2D TCR–pMHC–CD8 kinetics determines T‐cell responses in a self‐antigen‐specific TCR system

Abstract

Summary Two-dimensional (2D) kinetic analysis directly measures molecular interactions at cell-cell junctions, thereby incorporating inherent cellular effects. By comparison, three-dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T-cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T-cell receptors (TCR) interacting with a melanoma self-antigen peptide (gp100209–217) bound to major histocompatibility complex (pMHC) in the absence and presence of coreceptor CD8. We found that while 3D parameters are inadequate to predict T-cell function, 2D parameters (which do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T-cell function. Thus, our data support the general notion that 2D parameters of TCR–pMHC–CD8 interactions determine T-cell responsiveness and suggest a potential 2D-based strategy to screen TCRs for tumor immunotherapy.

Dual Biomembrane Force Probe Enables Single-Cell Mechanical Analysis of Signal Crosstalk between Multiple Molecular Species

“…The P a curve measured over a range of t c exhibits a two-stage pattern (Fig. 2C, red), similar to those observed in the studies of TCR/CD8 crosstalk [18][19][20] . Like the previous studies, this two-stage curve cannot be obtained by superposition of two single-stage curves each measured using the same batch of T cells contacted by RBCs coated with only one ligand species (Fig.…”

“…Interactions of two ligands (IgG1 and IgG2) to a common receptor (Fcγ receptor III B, FcγRIIIB) 16 or of two receptors (FcγRIIA and FcγRIIIB) to a common ligand (total IgG) 17 have been found to be concurrent and independent when the bond numbers are low enough for competition to be neglected, resulting in a total bond number that is equal to the sum of two bond species. By comparison, binding of a divalent ligand, pMHC or Thy-1, to the corresponding dual receptors, T cell receptor (TCR) and coreceptor CD8 [18][19][20] or integrin α 5 β 1 and Sydecan-4 21 , is cooperative and synergistic, resulting in an additional trimolecular bond species of greater number and longer duration than the sum of two bimolecular bond species separately formed between the ligand and the two receptors. Interestingly, the formation of the (TCR/CD8)-pMHC trimolecular bond exhibits a time delay and requires intracellular signaling via Src family kinases [18][19][20] whereas that of the (α 5 β 1 /Sydecan-4)-Thy-1 bond is elicited by pulling forces 21 .…”

“…By comparison, binding of a divalent ligand, pMHC or Thy-1, to the corresponding dual receptors, T cell receptor (TCR) and coreceptor CD8 [18][19][20] or integrin α 5 β 1 and Sydecan-4 21 , is cooperative and synergistic, resulting in an additional trimolecular bond species of greater number and longer duration than the sum of two bimolecular bond species separately formed between the ligand and the two receptors. Interestingly, the formation of the (TCR/CD8)-pMHC trimolecular bond exhibits a time delay and requires intracellular signaling via Src family kinases [18][19][20] whereas that of the (α 5 β 1 /Sydecan-4)-Thy-1 bond is elicited by pulling forces 21 . The mechanically based methods have also been combined with fluorescence imaging to enable concurrent observations of receptor-ligand binding and intracellular signaling so induced, to allow for correlative analysis over time at the single-cell level [22][23][24] .…”

Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence

“…Until recently, the precise quantification of TCR:pMHC kinetics at the surface of live T cells was technically challenging. Different techniques have been developed for TCR:pMHC kinetic determination based on micropipetting and thermal fluctuation assays (39,40). To our knowledge, the use of the novel NTAmer technology (15,18) allowed, for the first time, the real-time quantification of dissociation along a broad dynamic range of TCR avidities (.3 logs), on live human CD8 T cells and with monomeric resolution (Fig.…”

Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR–Ligand Off-Rates Measurements on Living Cells

“…However, the Streptamer assay (11) needs a significant lag time until monomeric TCR-pMHC dissociation starts to become detectable, limiting thereby off-rate analyses to antigen-specific T cells of relative long half-lives, typically found in immune responses against pathogens. Moreover, accurate measurements of TCR-pMHC binding parameters on living T cells (11)(12)(13)(14) require specialized equipment, which is currently not available for the high-throughput screen of antigen-specific T-cell populations.…”