Internalization of T-Cell Receptor Mimic Antibody RL6A by Brain Derived Endothelial Cells Analyzed by Flow Cytometry

Abstract T-cell receptor mimic monoclonal antibodies (TCRm) recognize peptide-MHC class I complexes. They combine the specificity of T-cell receptors with high binding affinities in the low nanomolar to picomolar range characteristic of monoclonal antibodies. The TCRm RL6A targets YLLPAIVHI peptide-HLA-A2 complexes, where the peptide is derived from the p68 RNA helicase protein. Previous work showed that this target is expressed by brain endothelial cells forming the blood-brain barrier (BBB). Upon binding, RL6A crosses the BBB via a receptor-mediated transcytosis mechanism. Confocal microscopy indicated that, after internalization, RL6A co-localized with the early endosomal marker (EEA1). Here we used conventional flow cytometry and imaging flow cytometry to quantitatively analyze the internalization process. Endothelial cell monolayers formed by hCMEC/D3 cells were incubated with unlabeled RL6A for time periods up to 120 min. Surface bound RL6A was detected by a secondary, PE-labeled antibody. RL6A disappeared in a biphasic pattern from the surface of hCMEC/D3 cells, with an initial half-life (t1/2) of internalization of about 3 min and a second, much slower phase (half life about 3 h) likely reflecting recycling to the membrane. In a complementary approach using imaging flow cytometry (Amnis ImagestreamX) and IDEAS analysis software, the internalization of AlexaFluor488-RL6A could be directly visualized and evaluated. The results confirmed that RL6A internalizes rapidly into hCMEC/D3 cells. In conclusion, flow cytometry is a suitable tool to analyze trafficking of specific peptide-HLA complexes in endothelial cells.