Dendritic cell-based cancer immunotherapy targeting MUC-1

Vaccination therapy using dendritic cells (DC) as antigen presenting cells (APC) has shown significant promise in laboratory and animal studies as a potential treatment for malignant diseases. Pulsing of autologous DCs with tumor-associated antigens (TAA) is a method often used for antigen delivery and choice of suitable antigens plays an important role in designing an effective vaccine. We identified two HLA-A2 binding novel 9-mer peptides of the TAA MUC1, which is overexpressed on various hematological and epithelial malignancies. Cytotoxic T cells generated after pulsing DC with these peptides were able to induce lysis of tumor cells expressing MUC1 in an antigen-specific and HLA-restricted fashion. Within two clinical studies, we demonstrated that vaccination of patients with advanced cancer using DCs pulsed with MUC1 derived peptides is well tolerated without serious side effects and can induce immunological responses. Of 20 patients with metastatic renal cell carcinoma, 6 patients showed regression of metastases with 3 objective responses (1 CR, 2 PR). Furthermore, we found that in patients responding to treatment T cell responses for antigens not used for treatment occurred suggesting that antigen spreading in vivo might be a possible mechanism of mediating antitumor effects. These results demonstrate that immunotherapy in patients with advanced malignancies using autologous DCs pulsed with MUC1 derived peptides can induce immunological and clinical responses. However, further clinical studies are needed to identify the most potent treatment regimen that can consistently mediate an antitumor immune response in vivo. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004.     

Breast carcinoma cell lysate-pulsed dendritic cells cross-prime MUC1-specific CD8+ T cells identified by peptide-MHC-class-I tetramers

For cancer immunotherapy the loading of dendritic cells (DCs) with whole tumor cell lysate preparations represents a simple and promising approach for presentation of tumor-associated antigens (TAAs), avoiding the disadvantages of HLA-matching and definition of TAAs. The aim of this study was to investigate whether lysate-pulsed DCs efficiently cross-prime CD8+ T cells and induce a strong T(H)1 cell response, as compared to DCs pulsed with specific peptides (FLU M1 and Melan-A/Mart-1). As a model system breast carcinoma cell lysate from either MCF-7 or MDA-MB-231 cell lines (both HLA-A*0201+) expressing the TAA MUC1 were selected. Both cell lines expressed MUC1, the epithelial mucin, which is a large molecular weight O-glycosylated protein expressed in the majority of breast, ovarian, and other epithelial malignancies and is under evaluation as a target antigen in cancer immunotherapy. We developed a simple lysate preparation method to solubilize all cell proteins without degradation. For loading of monocyte-derived dendritic cells, 100 microgmL(-1) of breast carcinoma cell lysate was used, accompanied by an adjuvant consisting of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin-E2. T cells were co-cultivated with lysate or peptide pulsed DCs and were restimulated weekly. Before cultivation, and after the 3rd stimulation, tetramer frequencies for the MUC1 epitopes M1.2 and F7 as well as for the FLU M1 and Melan-A/Mart-1 epitopes were determined. After stimulation with lysate, higher frequencies for M1.2-specific T cells were observed compared with the F7 epitope. Furthermore, we found expansion factors for M1.2-specific T cells that had been stimulated with MCF-7 lysate-pulsed DCs of up to 43-fold. The analysis of typical T(H)1/T(H)2 cytokines (IFN-gamma, TNF-alpha, IL-12p70, IL-2, IL-4, IL-5, and IL-10) revealed a strong T(H)1 response. These results provide evidence for a strong T(H)1 polarization and cross-priming of MUC1-specific CD8+ T cells and demonstrate the feasibility of using lysate-pulsed dendritic cells in breast cancer immunotherapy.     

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

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

MUC1 in human and murine mammary carcinoma cells decreases the expression of core 2 β1,6-N-acetylglucosaminyltransferase and β-galactoside α2,3-sialyltransferase

A good correlation between the expression of mucin1 (MUC1) and T antigen was found in breast cancer tumors and breast cancer cell lines, especially after treatment with neuraminidase. The association between the appearance of T antigen and the overexpression of MUC1 was further confirmed by transfecting MDA-MB-231 cells and murine 4T1 mammary carcinoma cells with cDNA for MUC1 and using an RNAi approach to inhibit the expression of MUC1 gene in T47D cells. Furthermore, we discovered that in 4T1 cells which express the sialyl Le(X) antigen, overexpression of MUC1 caused not only appearance of T antigen, but also loss of the sialyl Le(X) structure. As the observed changes in O-glycan synthesis can be associated with changes in the expression of specific glycosyltransferases, core 1 β1,3-galactosyltransferase, core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) and β-galactoside α2,3-sialyltransferase (ST3Gal I), we studied their expression in parental, vector-transfected and MUC1-transfected MDA-MB-231 and 4T1 cells as well as T47D cells transduced with small hairpin RNA targeted MUC1 mRNA. It was found that the expression of C2GnT1 and ST3Gal I is highly decreased in MUC1-expressing MDA-MB-231 and 4T1 cells and increased in T47D cells with suppressed expression of MUC1. Therefore, we found that changes in the structure of O-linked oligosaccharides, resulting in the occurrence of T antigen, are at least partially associated with MUC1 overexpression which down-regulates the expression of C2GnT1 and ST3Gal I. We showed also that the overexpression of MUC1 in 4T1 cells changes their adhesive properties, as MUC1-expressing cells do not adhere to E-selectin, but bind galectin-3.     

