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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 CD3 ε subunit (also known as a TRuC) and fully restored by exchanging the TCR α β 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.

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.

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 unclear. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds), and this was confirmed by molecular dynamic 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 this new force-shielding role for the adhesion receptors CD2 and LFA-1.One sentence summaryMechanical forces disproportionately accelerate the off-rates of higher-affinity antigens reducing T cell antigen discrimination

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.

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.

SARS-CoV-2 infection begins with the interaction of the SARS-CoV-2 Spike (Spike) and human angiotensin-converting enzyme 2 (ACE2). To explore whether population variants in ACE2 might influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities for Spike receptor binding domain through surface plasmon resonance (SPR). We discovered variants that enhance and reduce binding, including two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = ± 0.59 0.08 kcal mol−1) is often seen 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. Carriers of these alleles may be more susceptible to infection or severe disease and these variants may influence the global epidemiology of Covid-19. We also identified three rare 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) Spike binding. These variants may confer resistance to infection. Finally, we calibrated the mCSM-PPI2 ΔΔG prediction algorithm against our SPR data, give new predictions for all possible ACE2 missense variants at the Spike interface and estimate the overall burden of ACE2 variants on Covid-19 phenotypes.

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 domain are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the more transmissible B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD domain 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 variants first identified in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P1). 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 primarily enhance transmission, the K417N/T mutations facilitate immune escape, and the E484K mutation facilitates both transmission and immune escape.

SummaryThe scanning of surrounding tissues by T lymphocytes to detect cognate antigen is a highly demanding process that requires high rapidity, sensitivity and specificity. Co-receptors such as CD8 are known to increase detection performance, but the exact mechanism of this role remains incompletely understood. Here, we used interference reflection microscopy to image the initial spreading of 1G4 receptor transfected CD8+ and CD8− Jurkat cells dropped on surfaces exposing five cognate antigens of varying activating power, and we used a laminar flow chamber to measure the influence of CD8 on the kinetics of bond formation and rupture between cell-born T cell receptors (TCRs) and peptide-exposing major histocompatibility complex antigens (pMHCs) at the single molecule level. It is concluded that CD8 did not influence TCR-pMHC interaction during the first seconds following cell surface encounter, but it promoted the spreading responses during the first minutes, thus suggesting that CD8 was involved in early activation rather than binding. In addition, presented results were quantitatively compared with a recent report on the cell-free interaction between the same ligand-receptor couples : it is concluded that bond formation was strongly impaired by cell molecular environment, while bond rupture was comparable in both systems. Results from this and previous reports were used to propose a quantitative scheme of the strategy used by T lymphocytes to scan foreign surfaces. It is suggested that the understanding of the strategy used by cells to perform their basic functions may be a prerequisite to understand the function of molecular networks revealed by high throughput methods.

Protein-protein binding domains are critical in signalling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins have 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 signalling, which is a critical requirement of noise filtering mechanisms such as kinetic proofreading. Here, we use modelling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases when tandem domains bind by a kinetic, but not a static, avidity mode. We use surface plasmon resonance to show that ZAP70, a tandem SH2 domain-containing kinase, binds kinetically to biphosphorylated peptides from the T cell antigen receptor (TCR) and that the unbinding rate can be accelerated by the phosphatase CD45. An important functional prediction of regulated unbinding is that the intracellular ZAP70/TCR half-life in T cells will be correlated to 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 binding by kinetic avidity breaks 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 discriminated mediated by kinetic proofreading.

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., Author summary The interaction between T-cells and other cells is one of the most important interactions in the human immune system. If T-cells are not triggered major parts of the immune system cannot be activated or are not working effectively. Despite many years of research the exact mechanism of how a T-cell is initially triggered is not clear. One hypothesis is that conformational changes within the T-cell receptor (TCR) can cause further downstream signalling within the T-cell. In this study we computationally investigate the dynamics of four different TCRs in their free and bound configuration. Our large scale simulations show that all four TCRs react to binding in different ways. In some TCRs mainly the areas close to the binding region are affected while in other TCRs areas further apart from the binding region are also affected. Our results argue against a conserved structural activation mechanism across different types of TCRs.

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.

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.

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. Supplementary information Supplementary data are available at Bioinformatics online.

