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. 2017 Jun 20;9(8):1067–1087. doi: 10.15252/emmm.201707567

Figure 3. Identification of which T cells are responsible for BiTE‐mediated cytotoxicity.

Figure 3

  1. BiTE‐mediated T‐cell activation of CD4 and CD8 cells 24 h after co‐culture of CD3 T cells with DLD cells (5:1) and BiTE‐containing supernatant. Activation was assessed by surface expression of CD69 and CD25 and measured by flow cytometry.
  2. Proliferative response of CFSE‐stained CD4 and CD8 T cells in co‐culture with DLD cells and incubated with BiTE‐containing supernatants. Fluorescence was measured after 5‐day incubation, by FACS analysis.
  3. Degranulation of CD4 and CD8 cells following 6‐h co‐culture with DLD cells and BiTE‐containing supernatants. A CD107a‐specific antibody is added to the culture media for the duration of the co‐culture, and degranulation is assessed by flow cytometry.
  4. Cytotoxicity by either the CD4 or CD8 T‐cell subset is assessed by LDH release into supernatant, following 24‐h incubation of DLD cells with CD4‐ or CD8‐purified T cells (1:5) and BiTE‐containing supernatant.
Data information: Each condition was measured in biological triplicate and represented as mean ± SD. EpCAM BiTE treatment was compared to control BiTE unless stated otherwise, and significance was assessed using a one‐way ANOVA test with Tukey's post hoc analysis, **P < 0.01, ***P < 0.001. Source data are available online for this figure.