Effect of immobilized rULBP4 on human γδT cells. (A-B) Expansion of human γδT cells from (A) colonic carcinoma specimen or (B) ovarian epithelial carcinoma specimen TILs after stimulation with immobilized rULBP4 in vitro. Flow cytometric analysis by FITC-labeled anti-TCRγδ mAb shows the proportions of γδT cells (A-B upper panel). The phenotype of rULBP4-expanded γδ TILs was analyzed by anti-Vδ1 FITC/anti-Vδ2 FITC mAb/anti-Vδ3 FITC mAb (A-B lower panel). Immobilized anti-pan-TCRγδ mAb, immobilized rULBP3/LZ, immobilized rMICA, or tissue culture with IL-2 alone was used to expand γδT cells. (C) Cytokines secreted by OEC-Vδ2 T cells or PBMC-Vγ9/δ2 T cells before and after stimulation with rULBP4. Forty-eight–well plates were coated with anti–pan-γδTCR mAb, rULBP4, or control other human recombinant proteins (NG). PBMC-Vδ2 T cells or OEC-Vδ2 T cells (10⁶ cells/well) were cultured in a total volume of 500 μL complete medium plus 50 UI/mL IL-2. After 40 hours, cytokine in cell-free supernatants was determined by ELISA. Data represent means ± SD (error bars) of 3 independent experiments. (D) Anti-ULBP4 mAb blocks OEC-Vδ2 TIL cytotoxicity against the EL4-ULBP4 cells. In blocking assay, EL4-ULBP4 or EL4 cells were preincubated with anti-ULBP4 mAb 8C9.