Binding Kinetics of Proteins at Immune-Cell Contacts

Protein-protein interactions are crucial in numerous cellular functions and biological processes that take place inside our body. It is therefore not surprising that these interactions also govern the response of our body´s defence mechanism, the so-called immune system, towards an infection. Understanding how proteins interact entails studying the binding affinity (strength) and the lifetime (duration) of the protein-protein interaction to better decompose how an immune response is initiated and how we can explore this knowledge to treat diseases. In this thesis, total internal fluorescence microscopy (TIRF) and single-molecule imaging were used to observe and characterize protein-functionalized supported lipid bilayers (SLBs) interacting with immune cells to obtain the binding kinetics of various protein-protein pairs.In the first part of this thesis, the interaction between the rat CD2 (rCD2) adhesion protein and its ligand rat CD48T92A (rCD48T92A), a high-affinity mutant of the wild type rat CD48, was used to establish a new method of obtaining single-cell binding affinities of T cells interacting with SLBs using imidazole titrations. The results showed a relatively small spread in the rCD2-rCD48T92A binding affinity values despite the considerable spread of receptor densities within the cell population. The lifetime of the rCD2/rCD48T92A interaction was also investigated using single-molecule imaging and tracking displaying a similarly small lifetime spread within the cell population. Using both these methods, the single-cell binding affinity and lifetime of the cell population can be investigated and their spread can provide information concealed withpopulation-average techniques.The second part of the thesis focused on the CD4 co-receptor whose role in initiating an immune response is ambiguous. Even though the CD4 co-receptor increases the sensitivity of T cell signalling manyfold, it binds to its ligand, peptide major histocompatibility complex II (pMHCII), with the lowest binding affinity known to this day. The CD4-MHC II interaction is so weak that adhesion molecules are needed to ensure a successful CD4-MHC II contact formation. For this reason, the influence of an adhesion molecule, rat CD2, on the obtained binding kinetics of the human CD4 co-receptor was initially examined showing that the accumulation of CD4 was influenced when having a high concentration of bound CD2 inside the cell-SLB contacts. Later, the studies focused on the CD4-TCR-MHC II ternary complex where it was demonstrated that the presence of L3-12 TCR strongly supported the CD4-MHC II interaction by increasing the local density of MHC II inside the cell-SLB contacts. However, the presence of TCR did not seem to significantly influence the specific affinity for CD4 to MHC II. Lastly, CD4 binding studies showed that the co-receptor did not noticeably affect the TCR-MHC II binding at physiological levels of hCD4 in the SLB

Calcium Signaling in T Cells Is Induced by Binding to Nickel-Chelating Lipids in Supported Lipid Bilayers

Supported lipid bilayers (SLBs) are one of the most common cell-membrane model systems to study cell-cell interactions. Nickel-chelating lipids are frequently used to functionalize the SLB with polyhistidine-tagged ligands. We show here that these lipids by themselves can induce calcium signaling in T cells, also when having protein ligands on the SLB. This is important to avoid “false” signaling events in cell studies with SLBs, but also to better understand the molecular mechanisms involved in T-cell signaling. Jurkat T cells transfected with the non-signaling molecule rat CD48 were found to bind to ligand-free SLBs containing ≥2 wt% nickel-chelating lipids upon which calcium signaling was induced. This signaling fraction steadily increased from 24 to 60% when increasing the amount of nickel-chelating lipids from 2 to 10 wt%. Both the signaling fraction and signaling time did not change significantly compared to ligand-free SLBs when adding the CD48-ligand rat CD2 to the SLB. Blocking the SLB with bovine serum albumin reduced the signaling fraction to 11%, while preserving CD2 binding and the exclusion of the phosphatase CD45 from the cell-SLB contacts. Thus, CD45 exclusion alone was not sufficient to result in calcium signaling. In addition, more cells signaled on ligand-free SLBs with copper-chelating lipids instead of nickel-chelating lipids and the signaling was found to be predominantly via T-cell receptor (TCR) triggering. Hence, it is possible that the nickel-chelating lipids act as ligands to the cell’s TCRs, an interaction that needs to be blocked to avoid unwanted cell activation

Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces

Binding between ligands and receptors across cell contacts influences a range of biological processes including the formation of the immune synapse. The dissociation constant (Kd = 1/affinity) of the interaction corresponds to the concentration of ligands where half of the receptors in the contact have bound a ligand. In this chapter, we outline how to measure this two-dimensional affinity using model cell membranes called supported lipid bilayers (SLBs) functionalized with fluorescently labeled ligands that bind to cells containing the corresponding receptor. The affinity is calculated from the accumulation of ligands at the cell-SLB interface, while the use of different fluorescent tags, and/or unlabeled molecules, makes it possible to include various binding pairs in the contact to better mimic the conditions of binding in vivo

Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics

Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease