Abstract
The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/μm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/μm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.
Highlights
The interaction between T cell receptors (TCRs) on a T cell and their cognate antigen bound to major histocompatibility complexes (MHCs) on the surface of an antigen-presenting cell is the first step of an adaptive immune response
Jurkat T cells expressing rCD48T92A receptors were added to supported lipid bilayer (SLB) functionalized with different densities of fluorescently labeled rat CD2 (rCD2) ligands (Fig. 1 A)
After the cells adhered on the SLB and started forming a cell-SLB contact (Video S4), the density of bound ligands, B, almost immediately reached its steady-state value at 600–700 rCD2 molecules/mm2, whereas the cell-SLB contact area continued to expand for $30 min before leveling out (Fig. 1 C)
Summary
The interaction between T cell receptors (TCRs) on a T cell and their cognate antigen bound to major histocompatibility complexes (MHCs) on the surface of an antigen-presenting cell is the first step of an adaptive immune response. Several other interactions between receptors and ligands across cell-cell contacts have been found important for regulating the activation of the adaptive immune response [7,8]. The binding kinetics of these interactions have been estimated from measurements between receptors anchored to a biosensor surface with ligands binding from the bulk solution [9,10].
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