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

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.

The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.

Mast cells are well known for their harmful role in IgE-mediated hypersensitivity reactions, but their physiological role remains a mystery. Several recent studies have reported that mast cells play a critical role in innate immunity in mice by releasing tumor necrosis factor α (TNF-α) to recruit neutrophils to sites of enterobacterial infection. In some cases, the mast cell TNF-α response was triggered when these cells directly bound FimH on the surface ofEscherichia coli. We have identified CD48, a glycosylphosphatidylinositol-anchored molecule, to be the complementary FimH-binding moiety in rodent mast cell membrane fractions. We showed that (i) pretreatment of mast cell membranes with antibodies to CD48 or phospholipase C inhibited binding of FimH+E. coli, (ii) FimH+E. colibut not a FimH−derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) binding of FimH+bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected with CD48 cDNA, and (iv) antibodies to CD48 specifically blocked the mast cell TNF-α response to FimH+E. coli.Thus, CD48 is a functionally relevant microbial receptor on mast cells that plays a role in triggering inflammation.

References 1 Stern, D. et al. (2002) Receptor for advanced glycation endproducts: a multiligand receptor magnifying cell stress in diverse pathologic settings. Adv. Drug Deliv. Rev. 54, 1615–1625 2 Deane, R. et al. (2003)RAGE mediates amyloid-bpeptide transport across the blood–brain barrier and accumulation in brain. Nat. Med. 9, 907–913 3 Hofmann, M.A. et al. (1999) RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97, 889–901 4 Kerkhoff, C. et al. (1998) Novel insights into structure and functions of S100A8 and S100A9. Biochim. Biophys. Acta 1448, 200–211 5 Donato, R. (2003) Intracellular and extracellular roles of S100 proteins. Microsc. Res. Tech. 60, 540–551 6 Rammes, A. et al. (1997) Myeloid-related protein (MRP) 8 and MRP14, calcium-binding proteins of the S100 family, are secreted by activated monocytes via a novel, tubulin-dependent pathway. J. Biol. Chem. 272, 9496–9502 7 Boussac, M. and Garin, J. (2000) Calcium-dependent secretion in human neutrophils: a proteomic approach. Electrophoresis 21, 665–672 8 Schmidt, A.M. et al. (2001) The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J. Clin. Invest. 108, 949–955 9 Ridinger, K. et al. (1998) Clustered organization of S100 genes in human and mouse. Biochim. Biophys. Acta 1448, 254–263 10 Wicki, R. et al. (1996) Characterization of the human S100A12 (calgranulin C, p6, CAAF1, CGRP) gene, a new member of the S100 gene cluster on chromosome 1q21. Cell Calcium 20, 459–464 11 Kent, W.J. et al. (2002) The human genome browser at UCSC. Genome Res. 12, 996–1006 12 Schwartz, S. et al. (2003) Human–mouse alignments with BLASTZ. Genome Res. 13, 103–107 13 Blake, J.A. et al. (2003) MGD: the mouse genome database. Nucleic Acids Res. 31, 193–195 14 Foell, D. et al. (2003) Neutrophil derived human S100A12 (EN-RAGE) is strongly expressed during chronic active inflammatory bowel disease. Gut. 52, 847–853

Stochastic and spatial aspects are becoming increasingly recognized as an important factor in T-cell activation. Activation occurs in an intrinsically noisy environment, requiring only a handful of agonist peptide-major histocompatibility complex molecules, thus making consideration of signal to noise of prime importance in understanding sensitivity and specificity. Furthermore, it is widely established that surface-bound ligands are more effective at activation than soluble forms, while surface patternation has highlighted the role of spatial relocation in activation. Here we consider the results of a number of models of T-cell activation, from a realistic model of kinetic segregation-induced T-cell receptor (TCR) triggering through to simple queuing theory models. These studies highlight the constraints on cell activation by a surface receptor that recruits kinases. Our analysis shows that TCR triggering based on trapping of bound TCRs in regions of close proximity that exclude large ectodomain-containing molecules, such as the phosphatases CD45 and CD148, can effectively reproduce known signaling characteristics and is a viable 'signal transduction' mechanism distinct from oligomerization and conformation-based mechanisms. A queuing theory analysis shows the interrelation between sensitivity and specificity, emphasizing that these are properties of individual cell functions and need not be, nor are likely to be, uniform across different functions. In fact, threshold-based mechanisms of detection are shown to be poor at ligand discrimination because, although they can be highly specific, that specificity is limited to a small range of peptide densities. Time integration mechanisms however are able to control noise effectively, while kinetic proofreading mechanisms endow them with good specificity properties. Thus, threshold mechanisms are likely to be important for rapidly detecting minimal signaling requirements, thus achieving efficient scanning of antigen-presenting cells. However, for good specificity, time integration on a scale of hours is required.

