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  • 1
    Abstract: Dendritic cells (DCs) are key coordinators of the immune response, governing the choice between tolerance and immunity. DCs are professional antigen-presenting cells capable of presenting antigen on MHC molecules and priming CD4 and CD8 T-cell responses. They form a heterogeneous group of cells based on phenotype, location, and function. In this review, murine DCs will be discussed regarding their function with special emphasis on their tissue distribution. Recent findings on DC homeostasis during cancer progression will be presented. Finally, the developmental pathways leading to DC differentiation from their precursors will be summarized. Given the vital importance of immune system research, the gathering of clear, consistent, and informative protocols involving the study of dendritic cells is paramount. Bringing the popular first edition fully up to date, Dendritic Cell Protocols, Second Edition presents protocols from experts in the field that cover the basics and more complex forays into the exploration of DC development and function, both in mice and humans. The first section of the volume involving humans explores topics such as the isolation of blood DC subtypes, primary skin Langerhans cells, and the generation of gene-manipulated human DCs with the inclusion of more clinically relevant methods as well, while the second section involving rodent models delves into DC and precursor generation in vitro, isolation ex vivo, disease models, as well as DC functions and properties. Written in the highly successful Methods in Molecular Biology series style, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Dendritic Cell Protocols, Second Edition aims to become a bench-side handbook for both beginners and experts in the field of DC research and a long-term reference for some of the most popular methods put forward by those who lead the field.
    Type of Publication: Book chapter
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  • 2
    Keywords: PEPTIDE ; RECEPTOR ; CELLS ; EXPRESSION ; Germany ; PROTEIN ; DRUG ; MOLECULES ; LINES ; MICE ; COMPLEX ; murine ; primary ; ANTIGEN ; ANTIGENS ; BIOSYNTHESIS ; LYMPHOCYTES ; antigen presentation ; B-CELLS ; CLASS-II MOLECULES ; EXCHANGE ; H2-O ; HLA-DM ; HLA-DO ; IA MOLECULES ; INVARIANT CHAIN ; MHC MOLECULES ; MONOCLONAL- ANTIBODY ; PEPTIDE REPERTOIRE ; PEPTIDES ; T- CELLS
    Abstract: Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigenpresenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from A(b) molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor
    Type of Publication: Journal article published
    PubMed ID: 12645938
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  • 3
    Keywords: OPTIMIZATION ; PEPTIDE ; SPECTRA ; CELLS ; EXPRESSION ; INHIBITOR ; Germany ; PROTEIN ; PROTEINS ; MOLECULES ; MICE ; ACTIVATION ; COMPLEX ; COMPLEXES ; murine ; MEMBER ; MHC ; LYMPHOCYTES ; antigen presentation ; PEPTIDES ; STABILITY ; MHC class I ; DEGRADATION ; SUBUNITS ; TRANSLOCATION ; antigen processing ; CELL-LINE .220 ; HISTOCOMPATIBILITY COMPLEX ; LOADING COMPLEX ; MUTANT MICE ; NEWLY SYNTHESIZED PROTEINS
    Abstract: Tapasin is a member of the MHC class I loading complex where it bridges the TAP peptide transporter to class I molecules. The main role of tapasin is assumed to be the facilitation of peptide loading and optimization of the peptide cargo. Here, we describe another important function for tapasin. In tapasin- deficient (Tpn(-/-)) mice the absence of tapasin was found to have a dramatic effect on the stability of the TAP1/TAP2 heterodimeric peptide transporter. Steady-state expression of TAP protein was reduced more than 100-fold from about 3 x 10(4) TAP molecules per wild-type splenocyte to about 1 x 10(2) TAP per Tpn(-/-) splenocyte. Thus, a major function of murine tapasin appears to be the stabilization of TAR The low amount of TAP molecules in Tpn(-/-) lymphocytes is likely to contribute to the severe impairment of MHC class I expression. Surprisingly, activation of Tpn(-/-) lymphocytes yielded strongly enhanced class I expression comparable to wild-type levels, although TAP expression remained low and in the magnitude of several hundred molecules per cell. The high level of class I on activated Tpn(-/-) cells depended on peptides generated by the proteasome as indicated by blockade with the proteasome-specific inhibitor lactacystin. Lymphocyte activation induced an increase in ubiquitinated proteins that are cleaved into peptides by the proteasome. These findings suggest that in the presence of a large peptide pool in the cytosol, a small number of TAP transporters is sufficient to translocate enough peptides for high class I expression. However, these class I molecules were less stable than those of wild-type cells, indicating that tapasin is not only required for stabilization of TAP but also for optimization of the spectrum of bound peptides
