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Alexander Steinkasserer - One of the best experts on this subject based on the ideXlab platform.
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CD83 knockdown in monocyte derived dendritic cells by small interfering rna leads to a diminished t cell stimulation
Journal of Immunology, 2007Co-Authors: Alexander Prechtel, Nadine Turza, Alexandros A Theodoridis, Alexander SteinkassererAbstract:Mature human dendritic cells (mDCs) are the most powerful APCs known today, having the unique ability to induce primary immune responses. One of the best known surface markers for mDCs is the glycoprotein CD83, which is strongly up-regulated during maturation, together with costimulatory molecules such as CD80 and CD86. When CD83 surface expression was inhibited by interference with the messenger RNA export or by infection with certain viruses, DCs showed a dramatically reduced capability to induce T cell proliferation. However, in these cases side effects on other cellular functions cannot be excluded completely. In this study we present an efficient method to specifically influence CD83 surface expression by the use of RNA interference. We used small-interfering RNA targeted against CD83 and carefully evaluated an electroporation protocol for the delivery of the duplex into the cells. Furthermore, we identified freshly prepared immature DCs as the best target for the application of a CD83 knockdown and we were also able to achieve a long lasting silencing effect for this molecule. Finally, we were able to confirm that CD83 functions as an enhancer during the stimulation of T cells, significantly increases DC-mediated T cell proliferation, and goes hand in hand with clear changes in cytokine expression during T cell priming. These results were obtained for the first time without the use of agents that might cause unwanted side effects, such as low m.w. inhibitors or viruses. Therefore, this method presents a suitable way to influence DC biology.
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role of CD83 in the immunomodulation of dendritic cells
International Archives of Allergy and Immunology, 2002Co-Authors: Matthias Lechmann, Elisabeth Zinser, Antje Golka, Alexander SteinkassererAbstract:Glycoprotein CD83 is one of the best-known maturation markers for human dendritic cells (DCs). The fact that CD83 is strongly upregulated together with co-stimulatory molecules such as CD80 and CD86 d
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overexpression purification and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies
Protein Expression and Purification, 2002Co-Authors: Matthias Lechmann, Elisabeth Kremmer, Heinrich Sticht, Alexander SteinkassererAbstract:CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily. It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GST-hCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NMR and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.
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the extracellular domain of CD83 inhibits dendritic cell mediated t cell stimulation and binds to a ligand on dendritic cells
Journal of Experimental Medicine, 2001Co-Authors: Matthias Lechmann, Carl G Figdor, Gerold Schuler, Elisabeth Kremmer, Danielle J E B Krooshoop, Diana Dudziak, Christine Kuhnt, Alexander SteinkassererAbstract:CD83 is an immunoglobulin (Ig) superfamily member that is upregulated during the maturation of dendritic cells (DCs). It has been widely used as a marker for mature DCs, but its function is still unknown. To approach its potential functional role, we have expressed the extracellular Ig domain of human CD83 (hCD83ext) as a soluble protein. Using this tool we could show that immature as well as mature DCs bind to CD83. Since CD83 binds a ligand also expressed on immature DCs, which do not express CD83, indicates that binding is not a homophilic interaction. In addition we demonstrate that hCD83ext interferes with DC maturation downmodulating the expression of CD80 and CD83, while no phenotypical effects were observed on T cells. Finally, we show that hCD83ext inhibits DC-dependent allogeneic and peptide-specific T cell proliferation in a concentration dependent manner in vitro. This is the first report regarding functional aspects of CD83 and the binding of CD83 to DCs.
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human monocyte derived dendritic cells express functional p2x and p2y receptors as well as ecto nucleotidases
FEBS Letters, 1999Co-Authors: Susanne Berchtold, Alexandra Ogilvie, Cornelia Bogdan, Petra Muhlzurbes, Adaling Ogilvie, Gerold Schuler, Alexander SteinkassererAbstract:We investigated the expression and function of P2 receptors and ecto-nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT-PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA-2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor-α, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto-apyrase CD39 and the ecto-5′-nucleotidase CD73 as demonstrated by RT-PCR. Extracellular ATP was rapidly hydrolyzed by these ecto-enzymes as shown by separation of 3H-labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.
Yongan Zhang - One of the best experts on this subject based on the ideXlab platform.
