Modeling of B cell Synapse Formation by Monte Carlo Simulation Shows That Directed Transport of Receptor Molecules Is a Potential Formation Mechanism

The formation of the protein segregation structure known as the “immunological synapse” in the contact region between B cells and antigen presenting cells appears to precede antigen (Ag) uptake by B cells. The mature B cell synapse consists of a central cluster of B cell receptor/Antigen (BCR/Ag) complexes surrounded by a ring of LFA-1/ICAM-1 complexes. In this study, we used an in silico model to investigate whether cytoskeletally driven transport of molecules toward the center of the contact zone is a potential mechanism of immunological synapse formation in B cells. We modeled directed transport by the cytoskeleton in an effective manner, by biasing the diffusion of molecules toward the center of the contact zone. Our results clearly show that biased diffusion of BCR/Ag complexes on the B cell surface is sufficient to produce patterns similar to experimentally observed immunological synapses. This is true even in the presence of significant membrane deformation as a result of receptor–ligand binding, which in previous work we showed had a detrimental effect on synapse formation at high antigen affinity values. Comparison of our model’s results to those of experiments shows that our model produces synapses for realistic length, time, and affinity scales. Our results also show that strong biased diffusion of free molecules has a negative effect on synapse formation by excluding BCR/Ag complexes from the center of the contact zone. However, synapses may still form provided the bias in diffusion of free molecules is an order-of-magnitude weaker than that of BCR/Ag complexes. We also show how diffusion trajectories obtained from single-molecule tracking experiments can generate insight into the mechanism of synapse formation

Missing-in-Metastasis/Metastasis Suppressor 1 Regulates B Cell Receptor Signaling, B Cell Metabolic Potential, and T Cell-Independent Immune Responses

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cell activation by cognate antigens via B cell receptors (BCRs), or pathogen-associated molecules through pattern-recognition receptors, such as Toll-like receptors (TLRs), leads to transcriptional and metabolic changes that ultimately transform B cells into antibody-producing plasma cells or memory cells. BCR signaling and a number of steps downstream of it rely on coordinated action of cellular membranes and the actin cytoskeleton, tightly controlled by concerted action of multiple regulatory proteins, some of them exclusive to B cells. Here, we dissect the role of Missing-In-Metastasis (MIM), or Metastasis suppressor 1 (MTSS1), a cancer-associated membrane and actin cytoskeleton regulating protein, in B cell-mediated immunity by taking advantage of MIM knockout mouse strain. We show undisturbed B cell development and largely normal composition of B cell compartments in the periphery. Interestingly, we found that MIM-/- B cells are defected in BCR signaling in response to surface-bound antigens but, on the other hand, show increased metabolic activity after stimulation with LPS or CpG. In vivo, MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides the first comprehensive characterization of MIM in B cells, demonstrates its regulatory role for B cell-mediated immunity, as well as proposes new functions for MIM in tuning receptor signaling and cellular metabolism, processes, which may also contribute to the poorly understood functions of MIM in cancer

Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome

HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer

Notch signaling during human T cell development

Notch signaling is critical during multiple stages of T cell development in both mouse and human. Evidence has emerged in recent years that this pathway might regulate T-lineage differentiation differently between both species. Here, we review our current understanding of how Notch signaling is activated and used during human T cell development. First, we set the stage by describing the developmental steps that make up human T cell development before describing the expression profiles of Notch receptors, ligands, and target genes during this process. To delineate stage-specific roles for Notch signaling during human T cell development, we subsequently try to interpret the functional Notch studies that have been performed in light of these expression profiles and compare this to its suggested role in the mouse