    Type of Publication: Journal article published
    PubMed ID: 12594855
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  • 4
    Keywords: CELLS ; tumor ; carcinoma ; CELL ; COMBINATION ; Germany ; IN-VIVO ; MODEL ; THERAPY ; VIVO ; MOLECULES ; TISSUE ; TUMORS ; MICE ; ACTIVATION ; DNA ; OLIGODEOXYNUCLEOTIDES ; T cell ; T cells ; T-CELL ; T-CELLS ; TOLERANCE ; treatment ; MOLECULE ; MOUSE ; UP-REGULATION ; EFFICACY ; ADHESION ; CARCINOMAS ; STRATEGIES ; CD8(+) ; IMMUNE-RESPONSE ; vaccination ; MOUSE MODEL ; REJECTION ; DE-NOVO ; ADJUVANT ; EFFECTOR ; CROSS-PRESENTATION ; AGENT ; CELL CARCINOMA ; INFILTRATION ; T-CELL-ACTIVATION ; ANTIGEN-TRANSGENIC MICE ; ESTABLISHED TUMORS ; INDUCE REJECTION
    Abstract: In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4(+) and CD8(+) T cells. Indeed, this combination therapy p. roved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting
    Type of Publication: Journal article published
    PubMed ID: 15128765
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  • 5
    Keywords: CANCER ; Germany ; human ; IN-VIVO ; POPULATION ; PROTEIN ; PROTEINS ; MICE ; RESPONSES ; DNA ; INFECTION ; INDUCTION ; ANTIGEN ; ANTIGENS ; T-CELL ; E7 ; papillomavirus ; IMMUNE-RESPONSES ; antibodies ; antibody ; PARTICLES ; virus ; PROGRESSION ; VECTOR ; cervical intraepithelial neoplasia ; CERVICAL-CANCER ; FUSION ; human papillomavirus ; TYPE-16 ; VACCINES ; VIRUS-LIKE PARTICLES ; SURFACE ; MAMMALIAN-CELLS ; VACCINE ; STRATEGIES ; EPITOPES ; IMMUNE-RESPONSE ; IMMUNITY ; hepatitis B virus ; IMMUNOGENICITY ; TARGETS ; ADENOVIRUS ; FUSION PROTEIN ; T-cell response ; YOUNG-WOMEN ; IMMUNIZATION ; RECOMBINANT ; secretion ; ENHANCEMENT ; CARRIER ; CANCER PROGRESSION ; INDUCE ; HEPATITIS-B ; E7 PROTEINS
    Abstract: Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (10(6) infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen- specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8(+)-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein
    Type of Publication: Journal article published
    PubMed ID: 16188983
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  • 6
    Keywords: RECEPTOR ; EXPRESSION ; Germany ; MICE ; ACTIVATION ; RESPONSES ; DNA ; INDUCTION ; LYMPH-NODES ; oligonucleotides ; TOLL-LIKE RECEPTORS ; IMMUNE SUPPRESSION ; CATABOLISM ; LYMPH-NODE ; INDOLEAMINE 2,3-DIOXYGENASE ; BACTERIAL-DNA ; EXPANSION ; indoleamine 2,3-dioxygenase (IDO) ; local and systemic immunity ; MURINE DENDRITIC CELLS ; TRYPTOPHAN DEGRADATION
    Abstract: CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions
    Type of Publication: Journal article published
    PubMed ID: 16323249
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  • 7
    Keywords: RECEPTOR ; CELLS ; proliferation ; SURVIVAL ; CELL ; Germany ; IN-VIVO ; MODEL ; VIVO ; SUPPORT ; MICE ; TIME ; ACTIVATION ; LIGAND ; CD8(+) T-CELLS ; DENDRITIC CELLS ; chromosome ; MOUSE ; ACQUISITION ; PROMOTER ; EFFICIENT ; LYMPHOCYTES ; NATURAL-KILLER-CELLS ; NK cells ; MOUSE MODEL ; REVEALS ; IN-VIVO DEPLETION ; ORDER ; RE ; ABLATION ; LIFE ; dendritic cell ; immunology ; HOMEOSTASIS ; bacterial ; response ; NATURAL-KILLER-CELL ; BACTERIAL ARTIFICIAL CHROMOSOME ; KILLER-CELLS ; natural killer ; Natural killer cells ; NK-CELLS ; Diphtheria toxin receptor ; IL-15 ; INTERLEUKIN-15
    Abstract: Dendritic cells (DC) are known to support the activation of natural killer (NK) cells. However, little is known about the role for DC in NK-cell homeostasis. In order to investigate this question, a novel bacterial artificial chromosome transgenic mouse model was generated in which the diphtheria toxin receptor is expressed under the CD11c promoter. In these mice efficient DC depletion can be achieved over prolonged periods of time by multiple injections of diphtheria toxin. We show here that NK cells require DC for full acquisition of effector function in vivo in response to the bacterial-derived TLR ligand CpG. Importantly, DC were found to play an instrumental role for maintaining normal homeostasis of NK cells. This is achieved by IL-15 production by DC, which supports the homeostatic proliferation of NK cells
    Type of Publication: Journal article published
    PubMed ID: 18825750
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  • 8
    Keywords: CANCER CELLS ; CELLS ; GROWTH ; IN-VIVO ; KINASE ; PATHWAY ; METABOLISM ; MICE ; EPITHELIAL-CELLS ; PROTEIN-KINASE-C ; VITAMIN-D-RECEPTOR ; D-3 ; Intestine ; RESPONSE STEROID-BINDING ; TARGETED KNOCKOUT ; RECEPTOR/PDIA3/ERP57 ; 1.25D(3)-MARRS receptor/PDIA3/ERp57 ; Calcium absorption ; Phosphate absorption
