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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.

Summary Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling., Graphical Abstract, Highlights • Activating and inhibitory NK cell receptors have a distinct nanoscale organization • The transmembrane sequence of KIR controls their nanoscale organization • Nanoclusters of KIR2DS1 and its adaptor are juxtaposed but mix upon activation • Phosphorylation of ZAP-70 or SHP-1 is favored in larger receptor nanoclusters, Oszmiana et al. use different super-resolution microscopy techniques to compare the nanoscale organization of paired activating and inhibitory receptors at the human NK cell surface. They show that the size of receptor nanoclusters is controlled by the transmembrane sequence and establish that nanocluster size affects the strength of receptor signaling.

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.

THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation.

The T cell receptor (TCR) and CD8 bind peptide-major histocompatibility complex (pMHC) glycoproteins to initiate adaptive immune responses, yet the trimolecular binding kinetics at the T cell membrane is unknown. Using a micropipette adhesion frequency assay, we show that this kinetic has two stages. The first consists of TCR-dominant binding to agonist pMHC. This triggers a second stage consisting of a step increase in adhesion following a one second delay. The second-stage binding requires Src family kinase activity to initiate CD8 binding to the same pMHC engaged by the TCR. This induced-trimeric-cooperative interaction enhances adhesion synergistically to favor potent ligands, which further amplifies discrimination. Our data reveal a TCR-CD8 positive feedback loop involved in initial signaling steps that is sensitive to a single pMHC, is rapid, reversible, synergistic, and peptide-discriminative.

Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development. Refereed/Peer-reviewed

Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR) on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy., Author Summary Recognition of antigen by the T cell antigen receptor (TCR) is a central event in the initiation of adaptive immune responses and for this reason the TCR has been extensively studied. Multiple studies performed over the past 20 years have revealed intriguing findings that include the observation that the TCR has multiple phosphorylation sites that are sequentially phosphorylated by the kinase Lck and that ZAP-70, a cytosolic kinase, binds to these sites with different affinities. The purpose of multiple sites, their sequential phosphorylation by Lck, and the differential binding affinities of ZAP-70 are unknown. Using a novel mechanistic model that incorporates a high level of molecular detail, we find, unexpectedly, that all factors are critical for producing ultrasensitivity (switch-like response) and therefore this signaling architecture exhibits systems-level emergent ultrasensitivity. We use the model to study existing therapeutic chimeric antigen receptors and in the design of novel ones. The work also has direct implications to the study of many other immune receptors.

Based on studies in model systems it has been proposed that the cytoplasmic domains of T cell receptor signalling subunits that have polybasic motifs associate with the plasma membrane, and that this regulates their phosphorylation. Recent experiments in more physiological systems have confirmed membrane association but raised questions as to its function.

Successful T cell activation depends on the recognition of antigenic peptides by the T cell receptor (TCR) together with additional signals from co-stimulatory receptors such as CD28. To determine the requirement of matching TCR and CD28 ligand complexes in optimal co-stimulation, the length of CD80 was elongated by insertion of 2 or 4 immunoglobulin domains. Despite similar binding to soluble CD28 molecules, elongated CD80 molecules were much less effective at mediating co-stimulation to primary T cells. By splitting the TCR and CD28 ligands into 2 separate APCs, the trans-costimulation assay subsequently suggests that elongated CD80s are incapable of transducing co-stimulation signals into the T cell. In addition, confocal studies using CD80-GFP fusion protein indicated that large phosphatases such as CD45 co-localised with elongated CD80 in the T cell-APC interface. Together, these results demonstrated the importance of ligand dimensions in optimal T cell co-stimulation.

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.