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The integrin αLβ2 plays central roles in leukocyte adhesion and T cell activation, rendering αLβ2 an attractive therapeutic target. Compounds with different modes of αLβ2 inhibition are in development, currently. Consequently, there is a foreseeable need for bedside assays, which allow assessment of the different effects of diverse types of αLβ2 inhibitors in the peripheral blood of treated patients.; Here, we describe a flow cytometry-based technology that simultaneously quantitates αLβ2 conformational change upon inhibitor binding, αLβ2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low MW inhibitors, designated α I and α/β I allosteric αLβ2 inhibitors, were investigated. The first application revealed intriguing inhibitor class-specific profiles.; Half-maximal inhibition of T cell activation was associated with 80% epitope loss induced by α I allosteric inhibitors and with 40% epitope gain induced by α/β I allosteric inhibitors. This differential establishes that inhibitor-induced αLβ2 epitope changes do not directly predict the effect on T cell activation. Moreover, we show here for the first time that α/β I allosteric inhibitors, in contrast to α I allosteric inhibitors, provoked partial downmodulation of αLβ2, revealing a novel property of this inhibitor class.; The multi-parameter whole blood αLβ2 assay described here may enable therapeutic monitoring of αLβ2 inhibitors in patients' blood. The assay dissects differential effect profiles of different classes of αLβ2 inhibitors.
