Bleeding events are frequently encountered in hemato-oncology patients. To prevent this, in periods of thrombocytopenia patients receive prophylactic platelet transfusions, based on the platelet... Show moreBleeding events are frequently encountered in hemato-oncology patients. To prevent this, in periods of thrombocytopenia patients receive prophylactic platelet transfusions, based on the platelet counts. However, beside platelet counts many other patients factors likely contribute to the bleeding risk.In this thesis we focus on describing current clinical practice to prevent bleedings in a subpopulation of patients with persistent deep thrombocytopenia, risk factors for bleeding, and prediction of bleeding. We also describe a ongoing study which aims to identify and quantify risk factors in future.With this knowledge, in the ultimate goal is to predict bleeding more accurate based on patient characteristics and/or biomarkers. This could be a first step towards more personalized bleeding prevention strategies. Show less
Antibodies, the cardinal effector molecules of the immune system, are being leveraged to enormous success as biotherapeutic drugs. Adaptive immune responses consist of epitope-diverse polyclonal... Show moreAntibodies, the cardinal effector molecules of the immune system, are being leveraged to enormous success as biotherapeutic drugs. Adaptive immune responses consist of epitope-diverse polyclonal antibody mixtures that are capable of neutralizing their targets via binding interference and by mediating humoral and cellular effector functions. A mechanistic theme fundamental to virtually all aspects of antibody biology, including antibody-antigen binding, clonal selection and effector functions, is the utilization of avidity to drive and tune functional responses. Manipulating antibody avidity has since emerged as an important design principle for enhancing or engineering novel properties in antibody biotherapeutics. In the context of ‘classical’ effector functions, complement-dependent cytotoxicity (CDC) can be improved by single point mutations in the IgG Fc domain that increase intermolecular Fc-Fc interactions upon binding to membrane-bound targets, thereby facilitating enhanced IgG hexamer formation and C1q binding. Such engineering approaches illustrate the relevance of promoting avidity interactions such as antibody clustering to enhance effector functions. The aim of this thesis was to explore the role of antibody avidity interactions, and more specifically the importance of ‘ordered clustering’, in antibody mechanisms of action and to apply the knowledge obtained in designing novel and improved antibody-based therapeutics Show less
The interaction between tumor and immune cells in the microenvironment plays a key role in oncogenesis. This recognition can be mediated by changes in glycosylation on tumor cells which are sensed... Show moreThe interaction between tumor and immune cells in the microenvironment plays a key role in oncogenesis. This recognition can be mediated by changes in glycosylation on tumor cells which are sensed by lectins, such as the Macrophage Galactose-type Lectin (MGL), expressed on immune cells, resulting in immunosuppressive responses. MGL binding to the Tn-epitope on MUC1 triggers DCs to stimulate T-regulatory responses and tolerance against the tumor, while suppressing T-effector cells responsible for tumor eradication. This undesirable consequence in cancer led to interest in the characterization of targets of MGL. This thesis dealt with the analysis of glycoproteins binding to MGL expressed by different tumor cell models. For this purpose, a robust method to enrich MGL binding proteins, in combination with their mass spectrometry-based identification was established. Because of the high level of the Tn-antigen, due to a Cosmc mutation, the Jurkat cell line was initially used for this purpose (Chapter 2) but a slightly adjusted method was later applied to high- and low-MGL binding CRC cell lines as well (Chapter 3 and 5). These analyses led to the identification of hitherto unknown MGL binders. In Chapter 4, we focused on to the contribution of N-glycan MGL binding glycotopes in CRC cell lines, most probably corresponding to LacdiNAc structures, by implementing overall N-glycan release in our workflows. With this, we were able to show that N-glycoproteins represent a hitherto underestimated group of MGL binding protein in these cell lines. Also several secreted proteins from the CRC cell lines could bind to MGL (Chapter 5), indicating that the interaction with immune cells can also be mediated by this group of proteins. The results from previously published transcriptomics and N-/O-glycomic analyses could not explain the different expression of MGL binding proteins on the CRC cell lines used. For this reason, in Chapter 4, we extended our research with full comparative quantitative proteomics analyses, in an attempt to explain the differences in MGL binding, for example by different levels of MGL binding proteins or proteins involved in glycosylation pathways. Additionally, in Chapter 6, we used such a quantitative proteomics dataset also to test the suitability of a previously suggested mass spectrometry-based method to discriminate O-GalNAc (Tn) versus O-GlcNAc, which led to the first site-specific identification of O-glycosylation of both intracellular and secreted anterior gradient protein 2 (AGR2). Show less
Schuitema, I.; De Sonneville, L.; Kaspers, G.; Van der Pal, H.; Uyttebroeck, A.; Van der Bos, C.; Veerman, A. 2015
