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Regulation of expression of ULBP1, a ligand for the activating immunoreceptor NKG2D, and its influence on the cytotoxic function of natural killer cells against human leukemia

Himmelreich, Heike. Regulation of expression of ULBP1, a ligand for the activating immunoreceptor NKG2D, and its influence on the cytotoxic function of natural killer cells against human leukemia. 2012, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9937

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Abstract

Human natural killer (NK) cells are innate immunity CD56+CD3- lymphocytes, mediating spontaneous killing of tumor-transformed cells. The effector functions of NK cells are regulated by a balance of signals initiated from a variety of activating and inhibitory receptors. Recognition of HLA class I molecules on the surface of target cells by inhibitory NK cell receptors (e.g. KIR and NKG2A) ensures that healthy ,self’ cells are protected from NK cell lysis. The loss of HLA class I molecules as a consequence of tumor transformation renders cells susceptible to NK-cell mediated lysis. The cytolytic function of NK cells is dependent on activating receptors, which become engaged by specific cell surface molecules expressed on target cells.
The best characterized activating immunoreceptor is NKG2D, which triggers cellular cytotoxicity and cytokine production upon engagement with its ligands, ULBP and MICA/B molecules. NKG2D ligands (NKG2D-L) are rarely expressed on healthy cells, but frequently upregulated in response to cellular changes caused by malignant transformation. In healthy tissue, inappropriate overexpression of NKG2D-L can trigger autoimmunity. This indicates that NKG2D-L expression has to be tightly regulated to avoid destruction of untransformed tissue, but at the same time to allow recognition and elimination of tumor cells. The molecular mechanisms controlling NKG2D-L expression are poorly understood. Numerous studies demonstrated that different cells and tissues express NKG2D-L transcripts but lack any expression of the corresponding proteins on the cell surface. These findings suggest that NKG2D-L are, at least partly, regulated at post-transcriptional level.
In the first part of the thesis, we examined the involvement of post-transcriptional mechanisms in regulation of the NKG2D-L, ULBP1. Analysis of the 2.4 kb-long ULBP1-3’UTR revealed the presence of four ARE motifs and more then 200 putative microRNA binding sites, regulatory elements which mediate RNA degradation and translational repression. Using luciferase reporter assays, we showed that the full-length 3’UTR of ULBP1 is markedly involved in regulation of ULBP1 gene expression in human cancer cell lines and human primary foreskin fibroblasts. The involvement of ARE elements in negative regulation of ULBP1 gene expression was excluded by mutating ARE motifs in two regions of the ULBP1-3’UTR. The role of candidate microRNAs in regulation of ULBP1 was examined by mutating the putative microRNA binding sites, or by silencing or overexpression of candidate microRNAs. However, we could neither support nor disprove our hypothesis that microRNAs are involved in regulation of ULBP1 expression, and therefore further studies are needed to elucidate the role of microRNAs. Despite the inconclusive outcome on the microRNA studies, our work provided the first evidence that the regulation of ULBP1 expression takes place on a post-transcriptional level and involves the ULBP1-3’UTR as mediator of RNA destabilization and translational repression.
In the second part of the thesis, we investigated the effect of histone deacetylase inhibitors (HDACi) on surface expression of NKG2D-L in primary human fibroblast (HFF) and AML blasts. Treatment of HFF with trichostatin A (TSA) increased surface expression and transcript level of ULBP1. By using luciferase assay we revealed, that post-transcriptional mechanisms might participate in the upregulation of ULBP1 expression. Treatment of AML blasts with valproic acid (VA) also induced surface expression of NKG2D-L resulting in enhanced killing by NK cells. Efficient cytolysis of AML blasts was achieved by generating alloreactive NK cell lines with KIR-HLA class I mismatch, which allowed to circumvent the signaling by inhibitory NK cell receptors.
Taken together, these data demonstrate, that ULBP1 is regulated by post-transcriptional mechanisms and that the activation of NK cell can be augmented by pharmacological induction of NKG2D-L and the use of alloreactive NK cells.
Advisors:Palmer, Ed
Committee Members:Wodnar-Filipowicz, Aleksandra and Grosshans, Helge
Faculties and Departments:03 Faculty of Medicine > Bereich Medizinische Fächer (Klinik) > Nephrologie > Exp. Transplantationsimmunologie und Nephrologie (Palmer)
03 Faculty of Medicine > Departement Klinische Forschung > Bereich Medizinische Fächer (Klinik) > Nephrologie > Exp. Transplantationsimmunologie und Nephrologie (Palmer)
Item Type:Thesis
Thesis no:9937
Bibsysno:Link to catalogue
Number of Pages:153 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:49
Deposited On:25 Jul 2012 14:45

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