The human genome comprises of more than 22.000 genes. These genes encode for proteins playing a role in many general or tissue-specific biological processes, but for many of them the function is... Show moreThe human genome comprises of more than 22.000 genes. These genes encode for proteins playing a role in many general or tissue-specific biological processes, but for many of them the function is not yet unraveled yet. A way to study all the different genes at once in a certain biological process is by genome-wide screening. By considering all genes in a pathway, new players can be identified that would otherwise not have been linked to the studied process. In this thesis, I describe a siRNA-based genome-wide screen used to identify new proteins involved in MHC class II antigen presentation, expression and transport. The data obtained from this screen not only led to the identification of proteins involved in the MHC class II pathway specifically, but we also identified a regulator of a more general process namely intracellular endosome localization. This shows the importance of genome-wide screening in the identification of new players and regulators of certain biological pathways which may be considered as new drug targets to cure diseases. Show less
Major Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases.... Show moreMajor Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases. To better understand this process we performed a genome-wide RNAi screen. The effect of downregulation of each human gene on MHC class II expression and antigen loading was analyzed. Genes affecting MHC-II antigen presentation were tested for their involvement in MHC-II transcription and transport. Some genes might function in repositioning MHC-II towards the plasmamembrane upon Dendritic Cell maturation. This was confirmed by silencing these genes in immature Dendritic Cells. The novel GTPase ARF7, ARF7EP and MYO1E play a central role in this transport process. MHC-II antigen loading on intraluminal vesicles of the MHC-II compartment is facilitated by chaperone DM. In a confocal FRET study we investigated whether microdomain-forming tetraspanins CD63 and CD82, interacting with MHC-II and DM, could facilitate the MHC-II/DM interaction. MHC-II and DM preferably interacted with CD63 on the intraluminal vesicles, likely facilitating the MHC-II/DM interaction and antigen loading. MHC-II favoured interactions with CD82 on the limiting- and plasmamembrane suggesting a sorting role for CD82. This knowledge will contribute to development of new methodologies to influence MHC class II presentation in patients. Show less
Major advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. The notion that dendritic cells ... Show moreMajor advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. The notion that dendritic cells (DC) play a crucial role in the activation of T lymphocytes has made DC biology of central importance for vaccine development. Accordingly, efficient delivery of antigen to DCs is one of main objectives in vaccine development. In this thesis, antibody-mediated antigen targeting is evaluated as a potential antigen delivery strategy for therapeutic vaccination. Complexes of protein antigen and antigen-specific antibodies are natural formulations that bind to Fc__ receptors. Fc__R ligation on DCs leads to efficient uptake, DC maturation and presentation of the antigen to T lymphocytes. Interaction of Ag-Ab complexes with Fc__Rs on DCs provides a link between the humoral and cellular arms of the immune response. This thesis contains an extensive evaluation of Fc__R-mediated antigen delivery to dendritic cells in the context of T lymphocyte-mediated immunotherapy. In addition, it contains a detailed analysis of Fc__R function on DCs and addresses the kinetics of cross-presentation of antigen after Fc__R-mediated uptake. Show less
Intracellular proteins are degraded by the proteasome. The resulting protein fragments can be regarded as waste, but it is clear that peptides play an important role in several processes, like the... Show moreIntracellular proteins are degraded by the proteasome. The resulting protein fragments can be regarded as waste, but it is clear that peptides play an important role in several processes, like the immune response. Peptides are destroyed very rapidly and irrespectively of their sequence. A peptide's length appeared to be important as the half-life increases when additional amino acids are present at the amino-terminus. As exception dibasic N-terminal peptides appeared to be more stable than other peptides, resulting in overrepresentation by HLA-B27 molecules that bind preferably binds dibasic N-terminal peptides. Cross-presentation of peptides from infected cells by professional antigen presenting cells (APC) is crucial for a proper immune response because they express the required co-stimulatory molecules. The exact mode of antigen transfer is largely unknown, and we describe a novel pathway for cross-presentation, by passive diffusion of peptides through gap junctions from infected cells to APCs Apoptosis-derived antigens have been shown to be a major source of cross-presented antigens. Peptides from apoptotic cells can be presented by the cell itself, or transferred to healthy neighboring cells and be presented by MHC class I molecules of these cells, thereby facilitating cross-presentation, through gap junction mediated intercellular peptide transfer. Show less
Immune responsiveness is carefully regulated. Cells of the immune system have to respond adequately to invading micro-organisms and possibly to transformed cells, but tolerance for the own body... Show moreImmune responsiveness is carefully regulated. Cells of the immune system have to respond adequately to invading micro-organisms and possibly to transformed cells, but tolerance for the own body constituents needs to be preserved. Dendritic cells (DC) comprise a family of professional antigen presenting cells (APC) that play a central role in the regulation of the immune response. Immature DC, located in the periphery, can efficiently take up Ag, but lack the co-stimulatory signals for effective T-cell activation. Upon maturation, DC migrate to secondary lymphoid organs and increase the expression of co-stimulatory molecules and MHC molecules. Mature DC are very efficient in priming na____ve T-cells. In contrast to their T-cell priming capacity, DC in peripheral tissues constitutively process and present Ag in the absence of pathogen-related or endogenous inflammatory stimuli, and make a major contribution to peripheral tolerance by inducing unresponsiveness or deletion of specific T-cells. The central role of DC in controlling immunity makes these cells attractive tools for many clinical situations that involve T-cells: induction of tolerance in case of transplantation, allergy and autoimmune disease and induction of efficient T-cell responsiveness in case of infection and tumors. Many tumor components do not elicit Ag-specific T-cell responses in patients, which may be due to the absence of functional DC in tumors or the secretion of factors by tumor cells that reduce DC development and function. Application of tumor Ag to DC ex vivo and reinfusion of these DC leads to induction of specific immunity. In animals this strategy can lead to protection against tumors and even a reduction in size of established tumors. At present similar studies are carried out in patients. The research described in this thesis focuses on the requirements for induction of efficient cytotoxic T lymphocyte (CTL)- responses and tumor immunity by DC. Different modes of Ag presentation were studied for the induction of CTL-responses and tumor protection. Show less
