Insights from in situ analysis of TCR-pMHC recognition: response of an interaction network

Abstract

Recognition of peptide presented by the major histocompatibility complex (pMHC) molecule by the T-cell receptor (TCR) determines T-cell selection, development, differentiation, fate, and function. Despite intensive studies on the structures, thermodynamic properties, kinetic rates, and affinities of TCR-pMHC interactions in the past two decades, questions regarding the functional outcome of these interactions, i.e. how binding of the αβ TCR heterodimer with distinct pMHCs triggers different intracellular signals via the adjacent CD3 components to produce different T-cell responses, remain unclear. Most kinetic measurements have used surface plasmon resonance, a three-dimensional (3D) technique in which fluid-phase receptors and ligands are removed from their cellular environment. Recently, several two-dimensional (2D) techniques have been developed to analyze molecular interactions on live T cells with pMHCs presented by surrogate antigen-presenting cells or supported planar lipid bilayers. The insights from these in situ analyses have provided a sharp contrast of the 2D network biology approach to the 3D reductionist approach and prompted rethinking of our current views of T-cell triggering. Based on these insights, we propose a mechanochemical coupled triggering hypothesis to explain why the in situ kinetic parameters differ so much from their 3D counterparts, yet correlate so much better with T-cell functional responses.

Fig. 1. Analysis of molecular interactions at the T-cell surface

(A) Traditionally, TCR–pMHC and pMHC–CD4/8 interactions are analyzed in 3D by SPR using soluble molecules. (B) In the micropipette adhesion assay, live T cell is probed by a pMHC-coated RBC. (C) The interaction network being probed by the pMHC, including the TCR-CD3 complex, co-receptor, proximal signaling complexes formed after TCR ligand recognition. (D) Using mutant MHC to abolish co-receptor binding or null peptide to abolish TCR binding, independent TCR–pMHC and pMHC–CD4/8 bimolecular interactions can be analyzed separately. (E–G) Possible kinetic steps leading to TCR triggering. (H) Signaling-dependent cooperative binding among TCR, pMHC, and CD8. (I) Possible regulatory role of mechanical force. (J, K) Possible molecular organizations of the TCR in the membrane.

Fig. 2. Comparison of 2D and 3D binding parameters

Half-life t_1/2 (A), on-rate k_on (B), equilibrium dissociation constant K_d (C), and confinement length (D) plots are shown for indicated molecular systems. 2D data obtained using living cells (unless otherwise stated) by the fluorescent and mechanical methods are shown with open and closed symbols, respectively. Data for TCR–pMHC interactions are shown as open or closed triangles. (A) 2D data for 1G4 TCR interacting with a panel of pMHCs () (29) and PSGL-1 interacting with P- and L-selectin (♦) (33) were measured using cell-free systems by the flow chamber and BFP thermal fluctuation assays, respectively. (B) k_on was calculated from the data in (A) and (C) by k_on = 0.693/(K_d × t_1/2). (C, D) Black symbols are from (46), including micropipette data for CD16 isoforms interacting with IgG of different species (■) (72), and Golan-Zhu plot data for CD2 interacting with CD58 and CD48 (○) (36), CD28–CD80 (□) (73), and CD16b-IgG (◊) (74) interactions.

Fig. 3. Analysis of TCR–pMHC–CD8 trimolecular interactions

(A) Two-stage adhesion frequency vs. contact time data of T cells from OT1 TCR transgenic mice interacting with OVA pMHC in the absence (□) and presence (○) of PP2. Reproduced from (23) with permission. (B) Bimolecular interaction affinities measured under conditions in which only bimolecular interactions were allowed (open bars, denoted by resting interactions) or calculated under the assumption that the increased adhesion in the second stage was due to upregulation of the chosen interaction with all other interactions unchanged (closed bars, denoted by upregulated interactions). (C) Plots of normalized bonds, defined as the average number of bonds <n> at 5-s contact time, calculated from adhesion frequency P_a by <n> = − ln(1 − P_a) and divided by the pMHC density, of TCR–pMHC () and MHC–CD8 () bimolecular interactions and of TCR–pMHC–CD8 (◊) trimolecular interaction for the indicated peptides are plotted against 3D K_d.