Size-dependent protein segregation at the cell-cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK-cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL-2 stimulated C57BL/6 mouse-derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK-cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H-2K(b), a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H-2K(b) resulted in decreased inhibition of both lysis and IFN-gamma production by NK cells. These results establish that small ligand dimensions are important for both NK-cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.

The T-cell receptor (TCR) consists of a TCRαβ heterodimer, a TCRζ homodimer, and CD3γε and CD3δε heterodimers. The precise mechanism of T-cell triggering following TCR ligand engagement remains elusive. Previous studies reported that the cytoplasmic tail of CD3ε binds to the plasma membrane through a basic residue-rich stretch (BRS) and proposed that dissociation from the membrane is required for phosphorylation thereof. In this report we show that BRS motifs within the cytoplasmic tail of TCRζ mediate association with the plasma membrane and that TCR engagement results in TCRζ dissociation from the membrane. This dissociation requires phosphorylation of the TCRζ immunoreceptor tyrosine-based activation motifs by lymphocyte cell-specific protein tyrosine kinase (Lck) but not ζ-chain–associated protein kinase 70 binding. Mutations of the TCRζ BRS motifs that disrupt this membrane association attenuate proximal and distal responses induced by TCR engagement. These mutations appear to alter the localization of TCRζ with respect to Lck as well as the mobility of the TCR complex. This study reveals that tyrosine phosphorylation of the TCRζ cytoplasmic domain regulates its association with the plasma membrane and highlights the functional importance of TCRζ BRS motifs.

CD2 is a T cell surface molecule that enhances T and natural killer cell function by binding its ligands CD58 (humans) and CD48 (rodents) on antigen-presenting or target cells. Here we show that the CD2/CD58 interaction is enthalpically driven and accompanied by unfavorable entropic changes. Taken together with structural studies, this indicates that binding is accompanied by energetically significant conformational adjustments. Despite having a highly charged binding interface, neither the affinity nor the rate constants of the CD2/CD58 interaction were affected by changes in ionic strength, indicating that long-range electrostatic forces make no net contribution to binding.

Cellular signaling is initially confined to the plasma membrane, where the cytoplasmic tails of surface receptors and other membrane-anchored proteins are phosphorylated in response to ligand binding. These proteins often contain multiple phosphorylation sites that are regulated by membrane-confined enzymes. Phosphorylation of these proteins is thought to be tightly regulated, because they initiate and regulate signaling cascades leading to cellular activation, yet how their phosphorylation is regulated is poorly understood. Ultrasensitive or switchlike responses in their phosphorylation state are not expected because the modifying enzymes are in excess. Here, we describe a novel mechanism of ultrasensitivity exhibited by multisite membrane-anchored proteins, but not cytosolic proteins, even when enzymes are in excess. The mechanism underlying this concentration-independent ultrasensitivity is the local saturation of a single enzyme by multiple sites on the substrate. Local saturation is a passive process arising from slow membrane diffusion, steric hindrances, and multiple sites, and therefore may be widely applicable. Critical to this ultrasensitivity is the brief enzymatic inactivation that follows substrate modification. Computations are presented using ordinary differential equations and stochastic spatial simulations. We propose a new role, to our knowledge, for multisite membrane-anchored proteins, discuss experiments that can be used to probe the model, and relate our findings to previous theoretical work.

Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (Kd= 2–45 μM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8+T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity.