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identification of another primordial cd80 86 molecule in rainbow trout insights into the origin and evolution of cd80 and cd86 in vertebrates
Developmental and Comparative Immunology, 2018Co-Authors: Xujie Zhang, Xiang Yang Zhang, Peng Wang, Yongan ZhangAbstract:Abstract In mammals, the binding of distinct costimulatory ligands CD80 and CD86 to their receptors is essential for optimal T cell activation. Previous studies have shown that only a single CD80/86 gene exists in rainbow trout (termed rtCD80/86A) and other teleost fish, suggesting that CD80 and CD86 arose by gene duplication in the tetrapod branch, after the separation of fish and tetrapods. However, in this study, another CD80/86 gene has been cloned from rainbow trout, termed rtCD80/86B. The sequence identity between trout CD80/86 is significantly higher than that between CD80 and CD86 in tetrapods, indicating that CD80 and CD86 underwent divergent evolution in vertebrates, especially in tetrapods. Gene synteny analyses showed that the CD80 and CD86 genes are closely located in the same chromosome in tetrapods. However, CD80/86 genes are located in two distinct chromosomes in rainbow trout and Atlantic salmon, suggesting that salmonid CD80/86 genes arose by the salmonid-specific whole-genome duplication (WGD) event. Expression analysis showed that rtCD80/86A was more abundant and inducible than rtCD80/86B in various tissues, indicating the important role of rtCD80/86A in trout immunity. Interestingly, we found that head kidney B cells showed higher expression level of rtCD80/86A and rtCD80/86B when compared with the other leukocytes, suggesting a potential role for trout B cells as antigen-presenting cells (APCs).
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conservation of structural and functional features in a primordial cd80 86 molecule from rainbow trout oncorhynchus mykiss a primitive teleost fish
Journal of Immunology, 2009Co-Authors: Yongan Zhang, Junichi Hikima, Scott E Lapatra, Yanping Luo, Oriol J SunyerAbstract:In mammals, interaction of CD28 with CD80 or CD86 molecules provides costimulatory signals for T cell activation that leads to increased IL-2 gene and protein expression by activated T cells. Thus far, CD80 and CD86 have been cloned and functionally characterized only in mammals and birds. To shed light into the evolution of CD80 and CD86, we have cloned and functionally characterized a rainbow trout (rt) molecule (rtCD80/86) that shows the highest degree of sequence conservation and phylogenetic relationship with CD80 and CD86 molecules. Moreover, its genomic organization was almost identical to that of human CD86. Rainbow trout possess one membrane-bound and two soluble CD80/86 transcripts, all of which are derived from the same rtCD80/86 gene. The membrane-bound form exhibited its highest degree of expression in lymphoid tissues, particularly on B cells. Incubation of trout leukocytes with LPS and bacteria leads to up-regulation of rtCD80/86 gene expression. Importantly, we show that trout and other teleost fish contain a single CD80/86 gene, thus suggesting that this gene may represent the ancestor from which CD80 and CD86 arose by gene duplication in more evolved species. To gain further insights into the function of rtCD80/86, we have identified and cloned trout IL-2 and have shown that recombinantly produced trout CD80/86 up-regulates the expression of IL-2 in trout blood leukocytes. Significantly, this finding indicates that the capacity to modulate IL-2 expression is a primordial function that has been conserved both in fish and mammalian CD80/CD86 molecules throughout 350 million years of evolution.
Thomas F Tedder - One of the best experts on this subject based on the ideXlab platform.
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human blood dendritic cells selectively express CD83 a member of the immunoglobulin superfamily
Journal of Immunology, 1995Co-Authors: Liangji Zhou, Thomas F TedderAbstract:Dendritic cells are potent APC that initiate primary T cell-dependent immune responses. The lack of lineage-associated cell surface Ags for human dendritic cells has made characterization of this lineage difficult. In this study, analysis of leukocyte subpopulations isolated from human blood revealed that circulating or cultured B and T cells, NK cells, and monocytes did not express CD83, whereas CD83+ cells were predominantly found in the dendritic cell-enriched metrizamide low density fraction of plastic nonadherent blood mononuclear cells. Blood CD83+ cells had a cellular morphology characteristic of dendritic cells and a cell surface phenotype that did not correlate with that of T cells, B cells, NK cells, or cells of the myelomonocytic lineage. Analysis of CD83+ cells with a panel of mAbs that identify 126 leukocyte cell surface Ags revealed the CD83+ cells to be a phenotypically homogeneous and unique population of cells that expressed the highest levels of MHC class II molecules when compared with other leukocyte lineages. CD83+ cells were also the most potent stimulator cells in an allogeneic MLR when compared with other leukocyte lineages. Functional analysis of CD83+ cells revealed that MHC class II, CD11a, CD40 and CD86 played functionally dominant roles, whereas CD80 contributed minimally to the specialized costimulatory activity of these potent APC. Thus, CD83 serves as a useful and specific marker for this unique population of human blood dendritic cells.
