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

Abstract

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, 14±5 molecules/μm2, 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 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.

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

“…The slope of the Zhu-Golan plot corresponds to −1/K d of the CD2-CD48 interaction, where K d is the twodimensional dissociation coefficient, whereas the intersect with the x-axis gives the density of mobile CD48 receptors on the cell (Zhu et al, 2007). A linear fit to the data in Figure 2C gave a K d of 5 molecules per µm 2 , which agrees with what has previously been measured for this interaction (Junghans et al, 2020). From the x-intersect a density of mobile CD48 receptors of 36 molecules per µm 2 was obtained.…”

“…Cell Lines, Culture and Flow Cytometry E6.1 Jurkat T cells (ATCC) expressing non-signaling rat CD48 and human leukocyte antigen DQ8-glia-α1 (HLA-DQ8-glia-α1) were made as described by Junghans et al (2020). The cells were kept in RPMI 1640 medium (Sigma-Aldrich), which was supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 2% L-glutamine (Sigma-Aldrich), 1% sodium pyruvate (Sigma-Aldrich), 1% HEPES (Sigma-Aldrich), and 1% Penicillin-Streptomycin (Sigma-Aldrich).…”

“…Rat CD2 molecules containing a double polyhistidine-tag (12xH) at the C-terminus and the human L3-12 TCR containing one polyhistidine-tag (6xH) each on the C-terminus of the αand β-chain were made as described in Junghans et al (2020) and were fluorescently labeled using an Alexa Fluor 647 antibody labeling kit (#A20186, Invitrogen). Depending on the desired ligand density, between 0.3 and 2.5 µg/ml of CD2 in HBS buffer was incubated with the SLB for 15-30 min before rinsing.…”

“…Depending on the desired ligand density, between 0.3 and 2.5 µg/ml of CD2 in HBS buffer was incubated with the SLB for 15-30 min before rinsing. The density of the CD2 molecules on the SLB was obtained from the intensity of single CD2 molecules on glass as described previously (Junghans et al, 2018(Junghans et al, , 2020. The successful formation of a mobile SLB was confirmed by fluorescence recovery after photobleaching before each experiment (Jönsson et al, 2008).…”

“…The average intensity in each contact was saved yielding the sum of bound and free ligands in the contact. The intensity outside the contact gave the free ligand density, which was corrected for ligand exclusion in the cell-SLB contact by 25% (Junghans et al, 2020). The intensities were converted to protein densities using the single molecule intensity from one protein as described by Junghans et al (2018Junghans et al ( , 2020.…”