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1
Howarth, Mark. "The presentation of suboptimal peptides by MHC Class I". Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398092.
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Springer, Sebastian Hartmut. "The biochemistry of antigen presentation". Thesis, University of Oxford, 1996. http://ora.ox.ac.uk/objects/uuid:34d1afd2-fafc-4732-8e43-e00dcd8460d1.
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This thesis describes studies on the binding of peptides to the murine major histocompatibility complex (MHC) class I molecule H-2Db (Db). The expression of the recombinant soluble Db molecule in Chinese hamster ovary cells and its subsequent purification by nickel affinity chromatography, gel filtration, and preparative native isoelectric focusing are reported. The product is the correct molecule, homogeneous, a dimer of dimers, and free of endogenous peptide. A novel binding assay based on the enhancement of natural tryptophan fluorescence by the binding of peptide is introduced. This assay is used to determine melting curves of the empty and peptide-loaded protein, and to measure association rate constants by stopped-flow fluorescence spectroscopy. Radioligand binding measurements of equilibrium as well as association and dissociation rate constants and their temperature dependence are reported. In agreement with earlier observations, the ratio of association and dissociation rate constants is much larger than the equilibrium association constant. Fluorescence anisotropy decay spectroscopy gives evidence for conformational alterations in the Db molecule upon peptide binding. The data, possible errors and ways to avoid them, and mathematical models of binding are discussed to obtain an overall picture of the binding process.
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Winchester, Christopher Charles. "The roles of Hsp70 proteins in antigen processing and presentation". Thesis, University of Oxford, 1997. http://ora.ox.ac.uk/objects/uuid:567dff45-08ce-43b4-b011-d08afea42f76.
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The ability of members of the hsp70 family to bind to peptides in vivo and in vitro suggests that they may be involved in the processing of antigens for binding to Major Histocompatibility (MHC) class I and/or class n molecules. The aims of this thesis have been to provide evidence for the involvement of hsp70s in antigen processing and to characterise the binding of peptides by hspTOs by structural and functional studies. Firstly, the peptide-binding domains of two hsp70s, hsp70hom and PBP74, were expressed in isolation from the rest of the molecule for structure determination. Both of these hsp70s were implicated in antigen processing: hsp70hom in the class I pathway, due to its cytoplasmic localisation and constitutive expression, and the presence of its gene in the MHC; and PBP74 in the class n pathway because published work indicated that it was localised to endosomes and that antibodies against it inhibited antigen processing. The expression and purification of both peptide-binding domains was very successful, and one dimensional NMR experiments indicated that they were folded. However, it was not possible to determine their structures by NMR spectroscopy or X-ray crystallography because they aggregated in solution at high concentrations. Instead, the structure of the C-terminal region of hsp70hom, which includes its peptidebinding domain, was modelled based on the known structure of the equivalent portion of dnaK, the hsp70 of E.coli. The structure of hsp70hom is predicted to be very similar to that of dnaK, and modelling studies suggest that it is likely to bind peptides in a closely related fashion. The modelling of complexes between hsp70hom and two peptides suggest that the peptide-binding groove is very versatile, accounting for the broad peptide-binding specificity of hsp70s. The interactions of hsp70hom and PB74 with peptides were investigated using plate binding assays and isothermattitration calorimetry. A biotinylated peptide bound to the peptide-binding domain of hsp70hom, immobilised in plastic wells, with a Kd of <25 μM, which is within the range of Kds reported for other hsp70-peptide complexes (0.1-100 μM). In solution, isothermal titration calorimetry showed that the binding of peptides to the peptide-binding domains of hsp70hom and PBP74 was likely to be entropically rather than enthalpically driven, and, therefore, the interactions involved are likely to be predominantly hydrophobic. Secondly, PBP74, an hsp70 thought to be involved in the class II antigen processing pathway in endosomes, was localised by immunofluorescence microscopy. It was shown to be a mitochondrial protein, and is, therefore, unlikely to be involved in antigen processing. The presence of other members of the hsp70 family in lysosomes purified from a B cell line by Percoll density gradient centrifugation was investigated using antibodies that reacted with many Afferent members of the hsp70 family. No hsp70s were detected in these late endocytic compartments, even after heat shock or serum starvation. However, the presence of an hsp70 in endosomes, or of a member of this family not detected by the antibodies used, in lysosomes, cannot be ruled out. A third approach investigated the induction of the three hsp70 genes found in the MHC by four cytokines. The hsp70-l and hsp70-2 genes are induced at the mRNA level by IFN-γ and IL- 1, while TNF induces hsp70-2 alone. This data supports a role for the heat-inducible hsp70 in MHC class I antigen processing, as it appears to be coregulated with known members of this antigen processing pathway. The expression of hsp70hom was unaffected by any of the four cytokines examined. In addition, the mitochondrial hsp70 (which is not encoded in the MHC) appears to be induced by IFN-γ at the protein level. The research presented in this thesis provides a greater understanding of the peptide-binding properties of two hsp70s. Further work is necessary to show conclusively whether any of the hsp70s is involved in antigen processing.
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Farfán, Arribas Diego José. "On the Source of Peptides for Major Histocompatibility Class I Antigen Presentation: A Dissertation". eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/589.
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Peptides generated from cellular protein degradation via the ubiquitin-proteasome pathway are presented on MHC class I as a means for the immune system to monitor polypeptides being synthesized by cells. For CD8 + T cells to prevent the spread of an incipient infection, it appears essential they should be able to sense foreign polypeptides being synthesized as soon as possible. A prompt detection of viral proteins is of great importance for the success of an adaptive immune response. Defective ribosomal products (DRiPs) have been postulated as a preferential source which would allow for a rapid presentation of peptides derived from the degradation of all newly synthesized proteins. Although this hypothesis is intellectually appealing there is lack of experimental data supporting a mechanism that would prioritize presentation from DRiPs. In this dissertation I describe a series of experiments that probe the DRiPs hypothesis by assessing the contribution to class I presentation of model epitopes derived from DRiPs or from functional proteins. The results show that even at the early stages after mRNA synthesis DRiPs do not account for a significant fraction of the class I presented peptides. These observations suggest that the currently widespread model whereby a mechanism exists which selectively allows for DRiPs to preferentially contribute to class I antigen presentation, is incorrect. Rather, properly folded functional proteins can significantly contribute to class I antigen presentation as they are normally turned over by the ubiquitin-proteasome pathway.
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Lenz, Laurel L. "Presentation of formylated bacterial peptides to cytotoxic T cells by an MHC class Ib molecule /". Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/8339.
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Thomas, Lawrence James. "Proteolytic Cleavages of Molecules Involved in Antigen Processing and Presentation: A Thesis". eScholarship@UMMS, 1989. https://escholarship.umassmed.edu/gsbs_diss/97.
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The overall goal of my thesis research was to understand better the mechanisms that control antigen processing and presentation by class II MHC molecules. Towards this goal I investigated ways in which the physical structure and post-translational modifications of the class II MHC alpha and beta chains and associated molecules might serve to regulate antigen processing and presentation. Specifically, I investigated (1) a hypothesis that Ii might aid binding of foreign antigenic peptides to the class II MHC foreign antigen binding site (desetope), and the application of this hypothesis to the prediction of class II-presented peptides; (2) the proteolytic cleavage of Ii to p25; (3) the proteolytic cleavage of the class II MHC alpha and beta chains, and (4) the phosphorylation of Iiand the alpha and beta chains.
In exploring the hypothesis that amphipathic alpha helical peptides digested from foreign antigen, bind to the class II MHC desetope, to be presented to T cell receptors, we found such an extended, amphipathic helix in Ii (Phe146-Val164). A hypothesis was developed that this amphipathic alpha helix of Ii bound to the desetope of class II MHC molecules, and remained there from time of synthesis until catalyzing the charging of the desetope with a foreign peptide. This region of Iicould then be considered to be the prototypic T cell-presented peptide and the "strip-of-helix" algorithm was developed to search the sequences of proteins for similar amphipathic alpha helices. Such peptides might bind to the class II MHC desetope and have a high probability to be presented to the T cell.
The strip-of-helix algorithm calculated the mean hydrophobicity (from Kyte-Doolittle values; Kyte and Doolittle, 1982) of sets of amino acids in axial strips down sides of helices for 3 to 6 turns, at positions n, n+4, n+7, n+11, n+14, and n+18. Peptides correlating well with T cell responsiveness had: (1) 12 to 19 amino acids (4-6 turns of an alpha helix), (2) a strip with highly hydrophobic residues, (3) adjacent, moderately hydrophilic strips, and (4) no prolines to break the helix. This algorithm predicted 10 of 12 T cell-presented peptides in 7 well-studied proteins.
In a study of the post-translational modifications of Ii, an early proteolytic pathway of the destruction of Ii, resulting in the generation of p25, was described. This 25,000 dalton protein, seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii, was shown to be a C-termina1 fragment of a high mannose form of Ii. The evidence for this conclusion includes the following results. [35S]methionine-1abe1ed Ii and associated molecules were immunoprecipitated, denatured, resolubi1ized and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-termina1 peptides of Ii (183-193 and 192-211), but not an antiserum to an N-termina1 peptide (12-28), immunoprecipitated p25. A monoclonal antibody (mAb) to Ii immunoprecipitated [35S]methionine-1abe1ed p25 but not [35S]cysteine-1abe1ed p25, consistent with the loss of a portion of Ii containing the only cysteine in Ii, Cys28. [35S]methionine pulse-chase labeling demonstrated the maximal appearance of p25 at 20-40 min chase times. p25 molecules were reduced to about 10.5 kD by treatment with endoglycosidases F and H. p25 was, therefore, generated from a high mannose form of Ii in the ER or cis-Golgi. This finding could either implicate that site for class II MHC desetope charging with foreign peptides or reflect a mechanism for degradation of "excess" Ii molecules in the ER. Digestion of class II MHC antigen-Ii complexes with various proteases yielded fragments, migrating at and near p25 in 2-D electrophoretic gels, which were relatively resistant to further digestion. This observation was consistent with the presence of relatively protease-resistant secondary structures (domains) and a relatively protease-sensitive (IgG hinge-like) region in Iinear its insertion into the membrane.
