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. 2014 Nov 25;23(2):396–406. doi: 10.1038/mt.2014.215

Figure 2.

Figure 2

Relapsed tumors do neither demonstrate methylation of antigen gene nor Azacitidine-mediated upregulated expression of antigen protein. HLA-A2 tg mice bearing B16:A2-YLEP tumors were conditioned and treated with T cell receptors (TCR) or mock T cells as described in legend to Figure 1a. (a) Relapsed and progressed tumors derived from B16:A2-YLEP clones were analyzed for methylation of the antigen gene promoter. Following methylation-specific PCR, products were analyzed by gel electrophoresis. U and M denote PCR products specific for unmethylated and methylated promoter sequences, respectively. (b) Relapsed tumors derived from either B16:A2-YLEP clone or cell line (the latter corresponding to group 3, Figure 4) were analyzed for levels of A2-YLEP DNA and mRNA. In vitro cultured B16:A2-YLEP clone and cell line were included as controls. DNA and mRNA levels are presented relative to the endogenous reference gene TRP2, with n = 3–4 per group. (c) HLA-A2 surface expression was measured by flow cytometry following ex vivo treatment of tumor cells with the demethylating agent azacitidine (AZA) (n = 9), IFN-γ (n = 11), or prolonged culture times (n = 4). Data are presented as mean % positive cells in viable gate + SEM. Statistical significances were calculated with Student's t-tests: *P < 0.05.