Target-dependent T cell–mediated cytotoxicity of HER2-TDB.

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Supplemental Fig. 1: Characteristics of T cell activation and killing induced by HER2-TDB A) T cell activation was detected at various timepoints by staining.

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Target-dependent T cell–mediated cytotoxicity of HER2-TDB. Target-dependent T cell–mediated cytotoxicity of HER2-TDB. A, T-cell activation was detected by staining cells for CD8/CD69/granzyme B followed by FACS analysis. Effectors CD8+ T cells, target SKBR-3, E:T ratio 3:1, time point of 48 hours. Data presented as mean of two repeats. B, soluble granzymes and perforin were detected from the media using ELISA and cytotoxicity using LDH release assay. Effectors PBMC, target SKBR-3, E:T ratio 30:1, time point of 18 hours, Abs 10 ng/mL. C, elevated caspase activity (Caspase-3/7 Glo Assay) and apoptosis (Cell Death Detection ELISAplus assay) correspond with LDH release after treatment with 1 ng/mL bispecific antibody. Effectors PBMC, targets SKBR-3, E:T ratio 10:1, time point of 24 hours. Error bar, SD in C–F. D, cytotoxicity on HER2 (red) or vector-transfected (blue) 3T3. Effectors PBMC, E:T ratio 10:1, time point of 19 hours LDH release assay. E, HER2-arm binding was blocked using trastuzumab Fab (1 μg/mL, black) or soluble HER2 extracellular domain (1 μg/mL, blue). Effectors CD8+ T cells, target BT474, E:T ratio 5:1, time point of 24 hours, LDH release assay. F, killing activity of PBMC before and after depletion of CD3+ cells. Target SKBR3, E:T ratio 20:1, time point of 19 hours, FACS assay. Data points in the figure represent the mean of three samples; error bar, SD. Teemu T. Junttila et al. Cancer Res 2014;74:5561-5571 ©2014 by American Association for Cancer Research