In a study of the post-translational modifications of the class II MHC alpha and beta chains, well conserved pairs of basic amino acids in the sequences of these molecules were observed. It was hypothesized these could be sites for proteolytic cleavage, as precedented in other systems (i.e.proinsulin processing). These potential cleavage sites fall in significant locations with respect to the deduced structure of the class II MHC desetope, supporting the hypothesis that these cleavages might either aid or destroy antigen presenting functions. To test this hypothesis we looked for remnant polypeptides of the alpha and beta chains. Polypeptides were observed in gels of immunoprecipitated class II MHC complexes. To identify if such polypeptides were derived from the alpha and beta chains, immunoblotting to electrotransferred polypeptides was attempted, with antisera made to synthesized peptides that mimicked eight regions of the alpha and beta chains. These antisera were produced and characterized by dot blotting, ELISA, western blotting, and immunoprecipitation of native and denatured material. One antiserum, to an alpha chain peptide (77-88), blotted to a polypeptide immunoprecipitated by anti-class II MHC antiserum. This observation supported the hypothesis that the alpha and beta chains undergo proteolytic cleavages, possibly in the control of antigen presentation.
It was also demonstrated that Ii and the alpha and beta chains can be phosphorylated under varying culture conditions, but this project was not pursued.
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Parshotam, L. E. "Dynamic modelling of the processing of peptides for presentation on major histocompatibility complex class I proteins". Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1559176/.
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Antigen presentation is broadly implicated in disease and represents an important target for prophylactic and therapeutic treatments. A better understanding of the components of this system is fundamental to our understanding of disease path- ways and to treatment design. This thesis focuses on modelling the processing of peptides by enzymes in the cytosol and in the endoplasmic reticulum (ER) in the context of major histocompatibility complex class I (MHC) antigen presentation, and expounds upon current knowledge of the mechanistic details and specificity of both the proteasome and the endoplasmic reticulum aminopeptidase-1 (ERAP1). We use nonlinear ordinary differential equations to model the biochemical reaction pathways of amino-terminal peptide trimming by ERAP1 and distinguish parameter dependencies of two prevailing theories for the mechanism of ERAP1 trimming us- ing algebraic and numerical analysis. Importantly, we show that ERAP1 has a role in peptide optimisation when MHC acts as a template, but not when it trims free peptide using an internal molecular ruler. We present testable hypotheses that may elucidate the dominant trimming mechanism used by ERAP1 in vivo, which has been the subject of debate for more than 25 years. We show that all ERAP1 trimming mechanism hypotheses are able to predict the qualitative distribution of cell surface presentation of SIINFEKL derived from amino-terminally extended precursors. Notably, we find that the molecular ruler trimming mechanism is more robust than the MHC-as-template mechanism. Finally, we use neural networks to predict carboxyl-terminal cleavage by the proteasome, and demonstrate that we are able to distinguish between cleavage and non-cleavage sites on an unseen set of known peptide epitopes. Overall, this thesis contributes a more thorough quantitative and mechanistic understanding of the generation of peptides presented on MHC class I molecules.
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Eccleston, Ruth Charlotte. "A mechanistic model predicting cell surface presentation of peptides by MHC class I proteins, considering peptide competition, viral intracellular kinetics and host genotype factors". Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10038760/.
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Major histocompatability complex class I (MHC-I) proteins present short fragments of pathogenic or cancerous proteins (peptides) on the surface of infected cells for recognition by T lymphocytes which are stimulated upon recognition of foreign peptides. Due to the diversity of peptide sequences and the sequence-specificity of MHC-I alleles, being able to determine which peptides will be presented by which MHC-I alleles and in what proportion could be important for the development of vaccines and treatments based on the presented peptiodome. Machine learning tools, trained on experimental data, are widely used to predict immunogenic peptides. However they are unable to account for the impact the intracellular kinetics of the pathogenic or cancerous protein which will greatly influence the resultant peptidome. Here we describe a mechanistic model of peptide presentation, validated against experimental data, which accounts for intracellular peptide concentration, and can predict the relative cell surface presentation of competing peptides with varying affinities for MHC-I proteins. We demonstrate how combining this mechanistic model with the intracellular kinetics of HIV proteins can provide insight in to the experimentally reported immunogenicity of the viral protein Gag, and show how such a model can be used to predict the most abundant viral peptides presented on the cell surface. Similarly, we predict the HeLa cell peptidome and demonstrate how a simple metric can be used to approximate the abundance of a peptide based solely on protein synthesis and degradation, peptide-MHC affinity and proteasomal cleavage.
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McMurtrey, Curtis Paul. "Human leukocyte antigen class I presentation and immune recognition of West Nile virus peptide epitopes". Oklahoma City : [s.n.], 2009.
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Schirmacher, Anastasiya. "Modification of transmembrane peptides to probe SNARE-induced membrane fusion and cross-presentation of membrane-buried epitopes". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-1576-F.
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Lawand, Myriam. "Rôle des transporteurs de peptides dans la présentation antigénique par les cellules dendritiques". Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05T053.
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Les cellules dendritiques (DCs) sont des cellules spécialisées dans la présentation de l'antigène aux lymphocytes (CPAs), capables d'initier des réponses immunitaires adaptatives et ce sont également les acteurs majeurs de la présentation croisée des antigènes exogènes par le complexe majeur d’histocompatibilité de classe I (CMH-I). Les mécanismes moléculaires et cellulaires de la présentation croisée ont beaucoup été étudiés, mais des questions importantes restent à élucider. Notre laboratoire a précédemment montré que la pré-incubation à basse température des DCs déficientes pour TAP (transporter associated with antigen processing) normalise l’expression de molécules du CMH-I à la surface et la présentation croisée des antigènes phagocytés par une voie dépendante du protéasome, suggérant que les phagosomes pourraient être dotés d’un transporteur alternatif pour importer les peptides générés dans le cytosol par le protéasome. Comme la source de CMH-I chargés par cette voie reste incertaine, il est possible que le rôle de TAP dans la présentation croisée des antigènes phagocytés soit indirect et limité à fournir les molécules de CMH-I disponibles pour un chargement pendant leur recyclage. Ainsi, notre objectif était de déterminer le rôle exact de TAP dans le transport de peptides à l'intérieur du phagosome et d'évaluer le rôle de TAP-L (TAP-like), un transporteur lysosomal ATP-dépendant avec une fonction putative dans la présentation antigénique. Nous avons mis au point une technique de transport des peptides par cytométrie en flux (phagoFACS) et montré que TAP est présent dans les phagosomes des DCs et est capable de transporter des peptides ayant une forte affinité pour TAP d'une manière ATP-dépendante. Cette technique permet l'exclusion des phagosomes ayant un défaut d’intégrité membranaire, obtenus lors de la préparation des phagosomes, et apporte une preuve directe de l'accumulation du peptide à l'intérieur des phagosomes. Les paramètres affectant cette accumulation sont la maturation phagosomale et la présence de molécules CMH-I liant le peptide. De façon surprenante, en l'absence de TAP, le peptide SIINFEKL dérivé de l’ovalbumine ayant une affinité intermédiaire pour TAP est transporté de manière ATP-dépendante dans le phagosome. Ceci est cohérent avec l’hypothèse suggérant la présence d'un autre transporteur de peptide dans les phagosomes des DCs. Nous avons utilisé la même technique pour évaluer la fonction physiologique de TAP-L dans le transport de peptides et montré que TAP-L est présent dans les phagosomes et serait responsable de l’import de peptides dans ces vésicules. Nos résultats suggèrent aussi que TAP-L semble jouer un rôle dans la présentation croisée des antigènes phagocytés à basse température. Ceci a été observé dans des DCs déficientes pour TAP et TAP-L, indiquant que les deux transporteurs pourraient coopérer pour assurer l’import des peptides dans les phagosomes. Nous avons également pu démontrer un rôle de TAP-L dans la présentation de l’antigène par CMH-II. Ces résultats nous encouragent à explorer les mécanismes sous-jacents à ces fonctions pour comprendre la contribution relative de chaque transporteur de peptides dans la présentation antigéniqueDendritic cells (DCs) are potent antigen-presenting cells, capable of activating resting T cells and of initiating primary and stimulating memory immune responses. DCs can efficiently use internalized antigens for presentation by major histocompatibility class I (MHC-I) molecules: a phenomenon referred to as “cross-presentation.” Cross-presentation is important in priming of CD8+ T-cell responses to a variety of pathogens and to tumors as well as in immune tolerance to self and in autoimmunity. The molecular and cell biological mechanisms underlying cross-presentation have been studied intensively but important issues remain unclear. Our laboratory has previously shown that the pre-incubation of TAP-deficient DCs at low temperature normalized surface MHC-I expression and cross-presentation of phagocytosed antigens in a proteasome-dependent pathway. This suggested that phagosomes might harbor an alternative peptide transporter to import peptides generated by cytosolic proteasome complexes. As the source of MHC-I loaded in this pathway remains unclear, it is possible that the principal or partial role of TAP in proteasome-dependent cross-presentation of phagocytosed antigens is to provide recycling cell surface class I molecules. Our aim was to assess the exact role of TAP in peptide transport into phagosomes and to examine the role of the transporter associated with antigen processing-like (TAP-L), a lysosomal transporter with a putative function in antigen presentation. We have developed an assay of peptide transport using flow cytometry (phagoFACS) and shown that TAP is present in DC phagosomes and capable of transporting at least peptides with high affinity to TAP in an ATP-dependant manner. Using this assay, which allowed for eliminating background due to leaky vesicles, we were able to provide direct evidence of peptide accumulation inside phagosomes. ATP-dependant peptide accumulation inside phagosomes was affected by phagosomal maturation and by the presence of a peptide-binding MHC class I-molecule. Surprisingly, in the absence of TAP, another peptide transporter may be able to transport a peptide with intermediate affinity to TAP, namely the ovalbumin peptide SIINFEKL, in an ATP-dependant manner. We used the same technique to assess the function of TAP-L in peptide transport and found that TAP-L may be involved in peptide import into phagosomes. Additional results suggest that TAP-L plays a role in MHC-II presentation and cross-presentation of phagocytosed antigens at low temperature. The latter was shown in DCs lacking both transporters, suggesting that TAP and TAP-L might cooperate to ensure peptide import into phagosomes. The mechanisms underlying these functions should be explored to understand the relative contribution of each peptide transporter to antigen presentation
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Towne, Charles Fenton. "Analysis of the Role of Cytosolic Aminopeptidases in the Generation of MHC-Class I Presented Peptides: a Dissertation". eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/4.
