Flow cytometric analysis.

<p>(A) Reactivity of mAb and BsAb on hCXCR3 and hCCR6 L1.2 expressing cells. The hCXCR3 and hCCR6 L1.2 expressing cells were mixed and incubated with an isotype control antibody (grey line) or mX3 mAb IgG1 (blue line; top left panel); hX3 IgG1 (blue line; top middle panel); hX3 scFv (blue line; top right panel); mR6 IgG1 (green line; bottom left panel); hR6 IgG1 (green line; bottom middle panel) or BsAb (purple line; bottom right panel). The murine IgG1 were stained with an anti-mouse igG Fc specific PE-conjugated; the humanized scFv was stained with an anti-6HIS antibody followed by an anti-mouse igG Fc specific PE-conjugated antibody; the humanized IgG1 and BsAb were stained with an anti-human kappa PE conjugated antibody. (B) Reactivity of mAb and BsAb on purified human lymphocytes. Human lymphocytes were isolated from blood on a ficoll gradient and stained with an anti-human CD3-BV650 and an anti-human CD4 Pacific Blue conjugated antibody. The lymphocytes in the CD3+/CD4+ gate were stained with isotype controls (left panels); FITC conjugated hR6 mAb and hX3 (detected with anti-human Kappa PE-conjugated antibody) (middle panel) or BsAb (right panel) followed by an anti-human Kappa PE-conjugated antibody. (C) Intracellular staining for FoxP3 protein in lymphocytes isolated from human blood. Activated human lymphocytes were gated on CD3+/CD4+/hX3+ (left panel); CD3+/CD4+/hR6+ (left panel); CD3+/CD4+/BsAb+ (left panel) and stained intracellularly for FoxP3. (D) Intracellular staining for IFN-γ and IL-17A proteins in PMA/ionomycin-stimulated lymphocytes isolated from human blood. Activated human lymphocytes were gated on CD3+/CD4+/hX3+ (left panel); CD3+/CD4+/hR6+ (left panel); CD3+/CD4+/BsAb+ (left panel) and stained intracellularly for IFN-γ and IL-17A.