Figure 4

#21-3BiTE mediates T-cell activation and proliferation. (a) #21-3BiTE activates naïve T-cells by bridging T cells and target cells. PBMC from HLA-A*24⁺ healthy donor were mixed with or without WiDr cells at an E:T ratio of 10:1 in the presence of #21-3BiTE, 21-3 or control mouse IgG, and the reactions were allowed to proceed for 48 h. Cells were labeled with fluorescently coupled antibodies to CD4, CD8, and CD69 and were analyzed by FACS. Each FACS histogram shows CD69 binding. Representative FACS histograms (left) and frequency of CD69-positive cells in CD4⁺ or CD8⁺ cells are shown (Right). Data are representative of two independent experiments (n = 3). (b) T cells expanded from a HLA-A*24⁺ healthy donor were labeled with CellTrace-Blue. Labeled T-cells were mixed with or without WiDr cells at an E:T ratio of 10:1 in the presence of the indicated antibody, and the reactions were allowed to proceed for four days. CellTrace-Blue⁺ cells analyzed by FACS for proliferation are shown. Representative data from two independent experiments are shown. (c) Cell culture supernatants in (b) were analyzed for IFNγ by ELISA. Values are represented by the means of two independent experiments for two replicates/group.