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To detect viral infections and tumors, CD8 T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Cytosolic aminopeptidases such as leucine aminopeptidase (LAP), which is IFN-inducible, Bleomycin Hydrolase (BH), and puromycin-sensitive aminopeptidase (PSA) can trim precursor peptides to mature epitopes and have been thought to play an important role in antigen presentation. To examine the role of these aminopeptidases in generating MHC class I peptides in vivo, we generated mice deficient in LAP or PSA, as well as cell lines deficient in LAP, PSA, or BH. LAP mutant mice and cells are viable and grow normally, whereas PSA mutant mice are smaller than their wild-type and heterozygote littermates, are subfertile as adults, and are subviable as embryos.
The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full length antigen constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules, even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. Similarly, PSA deficient mice and BH deficient mice generate normal CTL responses to viral epitopes. These data demonstrate that LAP, BH, and PSA are not essential enzymes for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases in most cell types.
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Weimershaus, Mirjana Léona. "The Role of Insulin-Regulated Aminopeptidase in Cross-Presentation". Paris 5, 2011. http://www.theses.fr/2011PA05T036.
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L’aminopeptidase IRAP est localisée dans des endosomes du type de stockage dans divers types cellulaires. Dans les cellules dendritiques (CD), IRAP est impliquée dans l’élagage des ligands des molécules du CMH de classe I (CMH-I). L’absence d’IRAP provoque un défaut de la présentation d’antigènes internalisés sur le CMH-I (connue comme la “présentation croisée”). Par contre, IRAP n’est pas nécessaire dans les voies “classiques” de présentation antigéniques. Au vue de la colocalisation et interaction d’IRAP avec les CMH-I, nos observations suggèrent qu’IRAP marque un compartiment spécialisé à la dernière étape d’élagage et associations de peptides sur les CMH-I dans la présentation croisée. Nous avons pu mettre en évidence que ce compartiment, caractérisé par la présence de Rab14 et STX6, est fonctionnel dans les soustypes CD8+ et CD8- des CD. L’internalisation d’antigène est accompagné par le recrutement rapide des endosomes IRAP au phagosome. En l’absence d’IRAP la maturation phagosomale est accélérée, ce qui est manifesté par une acquisition plus rapide des marqueurs des endosomes tardifs et des lysosomes, ainsi qu’une dégradation plus importante de l’antigène. En conclusion, les endosomes d’IRAP ont au moins deux rôles distincts dans la physiologie des CD: l’elagage de peptides antigéniques et le ralentissement de la maturation phagosomale, un mécanisme connu pour être bénéfique pour la présentation croisée. La meilleure caractérisation des stimuli qui interviennent dans la mobilisation d’IRAP pourrait être pertinente pour mieux comprendre la régulation de différentes fonctions des CD, y compris la présentation antigéniqueInsulin-regulated aminopeptidase (IRAP) localizes to storage-type endosomes in many non-immune and immune cell types. In dendritic cells (DCs), IRAP functions as trimming peptidase to generate peptide ligands for MHC class I molecules (MHC-I). Absence of IRAP led to a defect in the presentation of internalized antigen on MHC-I (referred to as cross-presentation) in vitro and in vivo. Importantly, IRAP was not required for the “classical” presentation pathways (i. E. Presentation of extracellular antigens on MHC-II as well as presentation of intracellular antigens on MHC-I). Further supported by the colocalization and physical interaction of MHC-I with IRAP, these observations suggest that IRAP marks a cross-presentation compartment in which final trimming and loading on MHC-I can take place. We show that this compartment, marked by STX6 and Rab14, is functional in both CD8+ and CD8- DC subsets. In DCs, upon antigen internalization, IRAP vesicles are rapidly recruited to the phagosome. Additional to its function in antigen trimming, IRAP seems to regulate phagosome dynamics. In IRAP-deficient cells, phagosome maturation was accelerated, as shown by the rapid acquirement of late endosomal and lysosomal markers to the phagosome and increased antigen degradation. Thus, IRAP endosomes have at least two important roles in the DCs physiology: the trimming of antigenic peptides and the delay of phagosome maturation that is beneficial for cross-presentation. Understanding the stimuli that regulate IRAP mobilization, and possibly cross-presentation, could be relevant in order to understand if and how DCs control their different functions, including antigen presentation
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Schirmacher, Anastasiya [Verfasser]. "Modification of transmembrane peptides to probe SNARE-induced membrane fusion and cross-presentation of membrane-buried epitopes / Anastasiya Schirmacher". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1228364796/34.
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Serra, Vincent. "Influence de l'association C3b-toxine tétanique sur la production de peptides immunogéniques". Université Joseph Fourier (Grenoble), 1998. http://www.theses.fr/1998GRE10046.
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La proteine c3 est impliquee dans de nombreux mecanismes de defense de l'organisme contre des elements etrangers pathogenes. C3 participe a la reponse immune naturelle : element clef du syteme du complement, elle intervient dans l'elimination des complexes immuns. Cette proteine participe egalement a la reponse immune specifique : son influence dans l'activation b a clairement ete etablie. Un role dans l'appretement et la presentation de l'ag a egalement ete suggere. Mon projet de recherche a vise a determiner l'influence de c3b sur la generation de peptides antigeniques de la tt au cours de l'appretement dans la cpa. Je me suis d'abord interesse a mieux definir les effets de l'association de c3b a la toxine tetanique (tt) dans la presentation par les cpa aux cellules t. Les complexes tt-c3b activent tous les clones t specifiques des epitopes p2 et p30 a des doses 100 fois moins importantes, par rapport a la tt libre. Cette meilleure efficacite de presentation des cpa ne resulte pas, dans mon systeme experimentale, d'une modification de neosynthese de molecules hla-dr ou de b7. Mon etude s'est portee dans un deuxieme temps sur l'analyse de l'influence des complexes tt-c3b sur la stabilite en sds des molecules hla-dr. L'appretement de complexes tt-c3b permet la synthese de 2 fois plus de formes hla-dr1 compactes qu'avec la tt libre. Ces resultats demontrent que la proteine c3b modifie une ou plusieurs etapes de l'appretement de l'ag qui lui est associe. La production de formes hla-dr compactes en presence de complexes tt-c3b est preferentiellement observee dans les compartiments tardifs de la voie endocytaire, de type lysosomal. Je me suis enfin efforce de determiner les sequences des peptides de la tt naturellement generes par une cpa et associes aux molecules hla-dr, ainsi que d'analyser l'influence de l'association de c3b. L'appretement des complexes tt-c3b permet de generer des epitopes t differents de ceux de la tt, dont la quantite (ou l'immunogenicite) est responsable d'une meilleure activation des clones t utilises. Ces resultats suggerent donc un role direct de c3b dans la generation des epitopes t au cours de l'appretement de la tt, en favorisant la production de peptides immunogeniques differents, capables de se lier aux molecules hla-dr et d'activer de facon plus importante les clones t specifiques.
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Hearn, Arron R. "The delivery of exogenous peptides into the class I processing and presentation pathway using the B subunit of Escherichia coli heat labile enterotoxin". Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274823.
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MARTINEAU, PIERRE. "La proteine male d'escherichia coli k-12. Element du systeme de transport du maltose et vecteur de presentation de peptides du systeme immunitaire". Paris 7, 1991. http://www.theses.fr/1991PA077185.
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La proteine male d'e. Coli est necessaire pour le transport du maltose et pour la reponse chimiotactique vers ce sucre. Nous avons remplace par mutagenese dirigee les 8 trp et l'asn294 de la proteine par des alanines et etudie l'influence de ces mutations sur la fixation du sucre, le transport et la chimiotaxie. A partir de la structure tridimensionnelle de la proteine et des mutations decrites affectant les diverses interactions de male, nous avons propose un premier modele 3-d d'interaction de la proteine avec le complexe de la membrane interne malfgk et la proteine tar. Nous avons etudie dans le gene male deux sites internes permettant l'insertion d'un polypeptide etranger. A l'aide d'anticorps monoclonaux et polyclonaux diriges contre les sequences inserees, nous avons montre que les peptides sont soumis a des contraintes differentes suivant le site d'insertion. Les vecteurs male se sont reveles tres efficaces pour l'induction d'anticorps contre le peptide insere et contre la proteine native. Nous avons etudie le role du mode de presentation sur l'immunogenicite des proteines. Ces etudes permettent d'envisager d'utiliser ce systeme pour la construction de vaccins utilisant, soit la proteine recombinante purifiee, soit une bacterie attenuee comme vehicule
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DELEVIL, FABIENNE. "Caracterisation d'un compartiment d'association entre les molecules du cmh de classe ii et des peptides antigeniques. Inhibition de la presentation antigenique par le lps". Aix-Marseille 2, 1999. http://www.theses.fr/1999AIX22019.
