The life-long infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies employed by these viruses. Virus-derived peptides are... Show moreThe life-long infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies employed by these viruses. Virus-derived peptides are presented to cytotoxic T-lymphocytes by MHC I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC I molecules occurs. The UL49.5 protein of the varicelloviruse bovine herpesvirus 1 (BHV-1) is a potent inhibitor of TAP-mediated peptide transport. The viral protein prevents MHC I maturation by rendering the TAP complex in a translocation incompetent state. In addition, TAP proteins are degraded in the presence of BHV-1 UL49.5. The chapters 2 to 4 of this thesis focus on the functional dissection of BoHV-1 UL49.5 and aim to identify its target domain within the TAP complex. All herpesviruses sequenced to date code for a UL49.5 homolog. However, in chapter 5 and 6 we show that only a few viruses belonging to the genus Varicellovirus encode a TAP-inhibiting UL49.5. The applicability of the UL49.5 proteins to study and modify pathways of antigen presentation is demonstrated in chapter 7 and 8. Chapter 9 describes the identification of a TAP inhibitor in cowpox virus Show less
Stem cell transplantation (SCT) is an accepted treatment for patients with hematological malignancies. Stem cell donor-derived T cells mediate both beneficial Graft-versus-Leukemia (GvL) effects... Show moreStem cell transplantation (SCT) is an accepted treatment for patients with hematological malignancies. Stem cell donor-derived T cells mediate both beneficial Graft-versus-Leukemia (GvL) effects and detrimental Graft-versus-Host Disease (GvHD). Relapse of the original disease can be treated with infusions of donor lymphocytes (DLI). Like SCT, DLI elicits both GvHD and GvL. The risk of severe GvHD is especially high if the stem cell donor and patient are not fully matched for human leukocyte antigens (HLA). To minimize GvHD after HLA-mismatched SCT DLI should consist of pre-selected donor T cells that display reactivity restricted to the patient's residual leukemic or hematopoietic cells. This thesis focuses on exploring the alloHLA-A2 T cell repertoire and testing the feasibility of generating alloHLA-A2-restricted T cells specific for the hematopoietic system-restricted minor Histocompatibility antigens (mHags) HA-1 or HA-2. We describe several new methods of alloHLA-restricted antigen-specific T cell stimulation. However, we also show that alloHLA-recognition by T cells is inherently crossreactive. Unfortunately, currently available technologies can not readily distinguish between crossreactive and antigen-specific alloHLA-restricted T cells. We therefore conclude that generation of alloHLA-restricted antigen-specific T cells is as yet not feasibible in a clinical setting. Show less
Many cellular processes are studied by biochemical techniques. Usually, this involves experiments where large number of cells are lysed, protein content is subsequently isolated and studied using... Show moreMany cellular processes are studied by biochemical techniques. Usually, this involves experiments where large number of cells are lysed, protein content is subsequently isolated and studied using antibodies to detect changes in protein levels, post-translational modifications, pairing with partner molecules, etcetera. Although informative, these mass population analyses often lack the time resolution to study rapid alterations in protein state, and do not allow the characterisation of highly dynamic processes. Moreover, analysis of millions of cells at once evidently shows the average response in the population of cells, thereby obscuring cell-to-cell variation and the dynamic range of a process. With the availability of microscopic techniques in combination with genetically encoded fluorescent probes, many of these restraints have been overcome. Highly dynamic reactions can now be studied in detail in a relatively easy manner, and in the context of a living cell, hence "single cell biochemistry". In this way, we studied two different cellular processes, antigen presentation and drug resistance in unprecedented detail. Both parts seem at first unrelated, yet are interconnected through the use of similar techniques. Assessment of individual cells using sensitive microscopic measurements, has led to important and detailed understanding of the dynamic processes involved in both topics. Show less
