Vedolizumab (anti-α4β7-integrin)

Vedolizumab (anti-α4β7-integrin) binds to a subset of human peripheral blood memory CD4⁺ T lymphocytes including gut-homing interleukin 17 T-helper lymphocytes at the highest level, also binds to eosinophils at high levels, and to naive T-helper lymphocytes, naive and memory cytotoxic T lymphocytes, B lymphocytes, natural killer cells, and basophils at lower levels; Vedolizumab binds to memory CD4⁺ T and B lymphocytes with subnanomolar potency with EC50 of 0.3-0.4 nM.^[1]

PBMCs were resuspended in assay buffer for CD4 memory cell interleukin (IL) 17A intracellular staining. PBMCs were stimulated (or not stimulated, for the control samples) with 50 ng/ml phorbol 12-myristate 13-acetate and 1 µg/ml Ionomycin in the presence of 3 µM Monensin for 5 h at 37°C. After washing, cells were stained with fluorescent-labeled antibodies: vedolizumab, anti-α4 and anti-β7, anti-CD4, and anti-CD45RO, where applicable, at 4°C for 30 min. Cells were then washed with cold PBS and resuspended in fixation and permeabilization working solution staining buffer at 4°C for 30 min. After fixation and permeabilization, cells were washed with permeabilization buffer and blocked with 10% normal rat serum at 4°C for 10 min. After the blocking step, cells were stained with fluorochrome-conjugated anti_x0002_IL17A antibody at 4°C for 30 min. Finally, cells were washed with permeabilization buffer and analyzed.