Human leukocyte antigen (HLA) matched allogeneic stem cell transplantation (SCT) is an established curative treatment for hematopoietic malignancies and an investigative immunotherapeutic approach... Show moreHuman leukocyte antigen (HLA) matched allogeneic stem cell transplantation (SCT) is an established curative treatment for hematopoietic malignancies and an investigative immunotherapeutic approach for solid tumors. The curative effect of allogeneic SCT is based on so called graft versus-tumor (GvT) responses. The GvT effect in the HLA-matched setting is mainly driven by donor immune responses against so called minor histocompatibility antigens (mHags). Emerging data suggest that immunotherapy targeting mHags may eradicate cancer without severe side effects. This thesis investigated 1) the proof of concept for the efficacy of mHag specific immunotherapy in vitro and in vivo, 2) the optimal circumstances under which mHag specific cytotoxic T-cells (CTLs) are most effective against established leukemia and solid tumors, 3) new immune escape mechanisms in cancer that need to be considered when targeting mHags, 4) the optimal protocols for the in vitro expansion of mHags CTLs, 5) the optimal design of mHag peptide vaccines and 6) the optimal parameters for the clinical guidance of mHags specific immunotherapy of cancer. The data acquired in this thesis have provided relevant knowledge that is already partially implemented in the design of new HA-1 vaccination studies aiming at curing leukemia and solid tumors. Show less
TCR gene transfer is a strategy that enables the rapid engineering of anti-leukemic T-cells with defined specificity, resulting in a so called __off the shelf __ therapy. An elegant strategy to... Show moreTCR gene transfer is a strategy that enables the rapid engineering of anti-leukemic T-cells with defined specificity, resulting in a so called __off the shelf __ therapy. An elegant strategy to promote persistence of TCR modified T-cells may be TCR gene transfer into CMV- and EBV-specific T-cells, which exhibit proper memory and effector phenotypes. Furthermore, these virus-specific T-cells do not induce GvHD after HLA identical allo-SCT, and thus can be safely administered. For efficient anti-leukemic reactivity of the introduced TCR coinciding with enhanced in vivo survival, a balance between cell surface expression of the introduced and endogenous TCR is required. The aim of this thesis was to optimize the efficacy of TCR gene transfer, study possibilities and restrictions of virus-specific T-cells as host cells for TCR gene transfer and characterize the occurrence of potentially harmful mixed TCR dimers and strategies to prevent their formation. Show less
MHC class II antigen presentation by B cells is important to activate CD4+ T cells that stimulate the B cell to produce antibodies. Besides this, disruption of MHC class II antigen presentation... Show moreMHC class II antigen presentation by B cells is important to activate CD4+ T cells that stimulate the B cell to produce antibodies. Besides this, disruption of MHC class II antigen presentation could play a role in immune escape by tumor cells. This thesis describes MHC class II antigen presentation by B cells during bacterial infections and leukemia. We show that, in contrast to the current dogma, human B cells are able to phagocytose whole, living Salmonella bacteria via their B cell receptor (BCR). Salmonella survives intracellular and is transported by the B cell to the spleen where Salmonella can infect other cells. Although Salmonella uses the B cell to disseminate and escape the immune system, uptake of Salmonella by B cells also results in a good immune response consisting of specific antibody production, antigen presentation and activation of both CD4+ T helper cells and cytotoxic CD8+ T cells that are able to eliminate infected cells. Many leukemic cells express MHC class II. For AML (acute myeloid leukemia) we show that expression of the self-peptide CLIP could be a measure for immune escape because a high percentage of HLA-DR+/CLIP- blasts correlates with a longer disease-free survival. In B-CLL (chronic lymphocytic leukemia), the percentage of activated CD4+ and CD8+ T cells is enhanced and inversely correlated to CLIP. In addition, transcription of all MHC class II genes is increased and B-CLL patients with high HLA-DOA mRNA levels have a less favorable prognosis. Show less
One of the major problems in the treatment of patients with acute leukemia is failure of therapy due to acquired resistance, which may partially be caused by defects in the apoptotic machinery of... Show moreOne of the major problems in the treatment of patients with acute leukemia is failure of therapy due to acquired resistance, which may partially be caused by defects in the apoptotic machinery of these leukemic cells. Apoptosis is induced and regulated by a complex network of proteins connected via various signal transduction cascades, eventually leading to death of the target cell. The results described in this thesis demonstrate that apoptosis induction in leukemic cells after treatment with chemotherapy or (cellular) immunotherapy is very complex and frequently dependent on the target cell studied, and on the interaction between target and effector cell. G0 cells derived from patients with B-CLL compared to G0 cells from patients with acute leukemia responded differently to Ara-C treatment, which may be partially explained by the different mechanisms of action exerted by Ara-C. The role of the death receptor pathway in both chemotherapy-induced and CTL-induced apoptosis of leukemic cells was also unraveled in more detail. It will be worthwhile to focus future strategies in the treatment of leukemia on modulation or activation of proteins that are common in multiple apoptotic pathways, like caspase-8, since this approach will sensitize the leukemic cells simultaneously to chemotherapy and cellular immunotherapy. Show less
