Fig. 34 (abstract P297).

HLA-A2.1+ donor-derived CTLs specifically recognize HLA-A2.1+ K27M+ glioma cells in an HLA-class I-dependent manner. Peripheral blood mononuclear cells from an HLA-A2.1+ donor were stimulated in vitro with the H3.3.K27M peptide and evaluated for their reactivity against: a HLA-A2.1/H3.3.K27M-specific tetramer and anti-CD8 mAb, and (b) T2 cells pulsed with the mutant or non-mutated H3.3 peptide by IFN-γ ELISA. In (a), among the CD8+tetramer+ population (64.1% of total lymphocyte-gated cells), there is a tetramer^high subpopulation (2.4% of total lymphocyte-gated cells), some of which were used as CTL clones (Aim 2). In (b), the Cap1-6D peptide (tested at 5mcg/ml only) is a high avidity HLA-A2.1-binding epitope derived from CEA1 used as an irrelevant negative control. c The CTL line was evaluated for cytotoxicity against glioma cell lines T98 (HLA-A2.1+but K27M-negative), HSJD-DIPG-07 (HLA-A2.1-negative but K27M+), and HSJD-DIPG-13 (HLA-A2.1+ and K27M+) lines. CFSE-labeled target cells (10e4/well) were incubated with CTLs at the E/T ratio of 25 for 4 hours. To block the CTL cytotoxicity, anti-HLA-ABC 10μg/ml was added to one group. At the end of incubation, 7-AAD was added into each well and incubated for 10 minutes on ice. The samples were analyzed by flow cytometry, and the killed target cells were identified as CFSE+ and 7-AAD+ cells. The cytotoxicity was calculated as the percentage of CFSE+ and 7-ADD+ cells in total HLA-A2+ CFSE+ cells (*p<0.05 by Wilcoxon rank-sum tests)