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Blood, Vol. 113, Issue 12, 2715-2722, March 19, 2009
Hypomethylating drugs convert HA-1–negative solid tumors into targets for stem cell–based immunotherapy
Blood Hambach et al.
113: 2715
Supplemental materials for: Hambach et al
Files in this Data Supplement:
- Figure S1. CpG island analysis of the HA-1 locus (JPG, 41.3 KB)
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A 4 kb DNA sequence spanning from −2 kb and +2 kb of the HA-1 ATG was subjected to CpGisland searcher analysis (http://cpgislands.usc.edu/cpg.aspx). This analysis searches for the presence of CpG islands based on the criteria defined by Takai D and Jones PA (Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U.S.A. 2002;99(6):3740–5). A CpG island was identified between −821 to +311.
- Figure S2. Analysis of the methylation status of the HA-1 promoter in tumor cell lines and hematopoietic cells (JPG, 98.3 KB)
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Genomic DNA extracted from various HA-1neg tumor cell lines was subjected to sodium bisulfite treatment. (A) A long (−341 to +19) and a short (+47 to +222) DNA fragment were amplified by PCR and were subsequently analyzed by sequencing; y-axis: percentage of methylated cytosines in seven to ten independent DNA clones per cell line; x-axis: position of the CpGdi nucleotides with respect to the ATG translation start site (+1). (B) COBRA of tumor cell lines (upper panel) and hematopoietic cells (lower panel); PBMC: peripheral blood mononuclear cells; PHA: Phytohaemagglutinin; PBL: peripheral blood lymphocytes; M: methylation.
- Figure S3. HA-1 mRNA expression levels in response to 5-AZA-CdR (JPG, 46.8 KB)
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HA-1 mRNA expression levels were determined in different cell lines by qPCR after treatment with 10 µM 5-AZA-CdR for 72h; x-axis: tumor cell lines with non-methylated or methylatedHA-1 promoter; y-axis: increase of the HA-1 mRNA expression levels after treatment in relation to unstimulated conditions.
- Figure S4. CTL recognition EBV LCLs (JPG, 60.3 KB)
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All EBV-LCLs are HLA-A2pos. The allo HLA-A2 CTL clone 1E2 lyses all HLA-A2pos target cells. The HA-1 CTL 3HA15 lyses only HA-1pos or HA-1 peptide loaded target cells; x-axis, mean percent specific lysis (2 measurements per condition); y-axis, CTL clones each in 2 different effector–target ratios.
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