Research group at immunology department in University Medical Centre (UMC) Utrecht, Netherlands, developed a pre-clinical dendritic cell vaccine derived from cord blood CD34+ hematopoietic stem cells. The vaccine will be used in an upcoming clinical trial to prevent relapses in acute myeloid leukemia pediatric patients after receiving chemotherapy and cord blood stem cell transplantation. Since the oncoprotein Wilms’ tumor 1 (WT1) is overexpressed in more than 90% of pediatric leukemia cases, it will be the target of our vaccine strategy. Dendritic cells, loaded with WT1 antigen, either in the form of full length mRNA or HLA class I restricted Wilms’ tumor 1 peptides, cause induction of WT1 specific cellular immunity which enables CD8+ T lymphocyte mediated cytotoxic killing of leukemia blasts remaining in the patient after transplantation. Eradication of last leukemic cells is the key step to prevent relapses and high mortality in patients with acute myeloid leukemia.
The objective of our study was to elucidate the optimal antigen loading strategy of dendritic cells for the induction of WT1 specific CD4+ and CD8+ T lymphocytes, which can enhance the antigenic potential of the vaccine and consequently induce long-term immunological memory. As such, we optimised a 4 hour long cytotoxic assay using recombinant T cell receptor CD8+ and CD4+ T lymphocytes as effector cells, specific for Wilms’ tumor 1 126-134 epitope and human leukemia cell lines as target cells, labelled with CellTrace Violet fluorescent viability marker. With this method we compared the cytotoxic activity of tumor cells loaded with full length mRNA or 15 amino acid long peptides, where the latter seemed superior to elicit T cell mediated cytotoxic killing of loaded tumor cells. We performed a 24hour T cell activation assay as well as ELISA assay and came to the same conclusions. Afterwards we generated 4 DNA constructs coping for increasing sizes of amino acid sequences, where we took a known Wilms’ tumor 1 epitope ( a.a.126-134) RMFPNAPYL, which specifically binds to HLA-A2 molecules as the template of our designs. We successfully determined the flanking primers and amplified the DNA sequences using the PCR method. We ligated these fragments into the pcDNA 3.1 plasmid vector and transduced them into Escherichia Coli. Unfortunately, despite our best efforts and multiple trials, we could not detect the correct plasmid DNA fragments after isolation of grown bacterial colonies.
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