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. 2021 Mar 25;9(3):e002035. doi: 10.1136/jitc-2020-002035

Figure 1.

Figure 1

Tumor-associated antigen MAGE-A4 harbors an immunogenic candidate T-cell epitope. (A) Formalin-fixed paraffin-embedded human tumors of different histologic subtype (TNBC n=62, HNSCC n=172, NSCLC n=158, ovarian n=89) were stained with an anti-human MAGE-A4 antibody and evaluated by a study pathologist. The intensity of staining was scored based on a 3-point scale, the percentages of cells assigned to each score were estimated, and an H-score was assigned. Numbers below graph indicate the percentage of samples with an H-score ≥100. (B) CD8+ T cells from an HLA-A*02:01-positive healthy donor were expanded for 12 days by stimulation with autologous monocyte-derived dendritic cells electroporated with ivt-RNA encoding for MAGE-A4. Left dot plot depicts the gating strategy of expanded CD8+ T cells 16 hours after co-culture with MAGE-A4-negative A2+K562 cells for CD137-negative bulk sorting. CD137-negative sorted cells were co-cultured with A2+K562 cells transduced with MAGE-A4; 16 hours later, CD137-positive cells (right) were single cell sorted. (C, D) Single cell sort–derived T-cell clones were expanded for 15 days and tested for reactivity toward MAGE-A4. Shown are bar graphs displaying IFN-γ concentrations in supernatants of two positive T-cell clones 16 hours after co-culture with target cells for screening. (C) Co-culture with A2+K562 cells transduced with MAGE-A4 or an irrelevant protein (irrel. prot.). (D) Co-culture with A2+K562 cells pulsed with different candidate peptides.