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new cd from the b cell section of the fifth international workshop on human leukocyte differentiation antigens
Leukemia & Lymphoma, 1994Co-Authors: Pablo Engel, Thomas F TedderAbstract:This review summaries the expression and the molecular and biochemical characteristics of eight new Clusters of Differentiation (CD79-CD86) established by the B cell Section during the Fifth International Workshop on Human Leukocyte Differentiation Antigens. CD79 monoclonal antibodies (mAb) identify the mbl (CD79 alpha) and B29 (CD79 beta) components of the surface immunoglobulin (Ig) receptor complex. CD80 (B7/BB-1) is a costimulatory molecule that serves as the ligand for two molecules expressed on T lymphocytes, CD28 and CTLA-4. CD81 (TAPA-1) and CD82 (R2) are new members of the tetra-spans family of transmembrane proteins, which include CD9, CD37, CD53 and CD63. These proteins are postulated to be involved in signal transduction. CD83 (HB15) is a marker for human interdigitating reticulum cells, circulating dendritic cells and Langerhans cells. CDw84 and CD85 are new B cell-associated molecules that are also expressed by monocytes. CD86 is a new B cell activation antigen.
Brian J Nickoloff - One of the best experts on this subject based on the ideXlab platform.
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psoriatic skin derived dendritic cell function is inhibited by exogenous il 10 differential modulation of b7 1 cd80 and b7 2 cd86 expression
Journal of Immunology, 1995Co-Authors: R S Mitra, T A Judge, Frank O Nestle, Laurence A Turka, Brian J NickoloffAbstract:Regulation of immune responses depends on interactions between APCs and T cells. Such cellular interactions are mediated by surface molecules including MHC class II Ags (DR) and CD28 ligands B7-1 (CD80) and B7-2 (CD86). Recent evidence indicates that the presence or absence of costimulatory molecules on APCs significantly influences the qualitative and quantitative nature of an immune response. In this report, we analyze two relevant cytokines in skin immunobiology, granulocyte-macrophage (GM)-CSF and IL-10, and demonstrate their effects on cultured dendritic cells obtained from dermis (DDCs) of normal skin and psoriatic lesions. For comparison, the effects on these professional APCs were contrasted with cultured blood-derived monocytes. Normal and psoriatic skin-derived DDCs express high levels of CD86 over CD80, and the overall hierarchy is DR > CD86 > CD80, whereas cultured monocytes express low and equivalent levels of CD80 and CD86. If Ab is added to GM-CSF at the initial period of cultivation, DDCs that emigrate have lower levels of CD86 without any detectable effect on CD80 or DR expression and display a reduced capacity to stimulate either superantigen-driven or alloantigen-responsive T cells. Conversely, by adding GM-CSF to monocytes, CD86 levels are enhanced. When IL-10 was added at the beginning of culture, DDCs had significantly lower levels of CD86, without any effect on CD80 or DR expression, and like anti-GM-CSF-treated cells, these DDCs had approximately a 50% reduction in their T cell-stimulating capacity. In contrast, when monocytes were treated identically with exogenously added IL-10, they retained their relatively low levels of CD80 and CD86 with no detectable change in APC function. Blocking studies of DDC:T cell interaction indicated that CD86 was more important than CD80. Thus, differential expression patterns and functional cytokine responses involving these APC populations may be relevant to skin disorders such as psoriasis, in which discordant patterns of CD28 ligand expression and disordered cytokine networks are present.
Peter S Linsley - One of the best experts on this subject based on the ideXlab platform.
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both extracellular immunoglobin like domains of cd80 contain residues critical for binding t cell surface receptors ctla 4 and cd28
Journal of Biological Chemistry, 1995Co-Authors: Robert J Peach, Joanne L Greene, Jurgen Bajorath, Joseph Roy Naemura, Gina Leytze, Alejandro Aruffo, Peter S LinsleyAbstract:Abstract The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC′C″ β-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED β-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC′C″ face of the IgV domain and the ABED face of the IgC domain.
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human b7 1 cd80 and b7 2 cd86 bind with similar avidities but distinct kinetics to cd28 and ctla 4 receptors
Immunity, 1994Co-Authors: Peter S Linsley, Joanne L Greene, William Brady, Jurgen Bajorath, Jeffrey A Ledbetter, Robert PeachAbstract:B7-0 or B7-2 (CD86) is a T cell costimulatory molecule that binds the same receptors (CD28 and CTLA-4) as B7-1 (CD80), but shares with it only approximately 25% sequence identity and is expressed earlier during an immune response. Here we show that human CD86 maintains similar (within approximately 2- to 3-fold) overall receptor binding and T cell costimulatory properties as CD80. However, CD80 and CD86 did not bind equivalently to CTLA-4: CD80 bound Y100A, a form of CTLA4lg with a mutation in the CDR3-like region, > 200-fold better than did CD86; inhibition of CD80-mediated cellular responses required approximately 100-fold lower CTLA4lg concentrations; and CD80-CTLA4lg complexes dissociated 5- to 8-fold more slowly, Thus, CD80 and CD86 utilize different binding determinants and have different kinetics of binding to CD28 and CTLA-4.