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

Protein Dimensions and Interactions at Immune-Cell Contacts

Immune cells, such as B and T cells, fight pathogens infecting the human body. To fulfil this function the immune cells need to form a contact during which different membrane proteins play an important role. A key event in this contact is the interaction between the T-cell receptor (TCR) and an antigenic peptide-presenting major histocompatibility complex (MHC) protein. If this interaction is sufficiently strong then the T cell gets activated leading to an immune response. The TCR-MHC together with other partaking proteins are highly organised within the cell- cell contact in respect to their size and function. However, information about their lateral interactions, height- dependent orientation and the consequences of this for binding are largely missing, but important in order to understand how an immune response is initiated on a molecular scale. The aim of this thesis was to shed light on this. For investigating protein size and lateral interactions an important negative regulator of TCR signalling, the phosphatase CD45, was studied. It was postulated by the kinetic segregation model that the tall glycoprotein CD45 is excluded from the contact area. The segregation from the TCR in the contact results in a net positive signalling event and thus in T-cell activation. The size of CD45 is a key factor but complete structural information at the cell surface is lacking. By using a technique called hydrodynamic trapping I determined the dimensions of CD45 and quantified its lateral interactions with other CD45 molecules in a cell membrane model. This was done by trapping CD45 anchored to a supported lipid bilayer (SLB) causing high local protein densities below a micropipette through which a negative pressure was applied. Using this method, it was found that CD45 has a lower apparent height than estimated previously which is accounted for by a high flexibility of the protein on the model membrane.When investigating protein binding in this thesis the focus was on the binding strength (affinity) of TCR-MHC interactions. Each TCR has a specific affinity for their cognate peptide MHC, which varies from very weak to strong depending on the presented peptide. Agonist (pathogenic) peptides typically generate strong binding and an activation of the immune response. Measuring affinities in a cellular system is however complicated due to various protein interactions taking place at the same time. By using a model system in which cells bind to an SLB, the two- dimensional affinity of a TCR binding to an agonist MHC on the opposing cell was measured herein. It was found that high ligand densities affected the cells’ cytoskeletal reorganisation, observed as strong lamellipodia formation, which caused a significant increase in the amount of bound ligands. Additionally, it was observed that the binding strength of the TCR-MHC interaction was influenced by the presence of the auxiliary protein CD2 and decreased with increasing amounts of CD2 in the contact. Using the methods described in this thesis allows to measure and interpret weak interactions such as to ‘self’ (body’s own) peptide MHCs. In addition, my hydrodynamic trapping studies of proteins on functionalised SLBs could give new information about membrane protein behaviour, for example how protein flexibility can decrease the effective protein height and thus influence molecular exclusion at cell-cell contacts, and how glycosylation can increase the repulsion, which might prevent protein aggregation

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

Dimensions and Interactions of Large T-Cell Surface Proteins

The first step of the adaptive immune response involves the interaction of T cells that express T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes expressed by antigen-presenting cells (APCs). Exactly how this leads to activation of the TCR and to downstream signaling is uncertain, however. Recent findings suggest that one of the key events is the exclusion of the large receptor-type tyrosine phosphatase CD45, from close contacts formed at sites of T-cell/APC interaction. If this is true, a full understanding of how close contact formation leads to signaling would require insights into the structures of, and interactions between, large membrane proteins like CD45 and other proteins forming the glycocalyx, such as CD43. Structural insights into the overall dimensions of these proteins using crystallographic methods are hard to obtain, and their conformations on the cell surface are also unknown. Several imaging-based optical microscopy techniques have however been developed for analyzing protein dimensions and orientation on model cell surfaces with nanometer precision. Here we review some of these methods with a focus on the use of hydrodynamic trapping, which relies on liquid flow from a micropipette to move and trap membrane-associated fluorescently labeled molecules. Important insights that have been obtained include (i) how protein flexibility and coverage might affect the effective heights of these molecules, (ii) the height of proteins on the membrane as a key parameter determining how they will distribute in cell-cell contacts, and (iii) how repulsive interactions between the extracellular parts of the proteins influences protein aggregation and distribution

Hydrodynamic trapping measures the interaction between membrane-associated molecules

How membrane proteins distribute and behave on the surface of cells depends on the molecules’ chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method

Effects of a local auxiliary protein on the two-dimensional affinity of a TCR-peptide MHC interaction

The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding. The 2D Kd for L3-12 TCR binding to HLA-DQ8-glia-α1, of 14±5 molecules/μm2 (mean±s.d.), was only marginally influenced by including CD2 up to ∼200 bound molecules/μm2 but higher CD2 densities reduced the affinity up to 1.9-fold. Cell-SLB contact size increased steadily with ligand density without affecting binding for contacts at up to ∼20% of total cell area, but beyond this lamellipodia appeared, giving an apparent increase in bound receptors of up to 50%. Our findings show how parameters other than the specific protein-protein interaction can influence binding behavior at cell-cell contacts

HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma

Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours