T的比例。实验结果显示单独Tk不能刺激DC完全成熟,用Tk负载DC后必须联合外源性成熟刺激信号(如5C11),才能有效促进DC成熟,表现在CD80、CD83、CD86的上调表达,CD14下调表达,DC摄取FITC-Dextran的能力降低。与CD40信号单独诱导组相比,Tk联合CD40信号诱导的成熟DC表面PDL1分子呈下调表达,分泌IL-12的能力显著降低,明显提高T细胞中CD4~+IL-4~+T的比例。由此表明负载了Tk的成熟MoDC体外能有效介导Th2细胞分化。     

Interferon γ Stimulates Cellular Maturation of Dendritic Cell Line DC2.4 Leading to Induction of Efficient Cytotoxic T Cell Responses and Antitumor Immunity

Dendritic cells （DCs） are the most potent antigen-presenting cells （APCs） for the initiation of antigen （Ag）-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-α for 7-10 days and stimulated with lipopolysaccharide （LPS）. However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class Ⅱ and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli （IL-1β, IFN-γ, TNF-α CpG and LPS） in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-γ was able to stimulate the maturation of DC2.4 cells. The IFN-γ-activated ovalbumin （OVA）-pulsed DC2.4 cells have capacity to upregulate MHC class Ⅱ, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8^＋ T cell responses and antitumor immunity. Therefore, IFN-γ-activated immortal DC2.4 ceils may prove to be useful in the study of DC biology and antitumor immunity.     

Antigen loading of dendritic cells with whole tumor cell preparations

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.     

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.     

Chemokine programming dendritic cell antigen response: part II – programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype

In a companion article to this study,¹ the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre‐treatment of DCs with a specific ‘cocktail’ of select chemokines. Chemokine pre‐treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4⁺ T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre‐treated murine bone marrow‐derived DCs were examined in long‐term co‐culture with antigen‐specific CD4⁺ T cells to quantify how chemokine pre‐treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine‐treated DCs, OVA‐biased CD4⁺T‐cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon‐γ, interleukin‐1β, interleukin‐2 and interleukin‐10 in the CD4⁺ T cell : DC co‐culture (with or without chemokine pre‐treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC‐based vaccine efficiency.     

Differential capability for phagocytosis of apoptotic and necrotic leukemia cells by human peripheral blood dendritic cell subsets

CD11c+ dendritic cells (DC) and plasmacytoid DC (PDC) are the two major DC subsets in human peripheral blood. For the purpose of immunotherapy with DC, it is important to investigate the phagocytosis of killed tumor cells by different DC subsets. Using immature monocyte-derived DC (iMoDC) as reference, we have compared the ability of CD11c+ DC and PDC to phagocytose apoptotic and necrotic K562 leukemia cells. Freshly isolated CD11c+ DC phagocytosed apoptotic and necrotic K562 cells, whereas PDC did not show any evidence of uptake of dead cells. Blocking studies showed that CD36 is importantly involved in uptake of apoptotic and necrotic material. CD91 and CD11c were also involved. In addition, we found that beta5 integrin was expressed on CD11c+ DC but not in its classical association with alphaV. Uptake of apoptotic K562 cells by CD11c+ DC was increased following incubation with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, alone or in combination with transforming growth factor-beta1, to levels comparable with those observed for iMoDC. Phagocytosis of dead cellular material by the GM-CSF/IL-4-treated CD11c+ DC was largely restricted to a subset expressing low levels of human leukocyte antigen-DR and CD83. Thus, the relationship between phagocytosis of antigenic material and expression of maturation-related cell-surface molecules is similar for CD11c+ DC and MoDC. We conclude that CD11c+ DC in peripheral blood are precursor cells, which under the influence of cytokines, differentiate to cells with DC phenotype and function.