Logical Development of the Cell Ontology

<p>Abstract</p> <p>Background</p> <p>The Cell Ontology (CL) is an ontology for the representation of <it>in vivo </it>cell types. As biological ontologies such as the CL grow in complexity, they become increasingly difficult to use and maintain. By making the information in the ontology computable, we can use automated reasoners to detect errors and assist with classification. Here we report on the generation of computable definitions for the hematopoietic cell types in the CL.</p> <p>Results</p> <p>Computable definitions for over 340 CL classes have been created using a genus-differentia approach. These define cell types according to multiple axes of classification such as the protein complexes found on the surface of a cell type, the biological processes participated in by a cell type, or the phenotypic characteristics associated with a cell type. We employed automated reasoners to verify the ontology and to reveal mistakes in manual curation. The implementation of this process exposed areas in the ontology where new cell type classes were needed to accommodate species-specific expression of cellular markers. Our use of reasoners also inferred new relationships within the CL, and between the CL and the contributing ontologies. This restructured ontology can be used to identify immune cells by flow cytometry, supports sophisticated biological queries involving cells, and helps generate new hypotheses about cell function based on similarities to other cell types.</p> <p>Conclusion</p> <p>Use of computable definitions enhances the development of the CL and supports the interoperability of OBO ontologies.</p

Germinal center B cells recognize antigen through a specialized immune synapse architecture

B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we show that in contrast to naive and memory B cells, which gathered antigen towards the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β (PKC-β)–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T-cell dependent selection of high-affinity B cells in GCs

Monte Carlo Investigation of Diffusion of Receptors and Ligands that Bind Across Opposing Surfaces

Studies of receptor diffusion on a cell surface show a variety of behaviors, such as diffusive, sub-diffusive, or super-diffusive motion. However, most studies to date focus on receptor molecules diffusing on a single cell surface. We have previously studied receptor diffusion to probe the molecular mechanism of receptor clustering at the cell–cell junction between two opposing cell surfaces. Here, we characterize the diffusion of receptors and ligands that bind to each other across two opposing cell surfaces, as in cell–cell and cell–bilayer interactions. We use a Monte Carlo method, where receptors and ligands are simulated as independent agents that bind and diffuse probabilistically. We vary receptor–ligand binding affinity and plot the molecule-averaged mean square displacement (MSD) of ligand molecules as a function of time. Our results show that MSD plots are qualitatively different for flat and curved interfaces, as well as between the cases of presence and absence of directed transport of receptor–ligand complexes toward a specific location on the interface. Receptor–ligand binding across two opposing surfaces leads to transient sub-diffusive motion at early times provided the interface is flat. This effect is entirely absent if the interface is curved, however, in this instance we observe sub-diffusive motion. In addition, a decrease in the equilibrium value of the MSD occurs as affinity increases, something which is absent for a flat interface. In the presence of directed transport of receptor–ligand complexes, we observe super-diffusive motion at early times for a flat interface. Super-diffusive motion is absent for a curved interface, however, in this case we observe a transient decrease in MSD with time prior to equilibration for high-affinity values

VCAM-1 and VLA-4 Modulate Dendritic Cell IL-12p40 Production in Experimental Visceral Leishmaniasis

Vascular cell adhesion molecule-1 (VCAM-1) interacts with its major ligand very late antigen-4 (VLA-4) to mediate cell adhesion and transendothelial migration of leukocytes. We report an important role for VCAM-1/VLA-4 interactions in the generation of immune responses during experimental visceral leishmaniasis caused by Leishmania donovani. Our studies demonstrate that these molecules play no direct role in the recruitment of leukocytes to the infected liver, but instead contribute to IL-12p40-production by splenic CD8+ dendritic cells (DC). Blockade of VCAM-1/VLA-4 interactions using whole antibody or anti-VCAM-1 Fab′ fragments reduced IL-12p40 mRNA accumulation by splenic DC 5 hours after L. donovani infection. This was associated with reduced anti-parasitic CD4+ T cell activation in the spleen and lowered hepatic IFNγ, TNF and nitric oxide production by 14 days post infection. Importantly, these effects were associated with enhanced parasite growth in the liver in studies with either anti-VCAM-1 or anti-VLA-4 antibodies. These data indicate a role for VCAM-1 and VLA-4 in DC activation during infectious disease

Incunabular Immunological Events in Prion Trafficking

While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement