Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors.

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.

Ligand-induced segregation from large cell-surface phosphatases is a critical step in γδ TCR triggering.

Gamma/delta (γδ) T cells are unconventional adaptive lymphocytes that recognize structurally diverse ligands via somatically-recombined antigen receptors (γδ TCRs). The molecular mechanism by which ligand recognition initiates γδ TCR signaling, a process known as TCR triggering, remains elusive. Unlike αß TCRs, γδ TCRs are not mechanosensitive, and do not require coreceptors or typical binding-induced conformational changes for triggering. Here, we show that γδ TCR triggering by nonclassical MHC class Ib antigens, a major class of ligands recognized by γδ T cells, requires steric segregation of the large cell-surface phosphatases CD45 and CD148 from engaged TCRs at synaptic close contact zones. Increasing access of these inhibitory phosphatases to sites of TCR engagement, by elongating MHC class Ib ligands or truncating CD45/148 ectodomains, abrogates TCR triggering and T cell activation. Our results identify a critical step in γδ TCR triggering and provide insight into the core triggering mechanism of endogenous and synthetic tyrosine-phosphorylated immunoreceptors.

Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

Chimeric antigen receptors (CARs) can redirect T cells to target abnormal cells, but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a > 100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting cells (APCs) but nearly identical sensitivity when antigen is presented as purified protein. We next systematically measured the impact of engaging important T cell accessory receptors (CD2, LFA-1, CD28, CD27, and 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved the antigen sensitivity of the TCR by 125- and 22-fold, respectively, but improved CAR sensitivity by only < 5-fold. This differential effect of CD2 and LFA-1 engagement on the TCR vs. CAR was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3ε subunit (also known as a TRuC) and fully restored by exchanging the TCRαß variable domains for those of the CAR (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors and suggest approaches to increase sensitivity.

Mechanical forces impair antigen discrimination by reducing differences in T-cell receptor/peptide-MHC off-rates.

T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains debated. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity TCR/pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best-characterized catch bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA-1 in force-shielding the TCR/pMHC interaction.

Biomarkers of response to PD-1 pathway blockade.

The binding of T cell immune checkpoint proteins programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to their ligands allows immune evasion by tumours. The development of therapeutic antibodies, termed checkpoint inhibitors, that bind these molecules or their ligands, has provided a means to release this brake on the host anti-tumour immune response. However, these drugs are costly, are associated with potentially severe side effects, and only benefit a small subset of patients. It is therefore important to identify biomarkers that discriminate between responders and non-responders. This review discusses the determinants for a successful response to antibodies that bind PD-1 or its ligand PD-L1, dividing them into markers found in the tumour biopsy and those in non-tumour samples. It provides an update on the established predictive biomarkers (tumour PD-L1 expression, tumour mismatch repair deficiency and tumour mutational burden) and assesses the evidence for new potential biomarkers.

Missense variants in human ACE2 strongly affect binding to SARS-CoV-2 Spike providing a mechanism for ACE2 mediated genetic risk in Covid-19: A case study in affinity predictions of interface variants.

SARS-CoV-2 Spike (Spike) binds to human angiotensin-converting enzyme 2 (ACE2) and the strength of this interaction could influence parameters relating to virulence. To explore whether population variants in ACE2 influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities and kinetics for Spike receptor binding domain through surface plasmon resonance (SPR) at 37°C. We discovered variants that reduce and enhance binding, including three ACE2 variants that strongly inhibited (p.Glu37Lys, ΔΔG = -1.33 ± 0.15 kcal mol-1 and p.Gly352Val, predicted ΔΔG = -1.17 kcal mol-1) or abolished (p.Asp355Asn) binding. We also identified two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = 0.59 ± 0.08 kcal mol-1) is predominant in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (ΔΔG = 0.26 ± 0.09 kcal mol-1) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. We compared ACE2 variant affinities to published SARS-CoV-2 pseudotype infectivity data and confirmed that ACE2 variants with reduced affinity for Spike can protect cells from infection. The effect of variants with enhanced Spike affinity remains unclear, but we propose a mechanism whereby these alleles could cause greater viral spreading across tissues and cell types, as is consistent with emerging understanding regarding the interplay between receptor affinity and cell-surface abundance. Finally, we compared mCSM-PPI2 ΔΔG predictions against our SPR data to assess the utility of predictions in this system. We found that predictions of decreased binding were well-correlated with experiment and could be improved by calibration, but disappointingly, predictions of highly enhanced binding were unreliable. Recalibrated predictions for all possible ACE2 missense variants at the Spike interface were calculated and used to estimate the overall burden of ACE2 variants on Covid-19.

Dephosphorylation accelerates the dissociation of ZAP70 from the T cell receptor.

Protein-protein binding domains are critical in signaling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins has tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signaling, which is a critical requirement of noise-filtering mechanisms such as kinetic proofreading. Here, we use modeling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases. Using surface plasmon resonance, we show that the phosphatase CD45 can accelerate the unbinding rate of zeta chain-associated protein kinase 70 (ZAP70), a tandem SH2 domain-containing kinase, from biphosphorylated peptides from the T cell receptor (TCR). An important functional prediction of accelerated unbinding is that the intracellular ZAP70-TCR half-life in T cells will not be fixed but rather, dependent on the extracellular TCR-antigen half-life, and we show that this is the case in both cell lines and primary T cells. The work highlights that tandem SH2 domains can break the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discrimination mediated by kinetic proofreading.

Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics.

The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape.

As the COVID-19 pandemic has progressed, new variants of the virus SARS-CoV-2 have emerged that are more infectious than the original form. The variants known as Alpha, Beta and Gamma have mutations in a protein on the virus's surface that is vital for attaching to cells and infecting them. This protein, called Spike, carries out its role by binding to ACE2, a protein on the surface of human cells. Mutations on Spike are found on the region where it binds to ACE2. The interaction between these two proteins appears to be important to the behaviour of SARS-CoV-2, but the impact of individual mutations in Spike is unknown. In addition, some people have different variants of ACE2 with mutations in the region that interacts with Spike, but it is not known whether this affects these people's risk of contracting COVID-19. To answer these questions, Barton et al. measured the precise effect of mutations in Spike and ACE2 on the strength of the interaction between the two proteins. The experiments showed that three of the five common Spike mutations in the Alpha, Beta and Gamma SARS-CoV-2 variants strengthened binding to ACE2. The two mutations that weakened binding were only found together with other mutations that strengthened binding. This meant that the Spike proteins in all three of these SARS-CoV-2 variants bind to ACE2 more strongly than the original form. The experiments also showed that two common variants of ACE2 also increased the strength of binding to Spike. Interestingly, one of these ACE2 variants reversed the effect of a specific SARS-CoV-2 mutation, suggesting that carriers would be resistant to SARS-CoV-2 variants with this mutation. Identifying the precise effects of Spike mutations on ACE2 binding helps understand why new variants of SARS-CoV-2 spread more rapidly. This could help to identify concerning new variants before they spread widely and inform the response by health authorities. The finding that two common ACE2 variants bind more strongly to Spike suggests that people with these mutations could be more susceptible to SARS-CoV-2.

The discriminatory power of the T cell receptor.

T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity non-self peptides presented on major histocompatibility complex (pMHC) antigens. Although the discriminatory power of the TCR is widely believed to be near-perfect, technical difficulties have hampered efforts to precisely quantify it. Here, we describe a method for measuring very low TCR/pMHC affinities and use it to measure the discriminatory power of the TCR and the factors affecting it. We find that TCR discrimination, although enhanced compared with conventional cell-surface receptors, is imperfect: primary human T cells can respond to pMHC with affinities as low as KD ∼ 1 mM. The kinetic proofreading mechanism fit our data, providing the first estimates of both the time delay (2.8 s) and number of biochemical steps (2.67) that are consistent with the extraordinary sensitivity of antigen recognition. Our findings explain why self pMHC frequently induce autoimmune diseases and anti-tumour responses, and suggest ways to modify TCR discrimination.

CD8 Co-Receptor Enhances T-Cell Activation without Any Effect on Initial Attachment.

The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to measure at the single molecule level the kinetics of bond formation and rupture between TCR- transfected CD8+ and CD8- Jurkat cells and surfaces coated with five peptide-exposing major histocompatibility antigens (pMHCs) of varying activating power. We also used interference reflection microscopy to image the spreading of these cells dropped on pMHC-exposing surfaces. CD8 did not influence the TCR-pMHC interaction during the first few seconds following cell surface encounter, but it promoted the subsequent spreading responses, suggesting that CD8 was involved in early activation rather than binding. Further, the rate and extent of spreading, but not the lag between contact and spreading initiation, depended on the pMHC. Elucidating T-lymphocyte detection strategy may help unravel underlying signaling networks.

A generic cell surface ligand system for studying cell-cell recognition.

Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell-cell interface. Here, we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. These densities are robustly quantifiable, a major advance over previous studies. We validate the system for a range of immunoreceptors, including the T-cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. We also extend our work to the activation of chimeric antigen receptors. This novel system allows the effect of varying the surface density, valency, dimensions, and affinity of the ligand to be investigated. It can be readily broadened to other receptor-cell surface ligand interactions and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.

MHC binding affects the dynamics of different T-cell receptors in different ways.

