Figure 3
The combination of γ9δ2 T cells and CD19BiTE enhances the lysis of C19+ B-lineage cell lines in vitro. (a) Cytotoxic assays were conducted at an effector to target ratio (E: T) of 20:1, and the percentage of target cell lysis was determined by flow cytometry analysis. CD19-positive (Daudi, Raji, Rs4;11, VAL) and -negative (RPMI-8226) B-lineage tumor cell lines were labeled with PKH26 and CFSE fluorescence dyes and used as target cells, and the expanded γ9δ2 T cells (derived from three donors) were used as effector cells. Each labeled target cell line (1 × 104) was incubated with CD19BiTE (0.25 ng/µl), γ9δ2 T cells (2 × 105), or γ9δ2 T cells (2 × 105) + CD19BiTE (0.25 ng/µl) at a volume of 100 µl for 5 h, and then its lysis percentage was determined by flow cytometry. Data were plotted as mean ± s.d., and the statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. ***: p = 0.002, ****: p < 0.0001, n.s.: not significant. The replication number (n) in each treatment group was indicated. Black bar (■): tumor cell lines were incubated with CD19BiTE; White bar (□): tumor cell lines were incubated with γ9δ2 T cells; Gray bar (■): tumor cell lines were incubated with γ9δ2 T cells + CD19BiTE. (b ~ m) The cytotoxic activity of a donor’s expanded γ9δ2 T cells was examined by detecting the degranulation marker CD107a. At an effector to target ratio (E: T) of 1:1, three target cell lines (Daudi, Raji, RPMI-8226; 1 × 105) were incubated with γ9δ2 T cells (Daudi: b, c; Raji: f, g; RPMI-8226: j, k), or γ9δ2 T cells + CD19BiTE (0.25 ng/μl) (Daudi: d, e; Raji: h, i; RPMI-8226: l, m) at a volume of 100 µl for 6 h, and then the cell mixtures were subjected to flow cytometry analysis using a PE-labeled anti-CD107a and an FITC-labeled anti-Vγ9 antibodies. On the CD107a versus Vγ9 dot plots (b, f, j, d, h, l), Vγ9 positive counts were gated for evaluating the percentage of CD107a positive cells among Vγ9 positive cells on the forward scatter (FSC) versus CD107a dot plots (c, g, k, e, i, m). Images were created using FlowJo software 10.0.0r1.
