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Selected Publication:

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Type of publication: Master Thesis
Type of document:

Year: 2013

Authors: Betz, Nina

Title: Preparation of an HLA-A2 expressing variant of the TEL-AML1+ REH B cell precusor ALL cell-line.

Other title: Generieren einer TEL-AML1+ REH B precursor ALL Zelllinie die HLA-A2 exprimiert

Source: Master Thesis, Vet. Med. Univ. Wien, pp. 72.


Advisor(s):

Thompson Pamela
Saalm├╝ller Armin

Reviewer(s):
Sexl Veronika

Vetmed Research Units:
Institute of Immunology


Graduation date: 20.02.14


Abstract:
Leukaemia is the most common malignant disease of children in the United Kingdom, Europe and the USA. Acute lymphoblastic leukaemia (ALL) is the most frequent childhood leukaemia, with a 80-90% survival rate of 5 years. This project concers B cell precursor ALL (BCP-ALL) the most common variant of ALL cases. Fusion of the TEL (ETV6) and AML1 (RUNX1) giving rise to the TEL-AML1 chimaeric oncogene are found in ~25% childhood BCP ALL cases. Expression of a novel TEL-AML1 oncoprotein by TEL-AML1+ BCP-ALL cells make this a possible target for HLArestricted T cell immunity The aim was to generate tumour cell lines that express HLA-A*02:01 in order to generate a T cell response from HLA-A*02:01 restricted T cell receptors (TCRs). A cDNA encoding HLA-A*02:01 was cloned into the mammalian vector (pcDNA3.1V5 His-A) in order to express this HLA-A*02:01 molecule in the TEL-AML+ HLAA0201- BCP-ALL line REH and also control tumour cell lines including HT1080 (Fibrosarcoma) and LoVo (adenocarcinoma). Transfected cells were selected using the co-expressed neomycin phosphotransferase gene in combination with the neomycin analogue, G418. Initially, cell killing curves were generated to determine the sensitivity of each cell line to G418. In an alternative approach, a lentiviral vector expressing HLA-A*02:01 was also generated. This lentiviral vector was generated by transient co-transfection of 293T cells with the necessary packaging constructs and the generated vector used to transduce target cell lines. In order to confirm that the HLA-A*02:01 gene was expressed in a functional manner, primary human T cells were transduced to express a recombinant HLA-A*02:01 restricted T cell receptor and expanded in culture to perform functional assays against the HLA-A*02:01 expressing tumour cell lines. Preliminary studies have used well characterised melanoma targets to functionally validate the system (i.e. using the NY-ESO-1 target system). Future plans intend to exploit the HLA-A*02:01 REH cells as a model to investigate CD8+ restricted T cell responses against the TEL-AML1 antigen.


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