Faculty Bibliography

Molecular interactions at the interface between helper T cells and antigen-presenting B cells govern the ability to produce specific antibodies, which is a central event in protective immunity generated by natural infection or man-made vaccines. In order for a T cell to deliver effective help to a B cell and guide affinity maturation, it needs to provide feedback that is proportional to the amount of antigen the B cell collects with its surface antibody. This review focuses on mechanisms by which T and B cells manage to count the products of antigen capture and encourage B cells with the best receptors to dominate the response and make antibody-producing plasma cells. We discuss what is known about the proportionality of T cells responses to presented antigens and consider the mechanisms that B cells may use to keep count of positive feedback from T cells.

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Functional convergence of CD28 costimulation and TCR signaling is critical to T cell activation and adaptive immunity. These receptors form complex microscale patterns within the immune synapse, though the impact of this spatial organization on cell signaling remains unclear. We investigate this crosstalk using micropatterned surfaces that present ligands to these membrane proteins in order to control the organization of signaling molecules within the cell-substrate interface. While primary human CD4+ T cells were activated by features containing ligands to both CD3 and CD28, this functional convergence was curtailed on surfaces in which engagement of these two systems was separated by micrometer-scale distances. Moreover, phosphorlylated Lck was concentrated to regions of CD3 engagement and exhibited a low diffusion rate, suggesting that costimulation is controlled by a balance between the transport of active Lck to CD28 and its deactivation. In support of this model, disruption of the actin cytoskeleton increased Lck mobility and allowed functional T cell costimulation by spatially separated CD3 and CD28. In primary mouse CD4+ T cells, a complementary system, reducing the membrane mobility increased the sensitivity to CD3-CD28 separation. These results demonstrate a subcellular reaction-diffusion system that allows cells to sense the microscale organization of the extracellular environment.

T cells are among the most sensitive of cells, but in this issue of Immunity, Honda et al. (2014) demonstrate that effector T cells must lose their touch within hours to protect the host from immunopathology.

Ab-secreting cell (ASC) expansion and survival are important processes in optimizing vaccines and controlling autoimmunity. The microenvironment of the medullary cords is positioned to control these key processes. Previously, we imaged and characterized ASC differentiation and migration by intravital microscopy in the lymph node (LN) by transferring and activating B cells expressing yellow fluorescent protein only in the ASC compartment. In this study, we observed that yellow fluorescent protein(+) ASCs in the medullary cords migrated along myelomonocytic cells and arrested in contact with them. Acute ablation of myeloid cells using the human diphtheria receptor system (diphtheria toxin receptor [DTR]) expressed in Lysmd1-cre-positive cells increased ASC and Ab production by 2-fold. Increases in ASC numbers were associated with cell proliferation based on Ki-67 staining, rather than reduced apoptosis, or changes in egress from the LN. Using DTR-mediated ablation targeted to Ccr2-expressing myeloid cells also generated increases in ASCs. In contrast, neither the depletion of Gr-1-positive cells with an Ab nor the ablation of cells using a cd11c-DTR resulted in any change in ASCs. IL-6 cytokine signaling can enhance ASC production and has been implicated in dampening ASCs in lupus mouse models through myeloid cells. Using mixed bone marrow chimeras, we observed that IL-6 enhances ASC production, but IL-6 production was not required by myeloid cells to dampen ASCs in the LN. Inhibition of ASCs by these myeloid cells in the LN provides a new regulatory mechanism with implications for tuning Ab responses.

T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to the formation of an "immunological synapse" [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2,3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters.

Adhesion of cell membranes is ubiquitous in biological systems. Processes as diverse as adherens junctions between cells, intracellular junctions between endoplasmic reticulum and the plasma membrane, Golgi stacks, and exocytic vesicle fusion require stable contact between two apposing membranes. One well-studied example of such an interface is the immunological synapse, which enables communication between an antigen-presenting cell and a T-cell. During T-cell activation, adhesion molecules with large extracellular domains (such as LFA-ICAM) are separated from regions of short ligand-receptor protein pairs (such as TCR-pMHC), forming a striking 'bulls-eye' pattern of protein arrangement at the interface. Importantly, the large signaling phosphatase CD45 is segregated away from T-cell receptor clusters, allowing for stable phosphorylation and T-cell activation. To explain this protein sorting phenomenon, a passive mechanism known as kinetic segregation has been proposed in which long molecules are excluded from interfaces formed by short adhesive molecules. To test this model in a purely synthetic system, we developed a simplified membrane adhesion assay using giant unilamellar vesicles (GUVs) and synthetic proteins of different sizes and adhesion strengths. With this reconstituted membrane adhesion system, we show that proteins can indeed be sorted in a size-dependent manner without the active contribution of a cell. We find that proteins that are not involved in interface formation are excluded from the interface in a size-dependent manner, consistent with phosphatase segregation in the immunological synapse. In addition, we show that two adhesion protein pairs can form phaseseparated domains that minimize the membrane-bending penalty within the interface, but that these domains do not form concentric rings. This suggests a role for more complex, potentially active mechanisms in shaping the geometry of the immunological synapse. This system for forming synthetic membrane interfaces has the pot!

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca2+ ]i ) elevation. TCR activation triggers increased [Ca2+ ]i and can arrest T-cell motility in vitro. However the requirement for [Ca2+ ]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca2+ release-activated Ca2+ (CRAC) channel pathway required for [Ca2+ ]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naive CD4+ T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca2+ ]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen specific ORAI1-DN T cells had a two-fold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest