Abstract
Drug resistance activity of transporters such as P-glycoprotein within lymphocytes may have significant consequences to the pharmacologic treatment of HIV, immune suppression, autoimmune diseases, and leukemia, as many drugs are substrates of the MDR transporters. Variability in MDR activity may be a significant barrier in the evaluation of the predicted effect of a drug and on the therapeutic dosage necessary to overcome MDR activity. Current methodologies to measure MDR, e.g. FACS, do not provide kinetic measurements of dye efflux at the resolution of the individual cell thereby missing the existence of functionally different subpopulations. To overcome these limitations, we developed a new imaging device that is comprised of thousands of transparent pico-wells (20 micron in diameter) organized in a honeycomb structure that enables real-time visualization and sequential quantification of drug transport rate from thousands of individual living lymphocytes, thereby generating MDR kinetic data. Fluorescence microscopy was used to measure DiOC2 dye (a common fluorescent probe for P-glycoprotein transport) retention from individual human CD4+ or CD8+ lymphocytes over time. Kinetic analysis of dye export from CD4+ and CD8+ cells revealed the occurrence of subpopulations of cells with unique sensitivity to MDR inhibitors. When the MDR inhibitor Verapamil was used, CD8+ lymphocytes demonstrated a greater magnitude of inhibition compared to CD4+ lymphocytes. Remarkably, CD4+ lymphocytes contained a sizable subpopulation which was verapamil insensitive, suggesting the existence of a non-P-glycoprotein drug export activity in these cells. These findings indicate that functional MDR assays, which are based on transport kinetics, can efficiently dissect the heterogeneous lymphocyte cell populations and identify unique subsets of cells with increased activity.
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Disclosures: Molecular Cytomics.
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