The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness

Abstract

The T-cell receptor (TCR) interacts with peptide-major histocompatibility complexes (pMHC) to discriminate pathogens from self-antigens and trigger adaptive immune responses. Direct physical contact is required between the T cell and the antigen-presenting cell for cross-junctional binding where the TCR and pMHC are anchored on two-dimensional (2D) membranes of the apposing cells. Despite their 2D nature, TCR-pMHC binding kinetics have only been analysed three-dimensionally (3D) with a varying degree of correlation with the T-cell responsiveness. Here we use two mechanical assays to show high 2D affinities between a TCR and its antigenic pMHC driven by rapid on-rates. Compared to their 3D counterparts, 2D affinities and on-rates of the TCR for a panel of pMHC ligands possess far broader dynamic ranges that match that of their corresponding T-cell responses. The best 3D predictor of response is the off-rate, with agonist pMHC dissociating the slowest. In contrast, 2D off-rates are up to 8,300-fold faster, with the agonist pMHC dissociating the fastest. Our 2D data suggest rapid antigen sampling by T cells and serial engagement of a few agonist pMHCs by TCRs in a large self pMHC background. Thus, the cellular environment amplifies the intrinsic TCR-pMHC binding to generate broad affinities and rapid kinetics that determine T-cell responsiveness.

Figure 1

Micropipette and BFP. a and b, Micrographs of the micropipette (a) and BFP (b). A T cell (right) aspirated by a pipette was aligned with a RBC held stationary by another pipette (left) without (a, Movie M1) or with (b, Movies M2 and M3) a bead attached to the apex. c, RBCs or beads (left) were coupled by WT-SA or Di-SA with monomeric pMHC to interact with the TCR on T cells (right). d, Specificity controls of adhesion frequency measured at 5 s between OT1 T cells and unmodified RBCs, biotinylated RBCs without coating, biotinylated RBCs coated with BSA, null pMHC-I (VSV:H-2K^b), pMHC-II (MOG:I-A^b) or antigenic pMHC-I (OVA:H-2K^b), or between MOG CD4+ T cells and biotinylated RBCs coated with OVA:H-2K^b. e, Comparison between adhesion frequencies measured at 2 s using 7 and 5 μm^-2 pMHC respectively captured by WT-SA and Di-SA.

Figure 2

2D kinetics measurements. a and b, Adhesion curves for the OT1 TCR interacting with OVA (a) and R4 (b) measured by micropipette at 25 °C (a) or both 25 and 37 °C (b) at indicated site densities. c, Adhesion curves of the OT1 TCR interacting with OVA and G4 measured by BFP at 25 °C. The data (points) were fitted (color matched solid curves) by a model for 2D binding kinetics (a, b and c). d, Pooled ensembles of 239 (OVA) or 424 (G4) lifetimes of bonds between the OT1 TCR and OVA (□) or G4 (○) were respectively sorted according to their durations. For each peptide, the natural log of the number of events with a lifetime ≥ t_b was plotted vs. t_b and fitted by a straight line. The negative slope represents off-rate k_off (indicated). The goodness-of-fit was indicated by the R² values. Color matched dotted curves represent 95% confidence intervals of the best-fit curves obtaining by bootstrapping.

Figure 3

Comparison of 2D and 3D kinetics. Affinities (a and d), on-rates (b and e), and off-rates (c and f) of the OT1 TCR interacting with indicated pMHCs. The 2D data (a-c) were measured by the adhesion frequency assay and analyzed with a monomeric binding model. The 3D data (d-f) from Refs. 11,12 were measured by surface plasmon resonance and analyzed with the same monomeric binding model except for the OVA and A2 data at 37 °C, which were analyzed with a dimeric binding model (values of the second-step kinetics were plotted).

Figure 4

Correlation between 2D kinetics and T cell proliferation. The reciprocal concentration required to reach half-maximal T cell proliferation (1/EC₅₀) is plotted vs the effective 2D affinity (a and d), on-rate (b and e), and off-rate (c and f) measured at 25 °C (a-c) or 37 °C (d-f) for the indicated peptides. To quantify T cell proliferation, naïve OT1 splenocytes (3×10⁵/well) were cultured in 96-well plates with the indicated peptides at 37°C. After 48 h, 0.4 μCi/well of [³H] thymidine was added. After another 18 h, cells were harvested on a FilterMate harvester (PerkinElmer) and analyzed on a Matrix 96 Direct Beta Counter (PerkinElmer). EC₅₀ values were calculated using GraphPad Prism.