mAb 256 specifically recognizes the LILRB1/CD85j/ILT2 receptor. (A) Expression of Ag#256 was analyzed on 721.221 human B cells by flow cytometry. Ag#256⁺ and Ag#256⁻ B cells were next isolated by cell sorting and cloned. Values for the frequency of Ag#256 ⁺ and Ag#256⁻ cells within each cell subset are indicated in the quadrants. (B) Vγ8Vδ3 T cells (clone 73R9) were incubated together with Ag#256⁺ (39) and Ag#256⁻ (21) 721.221 B-cell clones (ratio: 1/1) in the absence (Ctrl, left) or presence (right) of mAb 256 (10 μg/mL). CD107a/b mobilization was measured by flow cytometry within activated γδ T cells. Values for the percentage of CD107a/b⁺ T cells are indicated in the quadrants. (C) Seven human B-cell lines and a negative control human renal carcinoma line (786–0) were stained with either α-ILT2 specific mAb (HP-F1, 10 μg/mL) or mAb 256 (10 μg/mL). Correlation coefficient (R²) calculation between ILT2 and Ag#256 mean fluorescence intensities (MFIs) obtained after analysis by flow cytometry is indicated on the graph. (D) Human osteosarcoma cells (U2OS) were stably transfected with either empty (left) or human ILT2 cDNA (right) plasmids, costained with α-ILT2 specific mAb (HP-F1, 10 μg/mL) and mAb 256 (10 μg/mL), and analyzed by flow cytometry. (E) The binding of both α-ILT2 specific mAb (HP-F1) and mAb 256 to recombinant human ILT2-Fc molecule (106 kDa) was analyzed by immunoblotting. Ctrl indicates secondary mAb alone. (F) Human B-LCL (Do#AD) and Ag#256⁺ 721.221 B cells (clone 39) were preincubated for 15 minutes with either α-ILT2 mAb (HP-F1, ●) or mAb 256 (○) at the indicated increasing concentrations. Vγ8Vδ3 T cells (clone 73R9) were next incubated for 4 hours together with B cells (ratio = 1:1). Values for the percentage of CD107a/b⁺ γδ T cells are indicated in the graph. Data are representative of at least 3 independent experiments.