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. 2016 Feb 18;5(5):e1138199. doi: 10.1080/2162402X.2016.1138199

Figure 4.

Figure 4.

CTLs re-directed with CD20p/HLA-A2-reactive TCRs target primary lymphoma and leukemia cells. PBMC were transduced with either A94mod or A23mod TCRs, expanded and color-coded as described in Fig. 2A. (A) CTLs were co-cultured with primary follicular lymphoma (FL) cells from 2 HLA-A2pos patients and one HLA-A2neg patient and degranulation responses were measured as described in Fig. 2A. (B) Bar graphs show frequencies of CD107a,bpos events among CD8pos T cells upon co-culture with patient-derived FL cells (left) or chronic lymphocytic leukemia (CLL) cells (right), corrected for TCR transduction efficiencies of 50–80%. Results from two separate experiments are shown (FL and CLL, respectively). Bars represent mean values of duplicate (CLL) or triplicate (FL) samples of one experiment (n = 1) with values for CTLs alone subtracted, and error bars represent SD. (C) Flow cytometric analysis strategy in cytotoxicity assay. Upper plot: A gate is set to include beads and tumor cells, which can be separated from CTV-labeled T cells (green dots). Middle plots: Gates identify SSChi FITC-labeled counting beads (red dots) used for quantification of target cells (black dots), including lysed and disintegrated target cells. Lower plots: Only tumor cells are shown. Gates identify the fraction of live tumor cells, which are negative for the apoptosis marker activated caspase-3 (FITC) and the dead cell marker live/dead aqua. (D) Percentage of CTL-induced lysis of the DLCL2 and FSCCL lymphoma cell lines after 4 h incubation with either A94mod or A23mod TCR transduced T cells at different effector cell/target cell ratios, as indicated. Samples were analyzed as shown in 4(C) and the % specific CTL-induced target cell lysis was calculated according to the formula in Materials and Methods. HLA-A2pos CD4pos T cells were used as a CD20neg control and were not lysed unless loaded with the CD20 peptide. Data represent mean±SD of triplicates of one experiment, representative of a total of five performed including DLCL2 and FSCCL cells.