Abstract
Haematopoietic stem cell transplantation (HSCT) often is a final treatment option for patients suffering from haematological malignancies and metabolic disorders. When HSCT is applied to treat leukaemia, relapse of the disease is a recurrent complication. Donor lymphocyte infusion (DLI) with T cells is then administered to eliminate these tumour cells.
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The disadvantage of this treatment is the increased risk of developing graft-versus-host disease (GVHD). Natural killer (NK) cells are lymphocytes capable of target cell lysis without prior sensitization. They specifically eliminate malignant cells that have downregulated their human leukocyte antigens (HLA) to escape lysis by T cells. The NK cell receptors responsible for detecting HLA downregulation are the Killer cell immunoglobulin-like receptors (KIRs). NK cells are considered a new treatment modality for prevention of relapse after HSCT, without inducing GVHD. In this thesis, various aspects of the complex characteristics of KIRs and the influence of interactions with their ligands were studied. The NK cell treatment for patients undergoing HSCT aims for the induction of an anti-leukaemic response without causing GVHD. For this purpose, the KIR repertoire of the donor is compared with the HLA repertoire of the patient. An inhibitory KIR, mismatched for its HLA ligand in the patient, will induce a NK cell alloresponse, a mechanism known as KIR-ligand incompatibility. The current criteria in the donor selection procedure do not account for NK cell-induced alloresponses. Two retrospective studies were conducted analysing the role of KIR genotypes and HLA repertoires on the clinical results after matched unrelated donor (MUD) HSCT and HLA-identical sibling HSCT. The MUD HSCT study was the first to evaluate not only the selected donor but also the potential, screened donor. It uniquely illustrates that the addition of KIR genotyping data to the current donor selection criteria, provides good evidence for the selection of another donor with improved clinical outcome for the patient. The data of the HLA-identical sibling panel reveal significant correlations for activating KIRs with overall survival and relapse. The activating KIRs, which are often not considered in NK alloreactivity prediction, should be added to the donor selection criteria because they significantly affect transplantation outcome. Recognition of HLA-C molecules by KIRs is an important mechanism in the regulation of NK cell activity. KIRs distinguish between two epitopes of the HLA-C molecule, the HLA-C group 1 and group 2 epitopes. A quick and reliable Q-PCR approach was developed which introduces an HLA-C epitope screening method to define the presence of ligand for the KIR-HLA-C interaction. KIR polymorphism contribute to KIR diversity which could affect reactivity of the NK cells. We addressed KIR2DL4 gene polymorphism by a newly developed DNA and cDNA based direct sequencing based typing (SBT) and cloning approach. This approach is applicable for routine KIR2DL4 allele typing and enables the characterisation of new KIR2DL4 alleles. Immunotherapy with NK cells requires transfusion of large quantities of the cells which obviates the need for an in vitro culture system for NK cells. This study showed that in vitro expansion of NK cells is dependant on the HLA-C environment. Cytolytic capacities of the cultured NK cells are maintained which supports the exploration of this method to expand NK cell numbers and the applicability of NK cell-based immunotherapy.
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