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Nous nous sommes interesses a la caracterisation d'un compartiment d'association entre les peptides antigeniques et les molecules de classe ii dans des lymphocytes b de souris. Nous avons mis en evidence par fractionnement subcellulaire l'existence d'un compartiment, le llc, apparente aux endosomes precoces et implique dans l'association de peptides issus de la proteine hel avec les molecules de classe ii neosynthetisees i-a k. Les molecules de classe ii transiteraient depuis le tgn dans les endosomes precoces ou la chaine invariante est degradee. Les molecules de classe ii seraient ensuite transportees dans le llc ou elles s'associeraient a un peptide avant d'etre adressees a la membrane plasmique. Afin de caracteriser un marqueur specifique du llc, nous avons produit des anticorps monoclonaux contre la fraction llc purifiee. Nous avons mis au point un criblage par dot blot permettant de selectionner les anticorps reconnaissant la fraction llc et pas les fractions endosomes precoces et tardifs. Sur 600 hybridomes cribles, 12 repondant aux criteres de selection etablis ont ete clones. Un seul hybridome, produisant l'anticorps 20c4, a ete caracterise a l'heure actuelle. Cet anticorps est dirige contre des molecules de classe ii matures dissociees de la chaine invariante. Dans la deuxieme partie de ce travail, nous nous sommes interesses a l'effet d'une molecules glycolipidique bacterienne, le lipopolysaccharide (lps) de brucella abortus, sur la capacite des macrophages a presenter des antigenes exogenes. Nous avons montre que le lps de b. Abortus inhibe la presentation de la proteine hel a un hybridome t sans perturber l'association des peptides du hel avec les molecules de classe ii, suggerant que le lps interagit avec les molecules de classe ii a l'exterieur du sillon a peptides. L'existence de macrodomaines contenant le lps et les molecules de classe ii a la surface des macrophages peritoneaux expliquerait que les complexes classe ii-peptides ne sont reconnus efficacement par les cellules t.
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Shen, Yuelei. "MHC Class I Antigen Presentation is Regulated by the SUMO-Conjugating Enzyme UBC9: a Dissertation". eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/111.
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CD8 T cells recognize complexes of MHC class I and peptide on the surface of target cells. MHC class I antigen presentation is a long pathway, in which proteins are degraded by proteasomes to generating oligopeptides, which may be further trimmed by aminopeptidases in the cytosol. Peptides are transported into the ER, where they may be further trimmed by ER lumenal aminopeptidases and bind to newly-synthesized MHC class I complexes. Proteins degraded by the proteasome are generally tagged with ubiquitin by a combination of ubiquitin-conjugating enzymes and ubiquitin ligases. UBC9 is one ubiquitin conjugating enzyme, which does not conjugate ubiquitin, but instead conjugates small ubiquitin-like molecules (SUMO) to target protein. UBC9 has been found to regulate the functions of many proteins in vivo, most importantly by modifying nuclear transportation and function. Curing [During] my thesis work, I studied the function of UBC9 in MHC class I antigen presentation.
UBC9 over-expression in COS cells co-expressing ovalbumin markedly increased presentation SIINFEKL (the immunodominant epitope from ovalbumin in the context of H-2Kb), and UBC9 overexpression increased cell surface H-2Kbin general, suggesting that Ubc9 increased MHC class I antigen presentation by increasing peptide supply.
UBC9 did not increase synthesis or degradation of ovalbumin. In transient transfection experiments, Ubc9 increased presentation of SIINFEKL precursors that did, and that did not, depend on proteasomes for processing, as well as SIINFEKL precursors targeted to the ER, bypassing cytosolic processing altogether. However, a C-terminal extended precursor of SIINFEKL, which requires only proteasomal processing before presentation, was the most markedly affected by UBC9 overexpression. This suggested that UBC9 was affecting the pattern of cleavages made by proteasomes in ways that enhance the generation of the C-terminus of SIINFEKL. Because presentation of SIINFEKL itself (which requires no further proteolytic processing) was also enhanced, UBC9 must also affect steps in the class I pathway that occur after the generation of the mature epitopes. UBC9 did not affect the rate of peptide degradation in cytosolic extracts or in intact cells.
These findings suggested that UBC9 might have multiple effects on the MHC class I antigen presentation pathway. Immunofluorescent microscopy demonstrated that UBC9 increased the expression of the beta subunits of immunoproteasomes (LMP2, LMP7, and MECL1) as well as of TAP1 and tapasin. In contrast, UBC9 expression did not increase levels of calnexin, calreticulin, ERp57, or Protein disulfide isomerase (PDI). Similarly, levels of leucine aminopeptidase were not increased in UBC9-transfected cells. Therefore, UBC9 overexpression increases the levels of some but not all components of the class I pathway.
UBC9 overexpression increased protein levels of MECL1, LMP2 or LMP7 that were under the control of viral promoters, and levels of MECL1 mRNA were similar in control vector and UBC9 transfected cells. Therefore, UBC9 did not increase the level of expression of these subunits through increased transcription. Pulse-chase experiments showed that UBC9 overexpression reduced the degradation of MECL1. Therefore, UBC9 increases the levels of at least some of these components of the MHC class I antigen presentation pathway by increasing their stability.
To know the biological significance of UBC9 in MHC class I antigen presentation, I used small interfering RNA (siRNA) to knock down UBC9. Though UBC9 can be successfully knocked down by siRNA, the UBC9-negative cells became very sick, and were not suitable for the study of MHC class I antigen presentation.
There are three forms of SUMO molecules in mammalian cells: SUMO-1, SUMO-2 and SUMO-3. My study suggested that SUMO-2 may be involved in UBC9's regulation of MHC class I antigen presentation, since mutant SUMO-2 blocked UBC9's ability to increase H-2Kb-SIINFEKL levels on the cell surface after the cells were loaded with ovalbumin.
To further study the function of UBC9, I mutated the active amino acid Cys 93 of UBC9 to Ser (UBC9OH). Unexpectedly, this mutant form (UBC9OH) has very similar effects as wild-type UBC9, increasing Kb-SIINFEKL levels at the cells surface. This suggested that UBC9 protein regulates MHC class I antigen presentation pathway proteins by direct or indirect protein interaction, rather than (or as well as) by SUMO conjugation. Taking account of SUMO-2 results, I propose that wild-type UBC9 (either transfected or endogenous) conjugates SUMO-2 to its substrates, and then UBC9 (wild-type or mutant) interacts with its sumoylated targets, thus affecting protein functions.
I also studied heat shock protein Hsp27, which is known to be a substrate for UBC9 in vivo. Hsp27 is expressed in a variety of tissues in the absence of stress, and may regulate actin dynamics.
Hsp27 overexpression decreased generation of H-2Kb-SIINFEKL complexes from SIINFEKL precursors that did, and did not, require proteasomes for processing, or that were targeted to the ER. Hsp27 over-expression did not affect protein synthesis, and globally decreased cell surface H2-Kb and H2-Dblevels, but did not affect HLA-A0302 level. Hsp27 overexpression inhibits the presentation of ER-localized SIINFEKL. Taken together, my data suggested that HSP27 may inhibit MHC class I antigen presentation by affecting MHC class I molecules itself rather than peptide supply.
After Hsp27 was eliminated with siRNA, the effects were very similar to those seen with Hsp27 overexpression. Levels of H-2Kb-SIINFEKL decreased, and overall cell surface H-2Kb and H-2Db levels decreased. It is possible that when Hsp27 is over-expressed, it acts as a dominant negative form, conferring a similar phenotype to Hsp27 knockdown. These observations suggest that Hsp27 plays an important role in MHC class I antigen presentation.
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TOURNE, SYLVIE. "Pl les peptides associes aux molecules de classe deux du complexe majeur d'histocompatibilite : voies de presentation et fonction dans la selection positive des lymphocytes t cd4 positifs". Université Louis Pasteur (Strasbourg) (1971-2008), 1996. http://www.theses.fr/1996STR13265.
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Le systeme immunitaire a pour fonction d'eliminer tout element qu'il reconnait comme etranger a l'organisme. Les lymphocytes t cd4 reconnaissent les antigenes associes aux molecules de classe deux du complexe majeur d'histocompatibilite (cmhii). Une reponse immunitaire mediee par les cellules t cd4 depend donc d'une presentation efficace des antigenes par les molecules du cmhii et de la selection positive des lymphocytes t cd4 specifiques des peptides antigeniques. Ce travail met en evidence l'importance des complexes h2-m dans la voie classique de presentation antigenique par les molecules du cmhii. Il montre egalement leur role dans les voies alternes et notamment dans celles des molecules du cmhii exprimees en absence de chaine invariante. Les transporteurs tap ne semblent par ailleurs pas importants dans ces voies de presentation. Ce travail montre aussi que les peptides associes aux molecules du cmhii ont un role specifique dans la selection positive des lymphocytes t cd4 mais ils agiraient avec une large degenerescence. Les mecanismes par lesquels ils interviennent demeurent incompris
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Maayouf, Hasna. "Développement de plateformes de signalisation dérivées de particules pseudo-virales pour contrôler les fonctions cellulaires". Electronic Thesis or Diss., Mulhouse, 2024. http://www.theses.fr/2024MULH7387.
