GÉTICA 2021

53 [ F I T C á n c e r - 7 ] Figure 1. Comparative in vitro study of engineered STAb-T19 and CAR-T19 cells. (a) Western blot detection of secreted 19-BiTE in the conditioned media from lentivirally-transduced human primary T cells (STAb-T19). Conditioned media from non-transduced T cells (NT-T) and blinatumomab (BLI) were used as negative and positive controls, respectively. One representative experiment is shown. (b) Represen- tative analysis of intracellular and cell surface-bound 19-BiTE, and cell surface-expressed 19-BBz CAR in NT-T and engineered CAR-T19 and STAb-T19 cells by flow cytometry. One representative experiment out of three independent experiments is shown. (c) Reporter protein ex- pression in STAb-T19 cells (tdTo) and CAR-T19 cells (GFP). One representative transduction out of three performed is shown. Percentage of CD4+ and CD8+ T cells (d) and naïve (TN), central memory (TCM), effector memory (TEM) and effector (TE) T cells (e) among NT-T, CAR-T19 or STAb-T19 cells 7 days after transduction (means ± SD of three independent experiments are shown). (f) Representative images of immu- nological synapse (IS) assembly by primary CAR-T19 and STAb-T19 cells stimulated for 15 minutes with CMAC (blue)-labelled CD19+ cells. Distribution of CD3ε (green channel) and actin (red channel) at the mature IS, as well as the merged images, are shown. The IS topology obtained from the 3D reconstructions of regions of interest in confocal stacks containing the red and the green channels is shown. (g,h) Real-time cell cytotoxicity assay: HEK-293CD19 target cells were co-cultured with NT-T, CAR-T19 or STAb-T19 cells at the indicated effec- tor: target (E:T) ratios, and cell index values were determined every 15 minutes for 65 hours using an impedance-based method. (g) Cell viability kinetics of HEK-293CD19 over time are shown, and (h) presented as percentage lysis normalized to NT-T cells as well (E:T ratio = 0.5:1). Results from one representative experiment performed in duplicate are shown. (i) Ability of secreted 19-BiTE to induce bystander T cell proliferation: decreasing numbers of cell trace violet (CTV)-stained activated T (A-T) cells (NT-T, CAR-T19 or STAb-T19) were co-cultured with NALM6 or HeLa target cells and increasing numbers of CFSE-stained freshly isolated non-activated T cells (NA-T). The total E:T ratio was constant (2:1), but the ratios A-T: target and A-T:NA-T varied as indicated. After 5 days, CTV or CFSE dilution was analyzed by flow cy- tometry to measure proliferation of A-T and NA-T cells, respectively. Each histogram displays the percentage of dividing cells. The number of cell divisions are indicated in parentheses. One representative experiment from three independent experiments is shown. (j,k) Cytotoxic activity and IFNg secretion of A-T cells (NT-T, CAR-T19 and STAb-T19). Decreasing numbers of A-T cells were co-cultured with NALM6Luc or HeLaLuc target cells and increasing numbers of NA-T cells from the same donor, maintaining a constant 2:1 E:T ratio. After 48 hours, (k) the percentage of cytotoxicity was calculated by adding D-luciferin to detect bioluminescence and, (j) IFNγ secretion was determined by ELISA. Data are mean ± SD of three independent experiments. (l) Similar co-cultures were performed in a non-contacting transwell system; 5 × 104 target cells (NALM6Luc or HeLaLuc) and 1 × 105 NA-T cells were plated in the bottom well and decreasing numbers (from 1 × 105 to 1 × 101) of A-T cells (NT-T, CAR-T19 or STAb-T19) in the insert well. After 48 hours, (m) the percentage of cytotoxicity was determined by luciferase assay, and IFNγ production was determined by ELISA (n). Data are shown as mean ± SD from four and three similar experiments, respectively. Significance was calculated by an unpaired Student t test.