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell-antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose-response curve in vitro by a factor of 3-10. In comparison, stimulation of T cells in the absence of lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction shifted the dose-response curve by a factor of 10, whereas absence of both CD2-CD48 and LFA-1-ICAM-1 interactions shifted the response by a factor of approximately 100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca(2+) fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

The structural analysis of surface proteins belonging to the CD2 subset of the immunoglobulin superfamily has yielded important insights into transient cellular interactions. In mice and rats, CD2 and CD244 (2B4), which are expressed predominantly on T cells and natural killer cells, respectively, bind the same, broadly expressed ligand, CD48. Structures of CD2 and CD244 have been solved previously, and we now present the structure of the receptor-binding domain of rat CD48. The receptor-binding surface of CD48 is unusually flat, as in the case of rat CD2, and shares a high degree of electrostatic complementarity with the equivalent surface of CD2. The relatively simple arrangement of charged residues and this flat topology explain why CD48 cross-reacts with CD2 and CD244 and, in rats, with the CD244-related protein, 2B4R. Comparisons of modeled complexes of CD2 and CD48 with the complex of human CD2 and CD58 are suggestive of there being substantial plasticity in the topology of ligand binding by CD2. Thermodynamic analysis of the native CD48-CD2 interaction indicates that binding is driven by equivalent, weak enthalpic and entropic effects, in contrast to the human CD2-CD58 interaction, for which there is a large entropic barrier. Overall, the structural and biophysical comparisons of the CD2 homologues suggest that the evolutionary diversification of interacting cell surface proteins is rapid and constrained only by the requirement that binding remains weak and specific.

We develop a model of tyrosine phosphorylation and activation of the T-cell receptor (TCR) by localization to regions of close membrane-membrane proximity (close contact) that physically exclude tyrosine phosphatases such as CD45. Phosphatase exclusion generates regions of low phosphatase and high kinase activity and thus our model provides a framework to examine the kinetic segregation model of TCR activation. We incorporate a sequence of activation steps modeling the construction of the signalosome with a final sequestered, or high-stability, signaling state. The residence time of unbound TCRs in tyrosine kinase-rich domains is shown to be too short for accumulation of activation steps, whereas binding to an agonist lengthens the localization time and leads to generation of fully active TCRs. Agonist detection depends only on this localization, and therefore kinetic segregation represents a viable ligand detection mechanism, or signal transduction mechanism across membranes, distinct from receptor oligomerization and conformational change. We examine the degree of discrimination of agonists from a background of null (self) peptides, and from weak agonists achievable by this mechanism.

Leukocytes play a critical role in recognizing and responding to infection and cancer. Central to this function is an array of cell-surface receptors that lack sequence homology. Many of these receptors have in common the fact that their signaling involves phosphorylation of cytoplasmic domains by extrinsic tyrosine kinases. These non-catalytic tyrosine-phosphorylated receptors (NTRs) share a number of other features, including small size and optimal stimulation by surface-associated ligands. We argue here that NTRs are also likely to share the same kinetic-segregation triggering mechanism, which involves segregation of the engaged NTR from receptor tyrosine phosphatases with large ectodomains such as CD45 and CD148. NTRs signal through tyrosine-containing cytoplasmic motifs, which recruit distinct cytoplasmic signaling proteins when phosphorylated, transducing activatory or inhibitory signals. They have two features that make them uniquely well suited to their role in immune recognition of infection and cancer. Their modular structure enables the coupling of many rapidly evolving receptors with diverse ligand specificities to the same conserved signaling machinery. Their similarity in size and shared signaling machinery enables them to colocalize at cell-cell interfaces when they engage ligands, facilitating the integration of activatory and inhibitory signals from multiple receptors at the cell surface.

Results from live-cell microscopy suggest that the behaviour of isolated components of the T-cell activation machinery in vitro does not represent the reality inside cells. Understanding the cellular-scale dynamics of microcluster migration can only be accomplished by in situ observation. Developments in 'super-resolution' microscopy have permitted investigators to move beyond tracking the movements of individual molecules, allowing the recognition of protein islands and nanodomains present in quiescent and active T cells. Many high-resolution techniques have their own susceptibilities to artefacts, so it is important to take a multifaceted approach to confirm results. A major challenge for the future will be to integrate all the new information into a coherent model of antigen recognition and T-cell activation.