    Abstract: We have used mice with a targeted knockout (KO) of the 1,25D(3)-MARRS receptor (ERp57/PDIA3) in intestine to study rapid responses to 1,25-dihydroxyvitamin D(3) [1,25D(3)] with regards to calcium or phosphate uptake. Western analyses indicated the presence of the 1,25D(3)-MARRS receptor in littermate (LM) mice, but not KO mice. Saturation analyses for [(3)H]1,25D(3) binding revealed comparable affinities for the hormone in lysates from female and male LM, but a reduced B(max) in females. Binding in lysates from KO mice was absent or severely reduced. Enterocytes from KO mice failed to respond to hormone with regard to either ion uptake, while cells from LM mice exhibited an increase in uptake. For calcium uptake, the protein kinase (PK) A pathway mediated the response to 1,25D(3). Enterocytes from LM mice responded to 1,25D(3) with enhanced PKA activity, while cells from KO mice did not, although both cell types responded to forskolin. Calcium transport in LM mice in vivo was greater than in KO mice. Cells from LM and KO mice had cell surface VDR; however, anti-VDR antibodies had no effect on ion uptake. Unlike chicks, the PKC pathway was not involved in phosphate uptake. As in chicks and rats, intestinal cells from adult male mice lost the ability to respond to 1,25D(3) with enhanced phosphate uptake, whereas in female mice, uptake in cells from adults was greater than that observed in young mice. Finally, when we tested phosphate uptake in vivo, we found that young female mice had a much greater rate of transport than young male mice.
    Type of Publication: Journal article published
    PubMed ID: 22546984
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  • 9
    Keywords: ANGIOGENESIS ; BONE-MARROW ; microenvironment ; inflammation ; INFILTRATION ; PANCREATIC-CANCER ; TUMORIGENESIS ; DESTRUCTION ; TUMOR-ASSOCIATED MACROPHAGES ; endothelium
    Abstract: Inefficient T cell migration is a major limitation of cancer immunotherapy. Targeted activation of the tumor microenvironment may overcome this barrier. We demonstrate that neoadjuvant local low-dose gamma irradiation (LDI) causes normalization of aberrant vasculature and efficient recruitment of tumor-specific T cells in human pancreatic carcinomas and T-cell-mediated tumor rejection and prolonged survival in otherwise immune refractory spontaneous and xenotransplant mouse tumor models. LDI (local or pre-adoptive-transfer) programs the differentiation of iNOS(+) M1 macrophages that orchestrate CTL recruitment into and killing within solid tumors through iNOS by inducing endothelial activation and the expression of TH1 chemokines and by suppressing the production of angiogenic, immunosuppressive, and tumor growth factors.
    Type of Publication: Journal article published
    PubMed ID: 24209604
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  • 10
    Keywords: ACTIVATION ; T-CELLS ; PROGRESSION ; P-SELECTIN ; PRECURSORS ; MULTIPLE-SCLEROSIS ; HEAT-STABLE ANTIGEN ; AUTOIMMUNE-DISEASES ; B-LYMPHOCYTES ; COSTIMULATORY MOLECULE
    Abstract: CD24 is an extensively glycosylated membrane protein that is linked to the membrane via a glycosyl-phosphatidylinositol (GPI)-anchor. In mice, CD24 is expressed by hematopoietic and non-hematopoietic cells. CD24-/- mice do not have gross immunological defects, but detailed analysis revealed strongly reduced responses in an experimental autoimmune encephalomyelitis (EAE) model and a massive proliferation of T cells under lymphopenic conditions. It was also demonstrated that preB cells from CD24-/- mice are impaired in alpha4-integrin-mediated cell binding. Here we report that CD24-/- mice have strongly reduced numbers of leukocytes in the colon compared to wildtype mice. The reduction comprized all subpopulations. Leukocyte counts in spleen, mesenteric lymph nodes or small intestine were not significantly different. We find that beside leukocytes, CD24 is widely expressed in EpCAM+ epithelial and CD31+ endothelial cells of colon and small intestine. However, in CD24-/- mice the number of CD31+ endothelial cells in colons was strongly reduced and the number of epithelial cells was augmented. Leukocyte transfer experiments provided evidence that the CD24 status of recipient mice, rather than of the transferred cells, is crucial for leukocyte recruitment to the colon. We hypothesize that CD24 on colonic epithelial and endothelial cells is required for the retention and positioning of leukocytes most likely by affecting integrin function.
    Type of Publication: Journal article published
    PubMed ID: 24956310
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