Faculty Bibliography

The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development.

Regulatory T cells (Tregs) are essential for tolerance to self and environmental Ags, acting in part by downmodulating costimulatory molecules on the surface of dendritic cells (DCs) and altering naive CD4 T cell-DC interactions. In this study, we show that Tregs form stable conjugates with DCs before, but not after, they decrease surface expression of the costimulatory molecule CD80 on the DCs. We use supported planar bilayers to show that Tregs dramatically slow down but maintain a highly polarized and motile phenotype after recognizing Ag in the absence of costimulation. These motile cells are characterized by distinct accumulations of LFA-1-ICAM-1 in the lamella and TCR-MHC in the uropod, consistent with a motile immunological synapse or "kinapse." However, in the presence of high, but not low, concentrations of CD80, Tregs form stationary, symmetrical synapses. Using blocking Abs, we show that, whereas CTLA-4 is required for CD80 downmodulation, CD28-CD80 interactions are critical for modulating Treg motility in the presence of Ag. Taken together, these results support the hypothesis that Tregs are tuned to alter their motility depending on costimulatory signals.

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.

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-gamma 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 gammat 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.

To determine the breadth and underpinning of changes in immunocyte gene expression due to genetic variation in mice, we performed, as part of the Immunological Genome Project, gene expression profiling for CD4+ T cells and neutrophils purified from 39 inbred strains of the Mouse Phenome Database. Considering both cell types, a large number of transcripts showed significant variation across the inbred strains, with 22% of the transcriptome varying by 2-fold or more. These included 119 loci with apparent complete loss of function, where the corresponding transcript was not expressed in some of the strains, representing a useful resource of "natural knockouts." We identified 1222 cis-expression quantitative trait loci (cis-eQTL) that control some of this variation. Most (60%) cis-eQTLs were shared between T cells and neutrophils, but a significant portion uniquely impacted one of the cell types, suggesting cell type-specific regulatory mechanisms. Using a conditional regression algorithm, we predicted regulatory interactions between transcription factors and potential targets, and we demonstrated that these predictions overlap with regulatory interactions inferred from transcriptional changes during immunocyte differentiation. Finally, comparison of these and parallel data from CD4+ T cells of healthy humans demonstrated intriguing similarities in variability of a gene's expression: the most variable genes tended to be the same in both species, and there was an overlap in genes subject to strong cis-acting genetic variants. We speculate that this "conservation of variation" reflects a differential constraint on intraspecies variation in expression levels of different genes, either through lower pressure for some genes, or by favoring variability for others.

The molecular interactions underlying regulation of the immune response take place in a nanoscale gap between T cells and antigen-presenting cells, termed the immunological synapse. If these interactions are regulated appropriately, the host is defended against a wide range of pathogens and deranged host cells. If these interactions are disregulated, the host is susceptible to pathogens or tumor escape at one extreme and autoimmunity at the other. Strategies targeting the synapse have helped to establish immunotherapy as a mainstream element in cancer treatment. This Masters' primer will cover the basics of the immunological synapse and some of the applications to tumor immunology. Cancer Immunol Res; 2(11); 1023-33. (c)2014 AACR.

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 naive 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.

The immune system uses much of the classic machinery of cell biology, but in ways that put a different spin on organization and function. Striking recent examples include the demonstration of intraflagellar transport protein and hedgehog contributions to the immune synapse, even though immune cells lack a primary cilium that would be the typical setting for this machinery. In a second example, lymphocytes have their own subfamily of integrins, the beta2 subfamily, and only integrins in this family form a stable adhesion ring using freely mobile ligands, a key feature of the immunological synapse. Finally, we showed recently that T-cells use endosomal sorting complexes required for transport (ESCRTs) at the plasma membrane to generate T-cell antigen receptor-enriched microvesicles. It is unusual for the ESCRT pathway to operate at the plasma membrane, but this may allow a novel form of cell-cell communication by providing a multivalent ligand for major histocompatibility complex-peptide complexes and perhaps other receptors on the partnering B-cell. Immune cells are thus an exciting system for novel cell biology even with classical pathways that have been studied extensively in other cell types.

Targeting pro-inflammatory cytokines is a promising approach to the treatment of certain autoimmune diseases. However, the signaling mechanisms involved in the initiation and effector phases of auto-aggressive pathways are still an enigma. Rho-associated kinase 2 (ROCK2) regulates the secretion of inflammatory cytokines interleukin (IL)-21 and IL-17 and the development of autoimmunity in mice. Our data from a phase 1 clinical trial demonstrates that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects significantly down-regulates the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g in response to TCR stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORgt protein levels in T cells derived from healthy subjects or rheumatoid arthritis (RA) patients. Simultaneously, ROCK2 inhibition induces phosphorylation of STAT5 and SMAD2/3, and subsequently increases the percentage of Foxp3+ T cells. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between pro-inflammatory and regulatory T cell subsets in man. Targeted inhibition of ROCK2 may therefore have a role in the treatment of autoimmune disease with disturbed immune homeostasis

T cells can form either a stable synapse or a mobile junction termed kinapse when interacting with antigen presenting cells during an immune response. Stable synapses can lead to long dwell times with antigen presenting cells, whereas mobile kinapses likely favor transient interactions with antigen presenting cells. The underlying cell-intrinsic mechanisms determining the mode of interaction as well as their specific immunological consequences are not known. We have implemented methods borrowed from computer-vision and machine-learning disciplines to accurately quantify the positional stability of T cell junctions formed on model antigen presenting surfaces coated with ICAM-1 and anti-CD3. We have found that human memory CD8 T cells form stable synapses whereas naive CD8 T cells form kinapses or transient contacts. This bias in synapse stability is neither observed in human CD4 T cell subsets nor in murine CD8 T cell subsets under the conditions we have tested. Increased surface expression of LFA1 in human memory CD8 T cells contributes to their increased synapse stability. Using patterned surfaces to simulate competition for antigen presenting cells, we show that synapse stability exhibited by memory CD8 T cells causes suppression of naive cell activation by preventing access to antigen. Increased synapse stability of memory CD8 T cells is expected to promote the development of CTL escape variants during hyper-mutable viral infections and thus prove detrimental to the host