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Diverses stratégies de fonctionnalisation de surface visent à améliorer la biocompatibilité des matériaux pour les dispositifs implantables, notamment en ingénierie tissulaire. Par exemple, le polydiméthylsiloxane (PDMS), bien qu’utilisé dans de nombreux domaines, présente des propriétés de surface défavorables à l’adhérence cellulaire. La fonctionnalisation par des protéines de la matrice extracellulaire (MEC) ou des peptides synthétiques dérivés de celles-ci permet d’améliorer l'adhérence des cellules. Bien que ces approches offrent certaines solutions, des défis tels que le coût de production et le contrôle de la présentation en 3D entravent leur manipulation. Pour répondre à ces défis, nous avons développé des particules pseudo-virales (VLPs) présentant des peptides bioactifs à leur surface. La protéine d’enveloppe CP3, dérivée du bactériophage à ARN AP205, a été modifiée génétiquement à ses extrémités N- et C-terminales pour produire des VLPs présentant des peptides d’adhésion (RGD et YIGSR) et ostéogéniques (BMP2). La bioactivité des VLPs a été testée sur du PDMS avec des cellules de myoblastes C2C12, montrant une stimulation de l'adhérence, de la migration, de la prolifération et de la différenciation cellulaires. Des VLPs hétéromériques co-exprimant les peptides RGD et YIGSR ou BMP2 ont montré une bioactivité combinée. Des comparaisons entre la fibronectine et les VLP-RGD ont révélé des similarités et des différences dans les interactions cellulaires et la formation des adhésions focales. Ces résultats démontrent que les VLPs d’AP205 peuvent servir de nano-plateformes de signalisation, avec des applications potentielles en nanomédecine et dans les biomatériauxScientists have explored various surface functionalization strategies to improve the biocompatibility of materials used in implantable devices, particularly in tissue engineering. For example, polydimethylsiloxane (PDMS), although used in many fields, has surface properties that are unfavorable for cell adhesion. Functionalization with extracellular matrix (ECM) proteins or synthetic peptides derived from ECM components improves cell adhesion. While these approaches offer some solutions, challenges such as production cost and control over 3D presentation limit their use. To overcome these challenges, we developed virus-like particles (VLPs) displaying bioactive peptides on their surface. The coat protein CP3, derived from the RNA bacteriophage AP205, was genetically modified at both its N- and C-termini to produce VLPs displaying adhesion peptides (RGD and YIGSR) and an osteogenic peptide (BMP2). The bioactivity of the VLPs was tested on PDMS with C2C12 myoblast cells, demonstrating enhanced cell adhesion, migration, proliferation, and differentiation. Heteromeric VLPs co-expressing RGD and YIGSR or BMP2 peptides showed combined bioactivity. By comparing focal adhesions formed by RGD VLPs and those formed by fibronectin, we elucidate both the similarities and the differences in cell interactions. These results demonstrate that AP205 VLPs can be used as nanoscale signaling platforms to stimulate multiple cell functions, with promising applications in nanomedicine and biomaterials
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ARNOLD, DANIELE. "Le controle genetique de la presentation de l'antigene par les molecules de classe i du complexe majeur d'histocompatibilite (cmh). Etude du transporteur de peptides (tap1/tap2) et des sous-unites proteasiques (lmp2/lmp7)". Université Louis Pasteur (Strasbourg) (1971-2008), 1993. http://www.theses.fr/1993STR13141.
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Les molecules de classe i du cmh presentent des peptides d'origine endogene a la surface de la cellule, dans le but d'assurer une surveillance par les cellules t#c#d#8#+. L'assemblage des molecules de classe i est une etape incontournable pour une migration du complexe de classe i vers la surface cellulaire. Ce mecanisme est interrompu si les peptides generes dans le cytoplasme sont incapables de traverser la membrane du reticulum endoplasmique (re), pour s'associer aux chaines de classe i naissantes. Par marche chromosomique et cartographie de deletions chez des mutants d'assemblage, nous avons clone et caracterise de nouveaux genes a l'interieur de la region de classe ii du cmh. Ces genes codent pour les sous-unites d'un transporteur putatif de peptides (tap1/tap2) et deux sous-unites (lmp2/lmp7) d'un complexe proteolytique, le proteasome, qui degrade les proteines cytoplasmiques. L'heterodimere tap1-tap2, qui est implique dans la translocation des peptides du cytosol vers la lumiere du re, est localise dans la membrane du re et controle ainsi le passage des peptides. En transfectant tap1 et tap2 dans la lignee cellulaire mutante 174, qui est deletee des quatre nouveaux genes, nous avons montre que seuls les genes tap1 et tap2 sont necessaires a restaurer une expression normale des molecules de classe i a la surface cellulaire. Nos resultats, qui vont a l'encontre des modeles recents ou lmp2 et lmp7 etaient directement impliques dans l'appretement de l'antigene, montrent que ces composants lmp2 et lmp7 ne sont pas requis dans cette voie de presentation, ni pour la production de peptides, ni pour un assemblage stable des molecules de classe i
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Barouch, Dan H. "Peptide binding and presentation by HLA-A2". Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294263.
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Elvin, John G. "Presentation of peptide antigens to cytotoxic T lymphocytes". Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314883.
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Young, Lesley Lynn. "Peptide presentation by the rat class I MHC molecule RT1.Aa". Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627518.
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MacEachern, Mary Christina. "Indirect presentation in allograft rejection and the potential for immune intervention". Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246249.
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Tiwari, Neeraj. "Characterization of antigen processing and presentation by peptide-linked MHC class I molecules". [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975866370.
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Taylor, Kathryn. "Structure-based refinement of peptide presentation on the surface of cowpea mosiac virus". Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389325.
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Haddad, Ziad. "Monocytic cell responses to Aspergillus fumigatus investigation of phagocytosis, gene expression and peptide presentation /". [S.l. : s.n.], 2006.
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Negroni, Maria P. "Studies in Antigen Presentation and Antigen Recognition at Different Interfaces of the Adaptive Immune System". eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/996.
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Antigen presentation and recognition are key processes of the immune system necessary to initiate the adaptive immune response. Longstanding goals of these fields have been to understand the molecular mechanism of MHC II-peptide binding, the way in which dysregulation of this process can lead to disease, and determining how γδTCRs recognize their ligands. To examine some of these outstanding questions, I designed photocleavable peptides that could bind HLA-DR1 and could be used to facilitate peptide exchange. I also performed studies to understand whether peptide exchange on HLA-DR1 can be affected by glycation modifications, which occur in hyperglycemic conditions such as diabetes. I observed that while glycation modifications on HLA-DR1 did not affect peptide exchange, these modifications decreased the catalytic effect of HLA-DM on this reaction, which could affect antigen presentation in diabetic patients. For studies on antigen recognition by γδTCRs, I focused on γδNKT cells, a subset of γδT cells known to play a role during Listeria infection. I used four different variants of the γδNKT TCR to study the restrictions on Vγ junctional region usage by this TCR for ligand recognition. I found that all the TCR variants I examined could recognize cells infected with Listeria, indicating that this TCR is not restricted by γ-chain usage in order to recognize ligand. My research generated reagents that could serve in future studies of HLA-DR1 peptide binding and contributed to understanding the effect of hyperglycemic conditions on antigen presentation, as well as provided greater understanding of γδTCR restriction for ligand recognition.
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Sjursen, Anne Marie. "TRP1 Peptide Requires Internalization and is Partially Dependent on GILT for Efficient Presentation on MHC Class II". Thesis, The University of Arizona, 2012. http://hdl.handle.net/10150/221412.
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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.Tyrosinase-related protein 1 (TRP1) is a melanosomal integral membrane protein and melanocyte differentiation antigen that contributes to the synthesis of melanin in melanocytes. Present in both benign and malignant melanocytes, it has been implicated in the autoimmune development of vitiligo and melanoma antitumor immunity. Since a naturally occurring MHC class II-restricted TRP1 epitope contains cysteine residues, we hypothesized that this epitope will require internalization and reduction by gamma-interferon-inducible lysosomal thiol reductase (GILT) for presentation on class II. GILT is known to catalyze the reduction of protein disulfide bonds in the endocytic pathway and contribute to antigen processing and presentation of certain MHC class I and class II epitopes. We have previously shown that GILT is required for efficient class II-restricted presentation of TRP1. Here, we found that TRP1 peptide presentation is partially dependent on GILT and that TRP1 peptide requires internalization for efficient presentation on class II. We also determined that antigen presentation increased with increasing peptide dose and increasing APC:T cell ratio. Compared to other TRP1-specific T cells, primary TRP1-specific T cells from TRP1BwRAG-/- 5 TRP1tg mice produce maximal IL-2 in response to presentation of TRP1. These results further illustrate the importance of GILT in the processing and presentation of TRP1. Thus, GILT may play a role in both the development of autoimmune vitiligo and anti-melanoma immune responses.
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Trenh, Peter. "An Examination of MHC, Peptide, and TCR Interactions". eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/989.
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T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV.
First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1.
Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops.
Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity.
Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation.
The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.
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Londono, Arcila Lida Patricia. "The use of hepatitis B cores for peptide presentation : delivery to the mouse immune system using live attenuated salmonella". Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297205.
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Rasche, Sarah S. "Processing and Presentation of Glutamic Acid Decarboxylase 65 T cell-Inducing Epitopes: Implications in the Non-Obese Diabetic Mouse Model of Type 1 Diabetes". University of Toledo / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1290046839.
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Nyambura, Lydon Wainaina. "Impact of monocyte differentiation and intracellular infection on processing and presentation of autoantigen". Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/19154.