T cells use their T-cell receptors (TCRs) to scan other cells for antigenic peptides presented by MHC molecules (pMHC). If a TCR encounters a pMHC, it can trigger a signalling pathway that could lead to the activation of the T cell and the initiation of an immune response. It is currently not clear how the binding of pMHC to the TCR initiates signalling within the T cell. One hypothesis is that conformational changes in the TCR lead to further downstream signalling. Here we investigate four different TCRs in their free state as well as in their pMHC bound state using large scale molecular simulations totalling 26 000 ns. We find that the dynamical features within TCRs differ significantly between unbound TCR and TCR/pMHC simulations. However, apart from expected results such as reduced solvent accessibility and flexibility of the interface residues, these features are not conserved among different TCR types. The presence of a pMHC alone is not sufficient to cause cross-TCR-conserved dynamical features within a TCR. Our results argue against models of TCR triggering involving conserved allosteric conformational changes.

pyHVis3D: visualising molecular simulation deduced H-bond networks in 3D: application to T-cell receptor interactions.

Motivation: Hydrogen bonds (H-bonds) play an essential role for many molecular interactions but are also often transient, making visualising them in a flexible system challenging. Results: We provide pyHVis3D which allows for an easy to interpret 3D visualisation of H-bonds resulting from molecular simulations. We demonstrate the power of pyHVis3D by using it to explain the changes in experimentally measured binding affinities for three T-cell receptor/peptide/MHC complexes and mutants of each of these complexes. Availability and implementation: pyHVis3D can be downloaded for free from http://opig.stats.ox.ac.uk/resources. Contact: science.bernhard.knapp@gmail.com. Supplementary information: Supplementary data are available at Bioinformatics online.

Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.

The contribution of major histocompatibility complex contacts to the affinity and kinetics of T cell receptor binding.

The interaction between the T cell antigen receptor (TCR) and antigenic peptide in complex with major histocompatibility complex (MHC) molecules is a crucial step in T cell activation. The relative contributions of TCR:peptide and TCR:MHC contacts to the overall binding energy remain unclear. This has important implications for our understanding of T cell development and function. In this study we used site directed mutagenesis to estimate the contribution of HLA-A2 side-chains to the binding of four TCRs. Our results show that these TCRs have very different energetic 'footprints' on HLA-A2, with no residues contributing to all TCR interactions. The estimated overall contribution of MHC side-chains to the total interaction energy was variable, with lower limits ranging from 11% to 50%. Kinetic analysis suggested a minor and variable contribution of MHC side-chains to the transition state complex, arguing against a two-step mechanism for TCR binding.

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response.

Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis.

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Costimulation of IL-2 Production through CD28 Is Dependent on the Size of Its Ligand.

Optimal T cell activation typically requires engagement of both the TCR and costimulatory receptors, such as CD28. Engagement of CD28 leads to tyrosine phosphorylation of its cytoplasmic region and recruitment of cytoplasmic signaling proteins. Although the exact mechanism of CD28 signal transduction is unknown, CD28 triggering has similarities to the TCR, which was proposed to use the kinetic-segregation (KS) mechanism. The KS model postulates that, when small receptors engage their ligands within areas of close (∼15 nm) contact in the T cell/APC interface, this facilitates phosphorylation by segregating the engaged receptor/ligand complex from receptor protein tyrosine phosphatases with large ectodomains, such as CD45. To test this hypothesis, we examined the effect of elongating the extracellular region of the CD28 ligand, CD80, on its ability to costimulate IL-2 production by primary T cells. CD80 elongation reduced its costimulatory effect without abrogating CD28 binding. Confocal microscopy revealed that elongated CD80 molecules were less well segregated from CD45 at the T cell/APC interface. T cells expressing CD28 harboring a key tyrosine-170 mutation were less sensitive to CD80 elongation. In summary, the effectiveness of CD28 costimulation is inversely proportional to the dimensions of the CD28-CD80 complex. Small CD28-CD80 complex dimensions are required for optimal costimulation by segregation from large inhibitory tyrosine phosphatases. These results demonstrate the importance of ligand dimensions for optimal costimulation of IL-2 production by T cells and suggest that the KS mechanism contributes to CD28 signaling.

SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.

The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ≤ n ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:ß2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.

Phenotypic models of T cell activation.

T cell activation is a crucial checkpoint in adaptive immunity, and this activation depends on the binding parameters that govern the interactions between T cell receptors (TCRs) and peptide-MHC complexes (pMHC complexes). Despite extensive experimental studies, the relationship between the TCR-pMHC binding parameters and T cell activation remains controversial. To make sense of conflicting experimental data, a variety of verbal and mathematical models have been proposed. However, it is currently unclear which model or models are consistent or inconsistent with experimental data. A key problem is that a direct comparison between the models has not been carried out, in part because they have been formulated in different frameworks. For this Analysis article, we reformulated published models of T cell activation into phenotypic models, which allowed us to directly compare them. We find that a kinetic proofreading model that is modified to include limited signalling is consistent with the majority of published data. This model makes the intriguing prediction that the stimulation hierarchy of two different pMHC complexes (or two different TCRs that are specific for the same pMHC complex) may reverse at different pMHC concentrations.