BTLA co-clustered with Vγ9Vδ2TCR after activation. (A) Distribution of BTLA and Vδ2TCR on resting (left panel) or BrHPP-activated γδ T cells (right panel). Cells were first fixed and permeabilized, and then stained with anti-BTLA (clone 7.1, IgG2b, red) and anti–TCRVδ2-FITC (IgG1, green) mAbs. The nucleus was stained with DAPI (blue). The second antibody used anti-mouse IgG2b-Cy5 for BTLA detection. Images were analyzed on a confocal microscope. Yellow indicates the overlay of red and green signals. Shown are representative images from 4 independent experiments. (B) Distribution of BTLA and Vδ2TCR on polarizing cells after interaction with HVEM-positive lymphoma cells (RL cells). Cells were first fixed and permeabilized and then stained with anti-BTLA 7.1 (pink) and anti–TCRVδ2-FITC (green) mAbs. The nucleus was stained with DAPI (blue). The second antibody used anti-mouse IgG2b-Cy5 for BTLA detection. Images were analyzed on a confocal microscope. The white arrows pointing to the white shading indicate the overlay of pink and green signals. (C) BTLA blockade (with full-length BTLA 8.2 or its Fab form) increases the phosphorylation of Zap-70 and Erk1/2. 1 × 106 purified γδ T cells derived from a HV were stimulated for 5 minutes with BrHPP (50 nM) and isotype control or anti–PD-1.3.1 mAb or anti-BTLA 8.2. Total cellular proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and revealed by Western blot analysis using a phospho-Zap-70 or phospho-Erk1/2 antibody. Quantification of phosphorylation was calculated as the ratio between the signals of phospho-protein and the corresponding total protein.
