Figure 3

T-cell mediated tumor cell killing elicited by hMSC-produced anti-CD33-anti-CD3 recombinant antibody is enhanced by co-stimulation from 4-1BBL. (a) For the analysis of the 4-1BBL transgene expression hMSCs were stained with PE-conjugated anti-CD137L antibody and the surface expression of the immunoligand as well as intracellular EGFP signal correlating with bsAb CD33–CD3 expression were analyzed by flow cytometry. Positive cells are shown as percentages of all analyzed cells. Dead cells were excluded by propidium iodide counter-staining. Quadrant position was placed based on isotype control staining (not shown). (b) In a flow cytometry-based cytotoxicity assay eFluor670 proliferation dye-labeled CD33⁺ OCI-AML3 cells were incubated with pan T cells at an e:t ratio of 1:1 for 24 h, 48 h (upper) and 96 h (lower) in the presence or absence of gene-modified hMSCs seeded at limiting densities 48 h before the experiment. Target cell numbers counted at the indicated time points were normalized to the control sample with only target cells. Data represent the means±s.d. of three different donors. Statistical significance was determined using one-way analysis of variance with Bonferroni multiple comparison test. **P<0.01.