Mucin-induced apoptosis of monocyte-derived dendritic cells during maturation

Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.     

Human tumor antigen MUC1 is chemotactic for immature dendritic cells and elicits maturation but does not promote Th1 type immunity

The immunostimulatory outcome of the interactions of many pathogens with dendritic cells (DCs) has been well characterized. There are many fewer examples of similar interactions between DCs and self-molecules, especially the abnormal self-proteins such as many tumor Ags, and their effects on DC function and the immune response. We show that human epithelial cell Ag MUC1 mucin is recognized in its aberrantly glycosylated form on tumor cells by immature human myeloid DCs as both a chemoattractant (through its polypeptide core) and a maturation and activation signal (through its carbohydrate moieties). On encounter with MUC1, similar to the encounter with LPS, immature DCs increase cell surface expression of CD80, CD86, CD40, and CD83 molecules and the production of IL-6 and TNF-alpha cytokines but fail to make IL-12. When these DCs are cocultured with allogeneic CD4+ T cells, they induce production of IL-13 and IL-5 and lower levels of IL-2, thus failing to induce a type 1 response. Our data suggest that, in vivo in cancer patients, MUC1 attracts immature DCs to the tumor through chemotaxis and subverts their function by negatively affecting their ability to stimulate type 1 helper T cell responses important for tumor rejection.     

Expression of the Thomsen-Friedenreich antigen and of its putative carrier protein mucin 1 in the human placenta and in trophoblast cells in vitro

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.     

Virology- and immunology-based gene therapy for cancer

Current strategies for cancer gene therapy consist mainly of direct inhibition of tumor cell growth and activation of systemic host defense mechanisms. Conventional chemotherapy and radiotherapy, even considered to be temporally suppressing tumor growth, suppress immune responses; therefore, we examined potential clinical feasibility of virus-mediated tumor destruction, which can rather enhance immunity. We showed that human tumors were more susceptible to adenoviruses (Ad) in which the E1A expression was controlled by a putative tumor promoter than normal cells, and that a replication of the Ad was greater in tumor cells than in normal cells. We also demonstrated that the intratumoral injection of the Ad bearing a tumor promoter inhibited the subsequent tumor growth in vivo. The E1A expression was detected in the tumors injected with the Ad but not in non-tumorous tissues of the same mice. The Ad modified to show the regulated E1A expression is thereby oncolytic in nature. Antitumor immune responses are initiated after the acquisition of putative tumor antigen(s) by dendritic cells (DCs); therefore, enhanced antigen presentation is a crucial step for the early phase of cell-mediated immunity. Destruction of tumors can release the tumor antigens and DCs come to recognize them thereafter. We found that the stimulation of Fas expressed on DCs with Fas ligand (FasL) did not induce apoptosis of DCs but rather enhanced the antigen presentation. Activation of DCs induced production of a number of cytokines, and we showed that the interleukin-12 family secreted from tumors could induce systemic antitumor immunity. We presume that the administration of oncolytic Ad, which can destroy local tumors and subsequently make the putative tumor antigen(s) released from the tumors, stimulation of DCs with the Fas/FasL signal pathway and secretion of DCs-derived cytokines coordinately produce synergistic antitumor effects and that a combinatory application of these procedures can be a possible therapeutic strategy for cancer treatment.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer” held in Shenzhen, China, on 9–11 December 2005.     

In vivo ablation of CD11c-positive dendritic cells increases susceptibility to herpes simplex virus type 1 infection and diminishes NK and T-cell responses

The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.     

Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

Interaction of mouse dendritic cells and malaria-infected erythrocytes: uptake, maturation, and antigen presentation

Consistent with their seminal role in detecting infection, both mouse bone marrow-derived and splenic CD11c+ dendritic cells (DCs) exhibited higher levels of uptake of Plasmodium chabaudi-parasitized RBCs (pRBCs) than of noninfected RBCs (nRBCs) as determined by our newly developed flow cytometric technique using the dye CFSE to label RBCs before coculture with DCs. To confirm that expression of CFSE by CD11c+ cells following coculture with CFSE-labeled pRBCs represents internalization of pRBC by DCs, we showed colocalization of CFSE-labeled pRBCs and PE-labeled CD11c+ DCs by confocal fluorescence microscopy. Treatment of DCs with cytochalasin D significantly inhibited the uptake of pRBCs, demonstrating that uptake is an actin-dependent phagocytic process. The uptake of pRBCs by splenic CD11c+ DCs was significantly enhanced after infection in vivo and was associated with the induction of DC maturation, IL-12 production, and stimulation of CD4+ T cell proliferation and IFN-gamma production. These results suggest that DCs selectively phagocytose pRBCs and present pRBC-derived Ags to CD4+ T cells, thereby promoting development of protective Th1-dependent immune responses to blood-stage malaria infection.     

Unique type I interferon responses determine the functional fate of migratory lung dendritic cells during influenza virus infection

Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103(+) DCs and CD11b(high) DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103(+) DCs allow the virus to replicate to significantly higher levels than do the CD11b(high) DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11b(high) DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103(+) DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11b(high) DCs. The attenuated IFNAR signaling by CD103(+) DCs correlates with their described superior antigen presentation capacity for na?ve CD8(+) T cells when compared to CD11b(high) DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The "interferon-resistant" CD103(+) DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination.     

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

Bordetella Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation,Migration and T Cell Stimulatory Capacity of Dendritic Cells

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.     

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.     

Costimulation of chemokine receptor signaling by matrix metalloproteinase-9 mediates enhanced migration of IFN-alpha dendritic cells

Type I IFNs induce differentiation of dendritic cells (DCs) with potent Ag-presenting capacity, termed IFN-alpha DCs, that have been implicated in the pathogenesis of systemic lupus erythematosus. In this study, we found that IFN-alpha DCs exhibit enhanced migration across the extracellular matrix (ECM) in response to chemokines CCL3 and CCL5 that recruit DCs to inflammatory sites, but not the lymphoid-homing chemokine CCL21. IFN-alpha DCs expressed elevated matrix metalloproteinase-9 (MMP-9), which mediated increased migration across ECM. Unexpectedly, MMP-9 and its cell surface receptors CD11b and CD44 were required for enhanced CCL5-induced chemotaxis even in the absence of a matrix barrier. MMP-9, CD11b, and CD44 selectively modulated CCL5-dependent activation of JNK that was required for enhanced chemotactic responses. These results establish the migratory phenotype of IFN-alpha DCs and identify an important role for costimulation of chemotactic responses by synergistic activation of JNK. Thus, cell motility is regulated by integrating signaling inputs from chemokine receptors and molecules such as MMP-9, CD11b, and CD44 that also mediate cell interactions with inflammatory factors and ECM.     

Synthetic glycopeptides for the development of tumour-selective vaccines.

Based on structural information reported for the tumour-associated epithelial mucin MUC1, glycopeptides have been synthesized which contain tumour-associated saccharide antigens. such as the Thomsen-Friedenreich (T), TN or sialyl TN antigen. in combination with peptide sequences of the tandem repeat region of MUC1. Solid-phase syntheses have been carried out using N-Fmoc protected O-glycosyl serine and threonine building blocks and an allylic anchor which is stable to basic and acidic conditions, but can be cleaved under neutral conditions in a palladium(0)-catalysed allyl transfer reaction. In addition. a (2-3)sialyl T antigen threonine building block was prepared by a chemoenzymatic strategy and used in the synthesis of an N-terminal glycopeptide antigen of leukosialin (CD43). The proliferation of cytotoxic T cells could be induced using a construct consisting of a MUC1-glycopeptide antigen and a T cell epitope.     

A high-affinity T-helper epitope enhances peptide-pulsed dendritic cell-based vaccine

The NV epitope, a dominant helper determinant from the circumsporozoite antigen of Plasmodium falciparum, is strongly immunogenic and can provide help for cytotoxic T-lymphocyte (CTL) activation. In this study, we evaluated whether the addition of NV peptide can augment the efficacy of peptide-pulsed dendritic cell (DC) immunization in vivo. Using B16 melanoma as tumor model, we demonstrated that DCs pulsed with both NV and gp100 (a melanoma-specific antigen) peptide enhanced immune priming and protection from tumor challenge in vivo. Further, we showed the mechanisms of the NV epitope that help CTL activation; MHC-II-restricted NV peptide induced dramatically more effective helper cells, with a higher level of CD40L expression and IFN-γ production, which, in turn, more effectively conditioned DCs for CTL activation. The improved helper cells also induced greater IL-12 production by DCs, accounting for the reciprocal T-helper polarization to Th1, and increased the expression of costimulatory molecules. Collectively, these findings demonstrate that NV peptide in addition to tumor antigen-pulsed DC immunizations augment helper cell activation, which in turn promotes maturation of DC, and enhance in vivo antitumor activity.