Background
T-Cell Receptor complex (TCR) is an immune structure aimed at antigen recognition and initiation of immune response. It is bound to three protein dimers, known as Cluster of Differentiation 3 (CD3), whose role is related to signal transduction. Since the immune response is precisely tuned, mobilization of the TCR complex is highly regulated, as the complex undergoes several rounds of positioning on the cell membrane, endocytosis, and recycling. This study was thus aimed at understanding the kinetics of recycling and internalization of the CD3 subunit, by targeting it with the antibody SPV-T3a. The final goal would be the development of a mathematical model of trafficking kinetics, which would not only help understanding the metabolic effect of endocytosis in inflammation processes, but also be a game changer in drug development.
Materials and Methods
Lymphoblastic cell line Jurkat overexpressing CD3, and K562 (negative control) were used for the experiments. Both were grown in RPMI1640 media with 10% HiFBS and 1% PeSt. The antibody SPV-T3a was labeled with either ATTO488 or the pH dependent dye pHrodo Green. Receptor trafficking was studied in real-time assays using LigandTracer Green and evaluated with TraceDrawer software.
Results
Binding of ATTO488-SPV-T3a to CD3 receptors showed a peculiar profile, which reflected an ongoing endocytosis pattern. The plateau phase of the curve at equilibrium was replaced by a section of linear growth in which the slope was constant and independent of antibody concentration. This slope is believed to reflect the kinetics of CD3 recycling on the cell surface. Dissociation was also ligand-independent, with a biphasic profile where a first initial detachment of the complex was followed by a stable signal, given by the fluorophores accumulated in the cells. Labeling SPV-T3a with pHrodo enabled a more precise analysis of the internalization pattern, which showed a linear intake. This together with the fluorescent signal growth in the dissociation phase verified how the internalized SPV-T3a gets degraded through time. A fast initial endocytosis was observed for higher antibody concentrations, that then slowed until a plateau phase before restarting linearly. When using lower concentrations, internalization happened in linear fashion. To investigate the endocytic pathway, cells were treated with various chemicals, in which a 1:1 binding curve was only obtained when treating the cells with 0.5 M sucrose.
Conclusions
The study showed that the recycling patterns of CD3 are mostly unmodified by the targeting process. The linear internalization kinetics indicates a fast receptor recycling and antibody degradation. Sucrose experiments showed that the clathrin dependent pathway is primal for CD3 internalization, but not exclusive, as more pathway-specific chemicals did not modify the shape of the curve.
P. Zucchetti: A. Employment (full or part-time); Significant; Ridgeview Instruments AB. S. Bondza: A. Employment (full or part-time); Significant; Ridgeview Instruments AB. S. Lundsten Salomonsson: A. Employment (full or part-time); Significant; Ridgeview Instruments AB.