B cell activation in human whole blood

The ability of rilzabrutinib to block BCR-driven activation of B cells was measured by expression of the surface maker CD69. Human whole blood (AllCells) were pretreated for 1 h with 5% CO₂ at 37°C with eleven 3-fold serial dilutions of compound starting at a concentration of 5 μM. Whole blood was then stimulated by addition of 50 μg/ml goat anti-human IgM F(ab’)2 (Southern Biotech, Birmingham, AL) to stimulate the BCR. Stimulated blood was incubated overnight for 18 h in 5% CO₂ at 37°C followed by staining for 30 min with labeled anti-CD20 and anti-CD69 (BD Biosciences). The RBCs were then lysed with PharmLyse (BD Biosciences), washed, and analyzed for percent CD69 expression on CD20⁺ B cells on a Cytek DxP flow cytometer. The log of the rilzabrutinib concentration was plotted as a function of the percent inhibition of the CD69 positive control value. IC₅₀ determination was performed using the Levenberg–Marquardt nonlinear least square–fitting algorithm implemented in the Dotmatics data package. The IC₅₀ values were determined by fitting the inhibition curves using a four-parameter sigmoidal dose–response model.