Abstract
Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fcγ receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7–0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.
Highlights
The human immune system is a complex network of cells, which communicate with each other in a highly organized manner to defend the body from pathogens and other potentially harmful substances
We describe and validate a method for enabling kinetic real-time measurements on living suspension cells in the time span of hours
The interaction between Rituximab and B-cells was analyzed in two different laboratories with different B-cell lines and different antibody concentrations
Summary
The human immune system is a complex network of cells, which communicate with each other in a highly organized manner to defend the body from pathogens and other potentially harmful substances. One way in which immune cells communicate is through secreted molecules that bind to designated receptors on target cells. A well-known example of this is the positive and negative selection of T-cells in the thymus; the fate of the thymocyte is dependent on how well the T-cell receptor binds self-antigens presented via Characterizing Interactions on Living Immune Cells the MHC of antigen presenting cells. A high affinity interaction with self-antigens will lead to apoptosis, whereas a weak affinity will induce survival signals and promote positive selection [1]. In this case, interactions of structurally very similar molecules can lead to completely opposing outcomes depending on the strength of the interaction. A detailed characterization and quantification of a molecular interaction is required for an in-depth understanding of immune cells interacting patterns
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