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Dendritische Zellen (DCs) und Makrophagen sind spezialisierte antigenpräsentierende Zellen, die eigene und fremde Antigene prozessieren und mittels Haupthistokompatibilitätsmoleküle, humane Leukozytenantige (HLA) im Menschen, T-Zellen präsentieren, um Toleranzen zu induzieren oder T-Zell-vermittelte Immunantworten zu initiieren. Abhängig von ihrer Differenzierung haben sie spezifische Phänotypen und Funktionen undunterschiedliche Interaktionen mit Pathogenen, in dieser Arbeit durch Leishmania donovani (LD) repräsentiert, welche in Phagolysosomen der Makrophagen propagieren. Der Einfluss der Differenzierungszustände und von intrazelluläre Infektionen auf die Antigenprozessierung und -präsentation waren weitgehend undefiniert. Um hier Einblick zu gewinnen, haben wir die HLA-I-präsentierten Selbstpeptidome von menschlichen unreifen und reifen DCs, die aus der MUTZ3-Zelllinie generiert wurden, und LD-infizierte bzw. nicht-infizierte aus der THP1-Zelllinie generierte Makrophagen mittels Flüssigchromatographie-Tandem-Massenspektrometrie (LC-MS/MS), sowie die Proteasom-Zusammensetzung per RT-PCR und die HLA-Expression und Aktivierungszustände der Zellen per Durchflusszytometrie analysiert und verglichen. Wir fanden, dass die HLA-I-Selbstpeptidome der Zellen heterogen und individualisiert waren, von Nonapeptiden dominiert wurden und ähnliche HLA-Bindungsaffinitäten und Ankerreste aufwiesen. Sie stammten aus Quellenproteinen aus fast allen subzellulären Lokalisationen und mit unterschiedlichen zellulären Funktionen in ähnlichen Anteilen und schlossen Tumor-assoziierter Antigene (TAAs) ein. Die Persistenz der LD hatte keinen Einfluß auf den Aktivierungszustand der Makrophagen, verursachte aber eine weitgehende Veränderungen des Peptidoms, der HLA-Bindungsaffinitäten und Ankerreste, der Quellproteine einschließlich TAAs und der HLA- und Proteasom-Expression.Dendritic cells (DCs) and macrophages are specialized antigen presenting cells that process self and foreign antigens and present them to T cells via major histocompatibility complex molecules, human leukocyte antigens (HLA) in humans, for induction of tolerance or initiation of T cell-mediated immune responses. Related to differentiation state, they have specific phenotypes and functions, and varied interactions with pathogens herein exemplified by Leishmania donovani (LD) that parasitize macrophages and propagate within their phagolysosomes. The impact of the differentiation state and intracellular infection on antigen processing and presentation by HLA class I remained undefined. To gain insight, we analyzed and compared the HLA-I self peptidomes of MUTZ3 cell line-derived human immature and mature DCs, and THP1 cell line-derived LD-infected and none-infected macrophages by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as proteasome compositions by quantitative RT-PCR, and HLA expression and cell activation states by flow cytometry. We found that the HLA I-presented self-peptidomes of the cells in the different states were heterogeneous and individualized, dominated by nonapeptides with similar HLA binding affinities and anchor residues. They were sampled from source proteins of almost all subcellular locations and from proteins involved in various cellular functions in similar proportion including tumour-associated antigens (TAAs). The persistence of LD within the macrophage, did not affect macrophage activation. However, its impact was observed in self-peptidome heterogeneity, HLA binding affinities, anchor residue preferences, source protein peptide sampling (including TAAs) and HLA and proteasome expression.
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Welsby, Iain. "PARP12, a novel interferon stimulated gene potentially involved in the control of protein translation and innate immunity". Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209714.
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Poly(ADP-ribose) polymerases belong to a family of proteins with 17 members in human beings. PARP1, the founding member of the family is a protein that synthesizes linear or branched polymers of ADP-ribose on itself or on target proteins. Different members of this family, that do not all possess ADP-ribosyl polymerase activity, are involved in the regulation of various cellular mechanisms. Some members of the family are particularly involved in the positive or negative control of the immune response. PARP1 is a key player in the regulation of inflammation, through its positive control of cell death and of proinflammatory cytokine production. On the other hand, the tankyrases (PARP5a and PARP5b) and PARP14 seem to regulate inflammatory responses in a negative fashion. PARP12 is a poorly characterized member of the family, whose expression is greatly increase following stimulation with type-I interferons, cytokines mainly involved in antiviral defences.PARP12 is a protein that possesses three main domains: A putative RNA binding N-terminal domain composed of tandem CCCH zinc-fingers, a central WWE domain and a C-terminal PARP catalytic domain. In this work, we have shown that the expression of PARP12 is strictly-dependent on type-I interferons, that it possesses ADP-ribosyl transferase activity and that in can regulate the translation of messenger RNA into proteins. PARP12 can be found in stress granules, sites of storage of untranslated mRNAs, and is capable of directly inhibiting the translation of a reporter mRNA when tethered to it, in a manner dependent on its catalytic activity. Furthermore overexpression of wild-type PARP12, in contrast to overexpression of a mutant with no detectable catalytic activity (PARP12-G575W), leads to a general arrest of most cellular translation.
On the other hand, we have shown that PARP12 can activate the transcription of genes under the control of an NFκB-dependent promoter, especially when its zinc-fingers are deleted or mutated (PARP12ΔZnF). PARP12ΔZnF is located in structures that can enclose TRIF, RIP1, NEMO, p62/SQSTM1 and ubiquitin. These proteins have all possess an important role in the activation of NFκB signalling cascades. Moreover, we have shown that endogenous PARP12 is situated in ALIS (Aggresome-Like Induced Structures) in LPS-stimulated macrophages. These structures have a possible role in the presentation of antigens on class I major histocompatibility complexes, implying that PARP12 may be involved in the regulation of antigen presentation.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Pernollet, Martine. "Modification de l'antigène toxine tétanique par des radicaux libres oxygénés et par des protéines à activité peptidyl-prolyl cis-trans isomérase : influence sur sa présentation à des lymphocytes T spécifiques". Université Joseph Fourier (Grenoble ; 1971-2015), 1994. http://www.theses.fr/1994GRE10238.
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L'influence du radical libre oxygene, le radical hydroxyle (oh) et des proteines a activite peptidyl-prolyl cis-trans isomerase sur le traitement de l'antigene toxine tetanique par des cellules presentatrices d'antigenes (lymphocytes b) a ete etudiee. En ce qui concerne le radical hydroxyle, sa production par des cellules de type macrophagique a ete reproduite a l'aide d'un systeme chimique. La toxine tetanique traitee par le radical oh subit un changement de conformation, et des liaisons bityrosine intramoleculaires resistantes a la proteolyse sont formees. La toxine ainsi modifiee est plus resistante a la proteolyse in vitro par des fractions endosomales isolees des cellules presentatrices. Cette diminution de proteolyse est correlee a une meilleure presentation par des cellules presentatrices fixees, a des lymphocytes t lorsque cet antigene est pre-proteolyse in vitro par des fractions endosomales. Ainsi, le radical oh favorise la presentation des epitopes de la toxine en les protegeant contre une proteolyse trop importante. La production du radical oh par un autre systeme chimique a permis de montrer l'existence d'un site de fixation pour le zinc a l'interieur de la chaine legere de la toxine, au niveau d'un epitope t. En ce qui concerne les activites peptidyl-prolyl cis-trans isomerases (ppiase), celles-ci ont pu etre mises en evidence dans les fractions endosomales de cellules presentatrices. L'addition a ces fractions endosomales de proteines a activite ppiase telles que la cyclophiline ou la fkbp augmente la proteolyse de la toxine tetanique. Il reste a tester les consequences de cet effet sur la presentation de cette derniere a des lymphocytes t. Un autre point de ce travail a ete la mise en evidence de la phosphorylation in vitro de la cyclophiline par la proteine kinase c purifiee. L'etude des consequences de cette phosphorylation sur l'activite ppiase de la cyclophiline est en cours
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Aun, Jason Paul. "Analysis and quantitation of the cross presentation of tumor antigens using the HIV protein transduction domain transactivating regulatory protein (TAT) to alter presentation". 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1462704.
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Huczko, Eric Lee. "The binding and presentation of peptide antigens by class I major histocompatibility molecules /". 1997. http://wwwlib.umi.com/dissertations/fullcit/9738893.
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Thesis (Ph. D.)--University of Virginia, 1997.Spine title: Binding of peptides by class I molecules. Includes bibliographical references (164-204). Also available online through Digital Dissertations.
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Fortin, Jean-Simon. "Antigen and superantigen presentation as defined by the MHCII-accessory proteins and associated-peptides". Thèse, 2013. http://hdl.handle.net/1866/12741.
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MHCII molecules expose a weave of antigens, which send survival or activation signals to T lymphocytes. The ongoing process of peptide binding to the MHC class II groove implicates three accessory molecules: the invariant chain, DM and DO. The invariant chain folds and directs the MHCII molecules to the endosomal pathway. Then, DM exchanges the CLIP peptide, which is a remnant of the degraded invariant chain, for peptides of better affinity. Expressed in highly specialized antigen presenting cells, DO competes with MHCII molecules for DM binding and favors the presentation of receptor-internalized antigens. Altogether, these molecules exhibit potential immunomodulatory properties that can be exploited to increase the potency of peptide vaccines.
DO requires DM for maturation and to exit the ER. Interestingly, it is possible to monitor this interaction through a conformation change on DOβ that is recognized by the Mags.DO5 monoclonal antibody. Using Mags.DO5, we showed that DM stabilizes the interactions between the DO α1 and β1 chains and that DM influences DO folding in the ER. Thus, the Mags.DO5+ conformation correlates with DO egress from the ER. To further evaluate this conformation change, directed evolution was applied to DO. Of the 41 unique mutants obtained, 25% were localized at the DM-DO binding interface and 12% are at the solvent-exposed β1 domain, which is thought to be the Mags.DO5 epitope. In addition, I used the library to test the ability of HLA-DO to inhibit HLA-DM and sorted for the amount of CLIP. Interestingly, most of the mutants showed a decrease inhibitory effect, supporting the notion that the intrinsic instability of DO is a required for its function. Finally, these results support the model in which DO competes against classical MHCII molecules by sequestering DM chaperone’s function.
