Specific binding of ULBP4 to TCRγ9/δ2-OT3-Fc chimeric protein or TCRγ9/δ2-OT3 expressed on transfected J.RT3-T3.5 cells. (A) EL4 cells (i-ii) or EL4-ULBP4 cells (iii-iv) pretreated with (gray filled histogram) or without (black line histogram) anti-ULBP4 mAb 8C9 were stained with human NKG2D-Fc or TCRγ9/δ2-OT3-Fc, followed by FITC-conjugated goat anti–human IgGFc(γ). Ig controls were shown as gray line histogram. PBMC-derived Vδ2 T cells (90% purity, v), pretreated with anti-NKG2D were incubated with rULBP4, followed by FITC-conjugated anti-Histag mAb (vi). TCR⁻ J.RT3-T3.5 cells and TCR transfectants were stained with FITC-labeled anti-TCRγδ mAb (black line histogram) or with control Ig (vii-viii). TCR⁻ J.RT3-T3.5 cells and TCR transfectants were stained with rULBP4 (black line histogram) or without rULBP4 (gray filled histograms), followed by FITC-conjugated anti-Histag mAb (ix-x). (B) Characterization of TCRγ9/δ2-OT3-Fc fusion protein. Daudi cells were stained with human TCRγ9/δ2-OT3-Fc, then followed by FITC-conjugated goat anti–human IgGFc(γ) (i-ii).TCRγ9/δ2-OT3-Fc fusion proteins were analyzed by reducing SDS-PAGE and visualized by Coomassie staining (iii). (C) IL-2 secretion by TCR⁻ J.RT3-T3.5 cells and TCR transfectants, with or without the stimulation of immobilized rULBP4. J.RT3-T3.5 and J.RT3-T3.5-TCRγ9/δ2-OT3 were preactivated with PMA, then incubated with rULBP4 or control protein (NG). After 24 hours, IL-2 in the supernatants was detected by ELISA (R&D Systems). Data represent means ± SD (error bars) of 3 independent experiments. (D) ELISA assay of rULBP4 binding to TCRγ9/δ2-OT3-Fc. Increasing concentration of TCRγ9/δ2-OT3-Fc or control human IgG was incubated with coated rULBP4.