Summary T cell receptor (TCR) binding to diverse peptide-major histocompatibility complex (pMHC) ligands results in various degrees of T cell activation. Here we analyze which binding properties of the TCR-pMHC interaction are responsible for this variation in pMHC activation potency. We have analyzed activation of the 1G4 cytotoxic T lymphocyte clone by cognate pMHC variants and performed thorough correlation analysis of T cell activation with 1G4 TCR-pMHC binding properties measured in solution. We found that both the on rate (kon) and off rate (koff) contribute to activation potency. Based on our results, we propose a model in which rapid TCR rebinding to the same pMHC after chemical dissociation increases the effective half-life or “confinement time” of a TCR-pMHC interaction. This confinement time model clarifies the role of kon in T cell activation and reconciles apparently contradictory reports on the role of TCR-pMHC binding kinetics and affinity in T cell activation., Highlights ► Potency of peptide-MHC as an agonist for TCR signaling is dependent on kon and koff ► A TCR-pMHC confinement time model is proposed to account for these parameters ► The model proposes that TCRs rebind the same peptide-MHC before dissociating ► The model reconciles binding kinetics and affinity in T cell activation

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

Udgivelsesdato: 2009-Mar Environmental factors account for 75% of the risk of developing multiple sclerosis (MS). Numerous infections have been suspected as environmental disease triggers, but none of them has consistently been incriminated, and it is unclear how so many different infections may play a role. We show that a microbial peptide, common to several major classes of bacteria, can induce MS-like disease in humanized mice by crossreacting with a T cell receptor (TCR) that also recognizes a peptide from myelin basic protein, a candidate MS autoantigen. Structural analysis demonstrates this crossreactivity is due to structural mimicry of a binding hotspot shared by self and microbial antigens, rather than to degenerate TCR recognition. Biophysical studies reveal that the autoreactive TCR binding affinity is markedly lower for the microbial (mimicry) peptide than for the autoantigenic peptide. Thus, these data suggest a possible explanation for the difficulty in incriminating individual infections in the development of MS.

Electrostatic interactions are important for molecular recognition processes including Ca2+-binding and cell adhesion. To understand these processes, we have successfully introduced a novel Ca2+-binding site into the non-Ca2+-dependent cell adhesion protein CD2 using our criteria that are specifically tailored to the structural and functional properties of the protein environment and charged adhesion surface. This designed site with ligand residues exclusively from the beta-sheets selectively binds to Ca2+ and Ln3+ over other mono- and divalent cations. While Ca2+ and Ln3+ binding specifically alters the local environment of the designed Ca2+-binding site, the designed protein undergoes a significantly smaller conformation change compared with those observed in naturally occurring Ca2+-binding sites that are composed of at least part of the flexible loop and helical regions. In addition, the CD2-CD48-binding affinity increased approximately threefold after protein engineering, suggesting that the cell adhesion of CD2 can be modulated by altering the local electrostatic environment. The study provides site-specific information for regulating cell adhesion within CD2 and gives insight into the structural factors required for Ca2+-modulated biological processes.

Human fibrillin-1, the major structural protein of connective tissue 10-12 nm microfibrils, contains multiple calcium binding epidermal growth factor-like domains interspersed with transforming growth factor beta-binding protein-like (TB) domains. TB4 contains a flexible RGD loop that mediates cell adhesion via alphaVbeta3 and alpha5beta1 integrins. This study identifies integrin alphaVbeta6 as a novel cellular receptor for fibrillin-1 with a K(d) of approximately 0.45 mum. Analyses of this interaction by surface plasmon resonance and immunocytochemistry reveal different module requirements for alphaVbeta6 activation compared with those of alphaVbeta3, suggesting that a covalent linkage of an N-terminal calcium binding epidermal growth factor-like domain to TB4 can modulate alphaV integrin binding specificity. Furthermore, our data suggest alpha5beta1 is a low affinity fibrillin-1 receptor (K(d) > 1 mum), thus providing a molecular explanation for the different alpha5beta1 distribution patterns seen when human keratinocytes and fibroblasts are plated on recombinant fibrillin fragments versus those derived from the physiological ligand fibronectin. Non-focal contact distribution of alpha5beta1 suggests that its engagement by fibrillin-1 may elicit a lesser degree and/or different type of intracellular signaling compared with that seen with a high affinity ligand.