B cell-expressed ILT2 acts as a ligand for a costimulatory receptor for the antigenic activation of Vγ8Vδ3 T cells. (A) The 73R9 γδ T-cell clone (Vγ8Vδ3) was incubated for 4 hours together with different target cells (721.221, B-LCL, Do#AD; Daudi, Raji, and Jurkat) in the absence (open bars) or presence (filled bars) of α-ILT2 mAb (256, 10 μg/mL). CD107a/b mobilization was measured by flow cytometry, and the values for the percentage of CD107a/b⁺ γδ T cells are indicated. − indicates no stimulation; CD3, α-CD3 mAb (UCHT1, 5 μg/mL); and PMA/Iono, phorbol myristate acetate and ionomycin control. (B) Target cells described in panel A were stained with α-ILT2 mAb (256, shaded histograms) or control mAb (IgG1, open histograms) and analyzed by flow cytometry. Values for MFI of ILT2⁺ cells are indicated. The frequency of ILT2⁺ cells within the 721.221 B-cell line is indicated. (C) J.RT3-T3.5 Jurkat T cells expressing the 73R9 TCR (Vγ8Vδ3, filled bars) or not (WT, open bars) were incubated for 4 hours with different human B-cell lines (B-LCL, Do#AD; Daudi, Raji, and 721.221). Surface expression of CD69 on Jurkat T cells was measured by flow cytometry. − indicates no stimulation; and CD3, α-CD3 mAb (UCHT1, 5 μg/mL). Data are MFI of triplicate samples (mean ± SD) and are representative of at least 3 independent experiments. *P < .05. **P < .005. (D) Vγ8Vδ3 (73R9) Jurkat T cells were incubated for 4 hours together with human B cells (721.221 or B-LCL, Do#AD) in the absence (Ctrl) or presence of α-ILT2 mAb (256, 10 μg/mL, ILT2). − indicates no stimulation; and CD3, α-CD3 mAb (UCHT1, 5 μg/mL). Surface expression of CD69 on Jurkat T cells was measured by flow cytometry. Values for the MFI of CD69 stainings are indicated. Data are representative of at least 3 independent experiments.