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

Figure 1. Characterisation of EpCAM BiTE and its effects on PBMC‐derived T cells.

Figure 1

  1. Schematic of the structure of the EpCAM‐targeted BiTE and non‐specific control BiTE. The VL and VH domains are connected with flexible peptide linkers (L) rich in serine and glycine for flexibility and solubility. SP, light chain immunoglobulin signal peptide; His, decahistidine affinity tag.
  2. Induction of activation markers (i) CD69 and (ii) CD25 on CD3‐purified PBMC cultured alone or with DLD cells (5:1) in the presence of BiTE‐containing supernatants. CD69 and CD25 were measured by flow cytometry after 24 h of co‐culture. Significance was assessed versus IgG isotype.
  3. Percentage of IFNγ‐positive T cells after 6 h in co‐culture with DLD cells (5:1) and BiTE‐containing supernatants.
  4. Proliferation, represented by division index and percentage of parental T‐cell population entering proliferation, of CFSE‐stained T cells in co‐culture with DLD cells (5:1) and BiTE‐containing supernatants. Fluorescence was measured by flow cytometry 5 days after co‐culture. Division index was modelled using FlowJo proliferation tool.
  5. Degranulation of T cells, measured by CD107a externalisation, in co‐culture with DLD cells (5:1) and BiTE‐containing supernatants. Externalisation was assessed by co‐culture with a CD107a‐specific antibody for 6 h followed by flow cytometry analysis.
  6. Cytokine levels were measured by LEGENDplex human Th cytokine panel using supernatants from co‐cultures of T cells with DLD cells (5:1) in the presence of BiTE‐containing supernatants for 48 h.
Data information: Each condition was measured in biological triplicate and data represented as mean ± SD. Significance was assessed versus untreated unless stated otherwise using a one‐way ANOVA test with Tukey's post hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001.Source data are available online for this figure.