MHCII molecules are also characterized by their ability to present superantigens, a group of bacterial or viral toxins that coerces MHCII-TCR binding in a less promiscuous fashion than what is observed in a canonical setting. While the mechanism of how bacterial superantigens form trimeric complexes with TCR and MHCII is well understood, the mouse mammary tumor virus superantigens (vSAG) are poorly defined. In the absence of a crystal structure, I chose a functional approach to examine the relation between vSAG, MHCII and TCR with the goal of uncovering the overall trimolecular architecture. I showed that TCR concomitantly binds both the MHCII α chain and the vSAG and that TCR-MHCII docking is almost canonical when coerced by vSAGs. Because many peptides may be tolerated in the MHCII groove, the pressure exerted by vSAG seems to tweak conventional TCR-MHCII interactions. Furthermore, my results demonstrate that vSAG binding to MHCII molecules is conformation-dependent and abrogated by the CLIP amino-terminal residues extending outside the peptide-binding groove. In addition, they also suggest that vSAGs cross-link adjacent MHCIIs and activate T cells via a TGXY motif.Les molécules du CMH présentent une panoplie d'antigènes qui, lorsque reconnus par un lymphocyte T spécifique, indique à ce dernier de survivre ou de s'activer. Le processus menant à la liaison d'un peptide à la poche du CMH de classe II, implique trois molécules accessoires, soit la chaine invariante, DM et DO. La chaine invariante replie et dirige les molécules du CMHII jusqu'à la voie endosomale. Ensuite, DM échange CLIP, peptide découlant de la dégradation de la chaine invariante, pour d'autres ayant une meilleure affinité. Exprimé seulement chez certaines cellules présentatrices, DO compétitionne avec les molécules du CMHII pour la liaison à DM et ainsi favorise la présentation d'antigènes internalisés par des récepteurs membranaires. Ensemble, ces protéines ont un potentiel immunomodulatoire pouvant être exploité afin d'augmenter l'efficacité de vaccin peptidique. DO requiert DM pour arriver à maturité et sortir du RE. Cette interaction, qui induit un changement de conformation dans la chaine β de DO, peut être sondée à l'aide de l'anticorps monoclonal Mags.DO5. En utilisant cet anticorps, nous avons montré que DM stabilise l'interaction entre les domaines α1 et β1 de DO et influence son repliement dans le RE. Donc, la conformation qui révèle l’épitope Mags.DO5 corrèle avec la migration de DO hors du RE. Afin d'étudier plus précisément ce changement de conformation, DO fut contraint à une ronde d’évolution dirigée. Des 41 mutants obtenus, 25% se retrouvent à l'interface DO- DM et 12% se retrouvent à la surface exposée au solvant du domain β1, région hypothétique de l'épitope Mags.DO5. De plus, la bibliothèque de mutants a été testée pour son habileté à inhiber l'activité de DM. La plupart des mutants montre une activité inhibitrice diminuée, ce qui supporte le model où DO compétionne les molécules du CMHII en séquestrant le rôle chaperon de DM. Les molécules du CMHII ont l'unique habileté de présenter les superantigènes, une famille de toxines virales et bactériennes qui force l'interaction CMHII-TCR de façon beaucoup moins spécifique qu'en contexte canonique. Alors que la façon dont les superantigènes bactériens s'assemblent avec le CMHII et le TCR soit bien comprise, la nature du complexe trimoléculaire découlant des superantigènes du virus de la tumeur mammaire de la souris (vSAG) reste mal définie. En l'absence d'une structure cristalline, une approche fonctionnelle a été choisie pour examiner la relation des vSAGs avec le CMHII et le TCR avec le but de dévoiler l'architecture de ce multi-complexe protéique. Je montre que le TCR lie parallèlement la chaine α du CMHII et vSAG, ce qui résulte en une interaction presque canonique. Puisque différents peptides peuvent être tolérés lors de cet ancrage, il semble que vSAG ajuste les interactions CMHII-TCR conventionnelles. En outre, mes résultats montrent que vSAG reconnait un épitope conformationnel et que cette liaison peut être abrogée par l'extension amino-terminale du peptide CLIP, laquelle s'étend en deçà de la niche. Finalement, mes résultats suggèrent que vSAG peut réticuler plusieurs CMHII adjacents et active les cellules T via son motif TGXY.
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Schmidt, Andreas [Verfasser]. "Nonsense mediated decay associated pioneer round of translation as source for peptides for presentation by MHC class I / vorgelegt von Andreas Schmidt". 2009. http://d-nb.info/993748112/34.
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Jackson, Angela M. "Development of a mass spectrometry based method for the identification of gp96-chaperoned peptides destined for presentation in MHC class I molecules". Thesis, 2006. http://hdl.handle.net/1828/2266.
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Theileria parva is an intracellular protozoan parasite and the causative agent of the lethal livestock disease East Coast fever (ECF). Research has shown that a protective cell-mediated immune response against parasite-infected lymphocytes is capable of clearing the host of T. parva (Pearson et al. 1979), leaving the host solidly immune to reinfection. The work presented in this thesis describes my attempts to develop a method for identification of major histocompatibility complex class I-associated T. parva peptides involved in eliciting this protective cell-mediated immune response.
The soluble chaperone gp96 interacts with peptides destined for association with major histocompatibily complex class I molecules and is therefore a source of T. parva peptides that interact with extracellular immune effectors. Using sensitive mass spectrometry methods the gp96-chaperoned peptide proteome from model parasite infected T lymphocytes was compared to an uninfected T cell line. With our findings we have demonstrated proof of concept for a highly sensitive method for the elucidation of potentially immunogenic peptides capable of initiating a protective immune response against the intracellular parasite T parva.
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de, Verteuil Danielle Angeline. "L’immunoprotéasome : producteur de peptides-CMH I et régulateur de l’expression génique". Thèse, 2014. http://hdl.handle.net/1866/10887.
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Le système ubiquitine-protéasome est le principal mécanisme par lequel les protéines intracellulaires sont dégradées. Le protéasome dit constitutif (PC) est donc essentiel à l’homéostasie mais aussi à la régulation de la majorité des processus cellulaires importants. La découverte d’un deuxième type de protéasome, appelé immunoprotéasome (IP), soulève toutefois de nouvelles questions. Pourquoi existe-t-il plus d’un type de protéasome ? L’IP a-t-il des rôles redondants ou complémentaires avec le PC ? L’IP étant présent principalement dans les cellules immunitaires ou stimulées par des cytokines, plusieurs groupes ont tenté de définir son rôle dans la réponse immunitaire. Or, l’implication de son homologue constitutif dans un éventail de processus non spécifiquement immunitaires nous laisse croire que l’IP pourrait lui aussi avoir un impact beaucoup plus large. L’objectif de cette thèse était donc de caractériser certains rôles cellulaires de l’IP dans les cellules dendritiques. Nous avons d’abord étudié l’impact global de l’IP sur la présentation antigénique de classe I. Ce faisant, nous avons pu déterminer ses deux contributions principales, soit l’augmentation drastique du nombre et de la diversité des peptides présentés sur les complexes majeurs d’histocompatibilité de classe I. Les différences de clivage entre le PC et l’IP pourraient expliquer en partie cette diversité du répertoire peptidique, notamment par l’affinité apparente de l’IP pour les régions protéiques non structurées. Dans un deuxième temps, nous avons dévoilé un nouveau rôle de l’IP sur un processus dépassant le cadre immunitaire : la transcription. Nous avons découvert que l’IP modifie l’abondance des ARNm en agissant principalement au niveau de leur synthèse. L’impact de l’IP sur le transcriptome est majeur et serait dû en partie à une dégradation différente de facteurs de transcription des familles IRF, STAT et NF-kB. Les cellules dendritiques IP-déficientes activent moins efficacement les lymphocytes T CD8+ et nous croyons que cette défaillance est causée (du moins en partie) par la perturbation transcriptomique provoquée par l’absence d’IP. Il importe donc de comprendre les différents rôles moléculaires de l’IP afin de mieux définir sa contribution globale au fonctionnement de la cellule et comprendre l’avantage évolutif, au niveau de l’organisme, procuré par une telle plasticité du système ubiquitine-protéasome.The ubiquitin-proteasome system is the major mechanism by which intracellular proteins get degraded. Constitutive proteasomes (CPs) are thus essential for cellular homeostasis but also to regulate the majority of important cellular processes. However, the discovery of a second type of proteasome, named immunoproteasome (IP), raises new questions. Why are there more than one type of proteasome? Does the IP perform redundant or complementary roles with the CP? The IP is predominantly expressed in immune or cytokine-stimulated cells and several groups worked at defining its role during the immune response. Yet, the implication of its constitutive homolog in a variety of processes suggests that the IP may also have a much broader impact. The objective was to characterize cellular roles of the IP in dendritic cells. We first studied the global impact of the IP on class I antigen presentation. We discovered that the IP drastically increases the number and the diversity of peptide presented by class I major histocompatibility complexes. Cleavage differences between the CP and the IP are likely part of the explanation for this peptide repertoire diversity, notably due to IP’s apparent affinity for unstructured protein regions. Second, we discovered a new role for the IP in a process unrestricted to the immune system: transcription. We found that the IP affects transcript abundance mostly at the level of mRNA synthesis. The impact of IPs on the transcriptome is major and would be partly based on a different degradation of IRF, STAT and NF-kB transcription factor family members by the two types of proteasomes. IP-deficient dendritic cells are less potent activators of CD8+ T cells and we believe that this defect is at least partly caused by the transcriptome alterations induced by the absence of IPs. It is therefore important to understand the different molecular roles of the IP in order to better define its global contribution to cellular functions and to understand the evolutionary advantage, at the level of the organism, brought by such plasticity of the ubiquitin- proteasome system.
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Bhatnagar, Aparna. "A unique APC phenotype in the non-obese diabetic (NOD) mouse : presentation of class II MHC-associated invariant chain-derived peptides (CLIP) by I-Ag7". Phd thesis, 2001. http://hdl.handle.net/1885/147142.
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Côté, Marie-Hélène. "Ciblage exosomal et effet de HLA-DM sur la présentation antigénique par le complexe majeur d'histocompatibilité de classe II". Thèse, 2008. http://hdl.handle.net/1866/2799.
