A dynamic CD2-rich compartment at the outer edge of the immunological synapse boosts and integrates signals.

The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse, with cellular or artificial APC, in a pattern we refer to as a 'CD2 corolla'. The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 co-stimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% as compared with central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2-mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8+ tumor-infiltrating lymphocytes displayed low expression of CD2 in the majority of people with colorectal, endometrial or ovarian cancer. CD2 downregulation may attenuate antitumor T cell responses, with implications for checkpoint immunotherapies.

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.

Dynamics and spatial organization of Kv1.3 at the immunological synapse of human CD4+ T cells.

Formation of the immunological synapse (IS) is a key event during initiation of an adaptive immune response to a specific antigen. During this process, a T cell and an antigen presenting cell form a stable contact that allows the T cell to integrate both internal and external stimuli in order to decide whether to activate. The threshold for T cell activation depends on the strength and frequency of the calcium (Ca2+) signaling induced by antigen recognition, and it must be tightly regulated to avoid undesired harm to healthy cells. Potassium (K+) channels are recruited to the IS to maintain the negative membrane potential required to sustain Ca2+ entry. However, the precise localization of K+ channels within the IS remains unknown. Here, we visualized the dynamic subsynaptic distribution of Kv1.3, the main voltage-gated potassium channel in human T cells. Upon T cell receptor engagement, Kv1.3 polarized toward the synaptic cleft and diffused throughout the F-actin rich distal compartment of the synaptic interface-an effect enhanced by CD2-CD58 corolla formation. As the synapse matured, Kv1.3 clusters were internalized at the center of the IS and released in extracellular vesicles. We propose a model in which specific distribution of Kv1.3 within the synapse indirectly regulates the channel function and that this process is limited through Kv1.3 internalization and release in extracellular vesicles.

The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.

A key role is emerging for the cytoskeleton in coordinating receptor signaling, although the underlying molecular requirements remain unclear. Here we show that cytoskeleton disruption triggered signaling requiring not only the B cell receptor (BCR), but also the coreceptor CD19 and tetraspanin CD81, thus providing a mechanism for signal amplification upon surface-bound antigen stimulation. By using superresolution microscopy, we demonstrated that endogenous IgM, IgD, and CD19 exhibited distinct nanoscale organization within the plasma membrane of primary B cells. Upon stimulation, we detect a local convergence of receptors, although their global organization was not dramatically altered. Thus, we postulate that cytoskeleton reorganization releases BCR nanoclusters, which can interact with CD19 held in place by the tetraspanin network. These results not only suggest that receptor compartmentalization regulates antigen-induced activation but also imply a potential role for CD19 in mediating ligand-independent "tonic" BCR signaling necessary for B cell survival.

Cutting Edge: Synapse Propensity of Human Memory CD8 T Cells Confers Competitive Advantage over Naive Counterparts.

Memory T cells are endowed with multiple functional features that enable them to be more protective than naive T cells against infectious threats. It is not known if memory cells have a higher synapse propensity (SP; i.e., increased probability to form immature immunological synapses that then provide an entry into different modes of durable interaction with APCs). In this study, we show that only human memory CD8 T cells have remarkably high SP compared with naive counterparts. Such a dichotomy between naive and memory cells is not observed within the human CD4 or murine CD8 T cell population. Higher SP in human memory CD8 T cells allows them to outcompete and prevent naive CD8 T cells from getting recruited to the response. This observation has implications for original antigenic sin and aging of the immune system in humans.

CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.

Here we describe the spatiotemporal architecture, at high molecular resolution, of receptors and signaling molecules during the early events of mouse B cell activation. In response to membrane-bound ligand stimulation, antigen aggregation occurs in B cell antigen receptor (BCR) microclusters containing immunoglobulin (Ig) M and IgD that recruit the kinase Syk and transiently associate with the coreceptor CD19. Unexpectedly, CD19-deficient B cells were significantly defective in initiation of BCR-dependent signaling, accumulation of downstream effectors and cell spreading, defects that culminated in reduced microcluster formation. Hence, we have defined the dynamics of assembly of the main constituents of the BCR 'signalosome' and revealed an essential role for CD19, independent of the costimulatory molecule CD21, in amplifying early B cell activation events in response to membrane-bound ligand stimulation.

The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.

Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igbeta as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses.

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22-/- T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22-/- T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22-/- T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22-/- bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.

Force and affinity in ligand discrimination by the TCR.

T cell recognition of antigen is a physical process that requires formation of a cell-cell junction that is rich in active force generation. Recently a biomolecular force probe was used to examine how the T cell receptor (TCR)-pMHC interaction responds to force and the consequences of force-dependent interactions for T cell activation. While adhesion and costimulatory molecules in the immunological synapse impact upon the overall force of the interaction, these results suggest that the TCR uses a force-dependent bond - a catch bond - and that it may therefore be important to consider the TCR-pMHC interaction in isolation in the early phases of the decision process. We discuss here these findings in the context of other work on the impact of forces on the TCR and the quantification of interaction in interfaces.

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells.

Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4(+) T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T cells via STAT3-dependent mechanism.

Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.

Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.

Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.

T cell activation is determined by the number of presented antigens.

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per µm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

Durable Interactions of T Cells with T Cell Receptor Stimuli in the Absence of a Stable Immunological Synapse.

T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses.

Spatial Control of Biological Ligands on Surfaces Applied to T Cell Activation.

In this chapter, we present techniques, based on molecular-scale nanofabrication and selective self-assembly, for the presentation of biomolecules of interest (ligands, receptors, etc.) on a surface with precise spatial control and arbitrary geometry at the single-molecule level. Metallic nanodot arrays are created on glass coverslips and are then used as anchors for the immobilization of biological ligands via thiol linking chemistry. The nanodot size is controlled by both lithography and metallization. The reagent concentration in self-assembly can be adjusted to ensure single-molecule occupancy for a given dot size. The surrounding glass is backfilled by a protein-repellent layer to prevent nonspecific adsorption. Moreover, bifunctional surfaces are created, whereby a second ligand is presented on the background, which is frequently a requirement for simulating complex cellular functions involving more than one key ligand. This platform serves as a novel and powerful tool for molecular and cellular biology, e.g., to study the fundamental mechanisms of receptor-mediated signaling.

Molecular Occupancy of Nanodot Arrays.

Single-molecule nanodot arrays, in which a biomolecule of choice (protein, nucleic acid, etc.) is bound to a metallic nanoparticle on a solid substrate, are becoming an increasingly important tool in the study of biomolecular and cellular interactions. We have developed an on-chip measurement protocol to monitor and control the molecular occupancy of nanodots. Arrays of widely spaced nanodots and nanodot clusters were fabricated on glass surfaces by nanolithography and functionalized with fluorescently labeled proteins. The molecular occupancy was determined by monitoring individual fluorophore bleaching events, while accounting for fluorescence quenching effects. We found that the occupancy can be interpreted as a packing problem, and depends on nanodot size and binding ligand concentration, where the latter is easily adjusted to compensate the flexibility of dimension control in nanofabrication. The results are scalable with nanodot cluster size, extending to large area close packed arrays. As an example, the nanoarray platform was used to probe the geometric requirement of T-cell activation at the single-molecule level.