Abstract B21: Dendritic cells loaded with amplified mRNA isolated from leukemic stem-cell-like cells induce specific T-cells
Authors: Bontkes Hetty J. , Ruben Jurjen M. , van den Ancker Willemijn A. , Westers Theresia M. , Ossenkoppele Gert J. , van de Loosdrecht Arjan A. 
Published: January 2011

Abstract
It is thought that recurrent acute myeloid leukemia (AML) originates from chemotherapy resistant quiescent leukemia initiating or leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38-LSC can be identified using malignant stem cell associated cell surface markers including C-type lectin-like molecule-1 (CLL-1) and lineage markers such as CD7 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. We have made use of the HLA-A2 positive CD38-AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ stem cell like cells as well as CLL1+CD34-CLL1-CD34+ and CLL1-CD34-cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Amplification of CLL1 and survivin transcripts was confirmed by RT-PCR. Mature monocyte derived DC (MoDC) were generated from HLA-A2 positive healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8 + T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed either by IFNγ production upon stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from total MUTZ-3 cells or sorted subpopulations, or by cytotoxicity towards MUTZ-3 subpopulations. The generated T-cells specifically produced IFN in response to MUTZ-3(subpopulation) mRNA loaded iDC but not in response to GFP mRNA transfected DC. Furthermore, the generated CTL killed the CLL1+CD34+ and CLL1+CD34-populations more efficiently compared to the CLL1-CD34+ population. We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the more mature subsets. The efficacy and feasibility of this approach is currently tested in an autologous AML setting in vitro.
Citation Information: Clin Cancer Res 2010;16(7 Suppl):B21