Fig. 3
ɑ-BTN2A1 mAbs antagonize Vγ9Vδ2 activation through different mechanisms, including blocking BTN2A1-TCR binding. (a) Inhibition of activation of G115-TCR expressing Jrt-3.5 cells after co-incubation with DaudiΔAAVS1 cells pre-incubated with 5 μM HMBPP and 6.8 nM (1 μg/mL) ɑ-BTN2A1 mAb or controls (MOPC isotype, 20.1 mAb, 103.2 mAb or Buffer + 5 μM HMBPP). CD69 expression was assessed by flow-cytometry (gated on isotype antibody) and normalized to Buffer + 5 μM HMBPP condition (100*Value-Baseline/Baseline). Mean + SD (n = 6, 2 independent experiments). Dunnett test pairwise comparison to – mAb control: ****p < 0.0001, ***p < 0.0002. (b) Inhibition of activation of Vγ9Vδ2 T cells expanded from primary PBMCs after co-incubation with DaudiΔAAVS1 cells pre-incubated with 5 μM HMBPP and increasing concentrations of ɑ-BTN2A1 mAb or controls (MOPC isotype, 103.2 mAb or Buffer + HMBPP). CD25 (top), CD107a (middle) and CD69 (bottom) expression were assessed by flow-cytometry (gated on isotype antibody). Mean + SD (n = 3). (c) Competition between ɑ-BTN2A1 mAbs and the Vγ9Vδ2 TCR for binding to BTN2A1 ectodomain assessed in High-Five cells expressing full-length BTN2A1 stained with 150 nM ɑ-BTN2A1 mAbs or controls (no TCR tetramer, MOPC isotype or TCR tetramer + Buffer followed by 120 nM fluorescently-tagged Vγ9Vδ2-TCR tetramers (G115 clone). TCR tetramer staining was assessed by flow-cytometry (gated on no TCR) and normalized to TCR tetramer, − mAb condition (100*Value-Baseline/Baseline). Mean + SD (n = 6, 2 independent experiments). Dunnett test pairwise comparison to Buffer control: ****p < 0.0001. (d) Correlation between ɑ-BTN2A1 mAb Vγ9Vδ2 antagonism (Baseline-corrected CD69 expression) and Vγ9Vδ2 TCR-competition (Baseline-corrected TCR tetramer staining) as calculated by simple linear regression (R2 = 0.136, p = 0.1944). Strong and weak TCR blocking designated as lower and upper half of the range of TCR tetramer staining, respectively. TCR inhibition classified based on p values from (a). (e) Vγ9Vδ2 TCR competition with ɑ-BTN2A1 Fab or control (20.1 Fab) for binding to BTN2A1 ectodomain assessed by BLI, normalized. Immobilized biotinylated BTN2A1 ectodomain was exposed to 1. 66 μM Fab followed by 2. 66 μM Fab + 80 μM TCR (G115 clone). Step 2 Binding (nm) of TCR to biotinylated BTN2A1 ectodomain with Fab was normalized to Fab alone binding (nm) at the end of Step 1. Bars and circles outlined in black indicate the representative reagent for each class of mAb.