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Les molécules du complexe majeur d'histocompatibilité de classe II (CMH II) sont exprimées exclusivement à la surface des cellules présentatrices d'antigènes et servent à stimuler les cellules CD4+ initiant une réponse immunitaire. Le chargement peptidique sur HLA-DR se produit dans les endosomes tardifs et les lysosomes sous l'action de HLA-DM. Cette molécule de classe II non-classique enlève les fragments peptidiques de la chaîne invariante (Ii) restés associés aux molécules de classe II (CLIP) et édite leur répertoire d'antigènes présentés. En utilisant une forme mutante de HLA-DM (HLA-DMy) qui s'accumule à la surface plasmique, nous avons observé que HLA-DMy augmente les chargements de peptides exogènes et aussi la réponse des cellules T en comparaison avec HLA-DM sauvage. Il a été démontré que des molécules chimiques, comme le n-propanol, pouvait avoir le même effet que HLA-DM en remplaçant les peptides associés aux molécules de classe II de la surface cellulaire. De plus, HLA-DMy et le n-propanol ont présenté un effet additif sur la présentation de peptides exogènes.
Certaines protéines de la voie endocytique, comme HLA-DR, HLA-DM, HLA-DO et Ii sont ciblés aux compartiments multivésiculaires (MVB) et peuvent être ciblées aux exosomes. Suite à une fusion entre les MVB et la membrane plasmique, les exosomes sont relâchés dans le milieu extracellulaire. Nous avons déterminé que le motif tyrosine de HLA-DMβ et son interaction avec HLA-DR n'affectaient pas le ciblage aux exosomes, sauf la molécule HLA-DO.
Cette étude nous a permis de démontrer que HLA-DMy augmente la quantité de peptides exogènes chargés sur les CPA et que HLA-DM et HLA-DMy sont incorporés dans les exosomes.Major histocompatibility class II (MHC II) molecules are expressed exclusively on the surface of antigen presenting cells and serve to stimulate CD4+ cells to initiate an immune response. Peptide loading on MHC II molecules occurs in late endosomal and lysosomal compartments by the catalytic action of HLA-DM. This non-classical class II molecule removes class-II-associated invariant-chain peptide (CLIP) from class II molecules and edits their repertoire of antigenic peptides loaded. Using a mutant form of HLA-DM (HLA-DMy) that is targeted to the plasma membrane, we observed in the case of HLA-DMy, there is an increase of the loading of exogenous peptides and also significantly increased T cell response in comparison with HLA-DM wild-type. It was found that some chemical molecules, like n-propanol, could mimic the effect of HLA-DM by removing peptides from cell surface class II molecules. Interestingly, HLA-DMy and n-propanol seem to have an additive effect on the exogenous peptide loading. Some proteins of the endosomal pathway, like HLA-DR, HLA-DM, HLA-DO and Ii are targeted to microvesicules-containing compartments called MVB and they can be introduced into exosomes. Following the fusion between MVB and plasma membrane, exosomes are released in extracellular environment. We have determined that tyrosine motif of HLA-DMβ and interaction with HLA-DR does not affect HLA-DM targeting to exosomes, except for the HLA-DO molecule. In conclusion, we showed that HLA-DMy increases the quantity and the quality of exogenous peptides loading on APC and HLA-DM and HLA-DMy are incorporated to exosomes.
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Granados, Diana Paola. "Effect of the unfolded protein response on MHC class I antigen presentation". Thèse, 2008. http://hdl.handle.net/1866/7632.
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Shen, Kuan-Yin y 沈冠印. "Anti-tumor Effects and Antigen Presentation Mechanisms of Toll-like Receptor 2 (TLR2) agonist-conjugated peptide". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/02005787959422829416.
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博士國防醫學院
生命科學研究所
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Dendritic cells (DCs) can process and present extracellular antigens on MHC class I molecules to activate cytotoxic T lymphocyte (CTL) responses through an important mechanism, antigen cross-presentation. However, toll-like receptor (TLR)-mediated cross-presentation of extracellular antigens by DCs remains unclear. In this study, models of synthetic di-palmitoylated peptides (Pam2IDG and Pam2EQL) explored the mechanisms of TLR2-mediated cross-presentation. We observed that TLR2 facilitated the internalization of di-palmitoylated peptides by bone marrow-derived DCs (BMDCs) via clathrin-mediated endocytosis. The immunization of di-palmitoylated peptide-pulsed BMDCs induced tumor regression through TLR2 signaling. These findings indicated that exogenous TLR2 agonist-conjugated peptide could be cross-presented to CTL. We further identified di-palmitoylated peptides-induced antigen-specific CTL responses was transporter associated with antigen processing (TAP) independent. In addition, endosomal acidification inhibitor (chloroquine) or a lysosomal degradation inhibitor (Z-FL-COCHO) could block the presentation of di-palmitoylated peptides by MHC class I molecules. The endocytosed di-palmitoylated peptide was delivered rapidly from early endosome antigen-1 (EEA1)-positive endosomes to RAS-related GTP-binding protein 7 (Rab7)-associated late endosomes compared with their non-lipidated counterparts. Furthermore, we found that the Rab7 expression co-related was up-regulated by di-palmitoylated peptide was via the TLR2/MyD88 pathway. Therefore, di-palmitoylated peptide could be cross-presented efficiently via vacuolar pathway to enhance CTL responses though TLR2 signaling. In conclusion, our data suggest that TLR2-mediated cross-presentation is through the upregulation of Rab7 and a vacuolar pathway to prime CTL responses.
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Tiwari, Neeraj [Verfasser]. "Characterization of antigen processing and presentation by peptide-linked MHC class I molecules / presented by Neeraj Tiwari". 2005. http://d-nb.info/975866370/34.
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Sever, Lital. "A Functional Study of the Major Histocompatibility Class I Antigen Presentation Pathway in Rainbow Trout (Oncorhynchus mykiss)". Thesis, 2014. http://hdl.handle.net/10012/8135.
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Major Histocompatibility Complex (MHC) class I receptors are glycoproteins which play a critical role in anti-viral immunity by displaying foreign peptides to cytotoxic T cell lymphocytes. The loading of high affinity peptides into the MHC class I receptor in mammals is coordinated by a multiple proteins that are collectively referred to as the peptide loading complex (PLC). To date, the composition of the peptide loading complex in fish is unknown and therefore the characterization of the molecules which may exist in this putative complex was pursued.
This thesis includes the cloning and functional characterization of ERp57 and calnexin in rainbow trout which, in mammals, are known to interact with the MHC class I receptor either during its early biogenesis or later in the assembly of the PLC. Trout ERp57 and calnexin cDNA sequences are ubiquitously expressed in trout tissues and both the ERp57 and calnexin genes appear in at least two copies each in the trout genome. Interestingly, despite their high sequence identity with their mammalian homologues, some structural discrepancies were identified. ERp57 does not contain an endoplasmic reticulum (ER) retention signal or a nuclear localization signal, while one of the two isolated cDNA clones for calnexin does not contain an ER (endoplasmic reticulum) retention signal and lacks a conserved C-terminal serine phosphorylation site. These findings suggest that in trout, there may be unique versions of these proteins that have acquired different cellular functions. Through the production of polyclonal antibodies against trout ERp57, the conserved protein induction of ERp57 during ER stress was demonstrated concurrently with calnexin.
In addition, this study shows for the first time that ERp57 can be induced transcriptionally by phytohemagglutinin and synthetic double stranded RNA, which implies its possible regulatory role during viral infection and the activation of the immune response. Furthermore, the functional characterization of the MHC class I specific chaperone tapasin, a key element in the PLC of mammals was pursued. Tissue and cell line distribution revealed that tapasin is expressed in high levels in immune system organs and in the rainbow trout macrophage cell line RTS11, at a relative molecular weight of 48 kDa with an additional 20 kDa band detected by the tapasin antibody. Tapasin protein was significantly up regulated upon exposure to synthetic double stranded RNA and during infection with two fish viruses: chum salmon virus and viral hemorrhagic septicemia virus genotype IVa, whereas the expression of the 20 kDa band was not affected by these stimuli.
This study also examined the regulation of the MH class I heavy chain,β2 microglobulin and their associated machinery upon exposure to viral hemorrhagic septicemia virus genotype IVa at permissive and non-permissive temperatures. β2 microglobulin secretion into the cell media, a marker of MH class I receptor turnover, was detected in the conditioned media of RTS11 cells under normal conditions and was shown to be significantly enhanced during viral hemorrhagic septicemia virus genotype IVa infection. Furthermore, when RTS11 cells were maintained at cold temperatures, the secretion of β2 microglobulin was significantly reduced in both infected and non-infected cultures, while the cellular levels of β2 microglobulin remained unchanged. These results suggest that cold temperature can alter the expression of the MH class I molecule on the cell surface and therefore may be contributing to host susceptibility to viral hemorrhagic septicemia virus genotype IVa during the winter.
Lastly, Co-immunoprecipitation demonstrated the interaction of the lectin chaperones: calnexin and calreticulin with the glycosylated MH class I receptor supporting their conserved role during MH class I receptor folding in fish. Concurrently, tapasin's interaction with transporter associated with antigen processing (TAP) and with the glycosylated form of the MH class I was revealed for the first time in fish, which supports their role in antigen presentation as in mammals. This study demonstrated that ERp57 and tapasin form a conserved disulfide linked heterodimer of 110 kDa, however unlike mammals, an additional 75 kDa heterodimer was detected which suggests a possible novel interaction of ERp57 with a 20 kDa tapasin version alternately regulating antigen presentation in fish.
Overall, this study suggest that the interactions involved in antigen presentation in mammals are conserved in fish, however the presence of different protein versions of calnexin, ERp57 and tapasin might dictate a different mode of regulation for MH class I assembly in fish, as opposed
to mammals. Elucidating these interactions during various viral infections in fish can help to uncover possible viral strategies to manipulate the host immune response and will provide information needed to assist in designing novel tools to prevent fish viral diseases.