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

Abstract

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.

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

“…Interestingly, the 2D affinities measured by Huppa et al were of similar magnitude at 24 and 37 °C (Huppa et al, 2010), a correlation that has also been observed by us for CD4 binding pMHC class II molecules (Jönsson et al, 2016) and for rat CD2 binding rat CD48 T92A (Junghans et al, 2020;Dam et al, 2021). Nonetheless, Huppa et al (2010) observed a monotonic decrease from 1.7 to 0.1 s in 2D lifetimes as the temperature was increased from 24 to 37 °C.…”

“…Care should thus be taken on the state of the T cells when measuring ligand-receptor affinity. This can in particular be a problem when using an SLB containing nickel-chelating lipids that on their own have been shown to induce cell activation unless effectively blocked (Dam et al, 2021). In order to minimize intracellular interactions upon cell activation, it is also possible to take advantage of protein design, for example by only expressing the extracellular region of the studied receptor on the cell surface (Junghans et al, 2020).…”

“…For all these methods, care must be taken such that the cells do not bind non-specifically to the model membranes. This has been shown to be particularly important when using nickel-chelating lipids, which can bind to cell receptors on the approaching cell and potentially cause cell signaling (Dam et al, 2021). However, by using different approaches to passivate the SLB, such as adding bovine serum albumin or polyethylene glycosylated lipids in the SLB, the unspecific binding events can be minimized (Taylor et al, 2017;Dam et al, 2021).…”

“…This has been shown to be particularly important when using nickel-chelating lipids, which can bind to cell receptors on the approaching cell and potentially cause cell signaling (Dam et al, 2021). However, by using different approaches to passivate the SLB, such as adding bovine serum albumin or polyethylene glycosylated lipids in the SLB, the unspecific binding events can be minimized (Taylor et al, 2017;Dam et al, 2021). It should be stressed that the sample preparation is the first crucial step in 2D binding kinetic studies using SLBs and thus verifying the properties of the deposited SLB should be done before performing any interaction experiment.…”

Editorial: The Role of Biomembranes and Biophysics in Immune Cell Signaling

“…Apparently, structural features of microvilli favor this segregation. Of particular interest is the piece by Dam et al that discussed a phenomenon related to this topic (5). Supported lipid bilayers are commonly used a surrogate of MHC/peptide to interact with TCR.…”