NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization

Natural killer cells play an important role in first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell-surface expressed inhibitory and activating receptors. NKp46 is a major NK cell activating receptor that is involved in the elimination of target cells. NK cells form different types of synapses that result in distinct functional outcomes: cytotoxic, inhibitory, and regulatory. Recent studies revealed that complex integration of NK receptor signaling controls cytoskeletal rearrangement and other immune synapse-related events. However the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study we determined that NKp46 forms microclusters structures at the immune synapse between NK cells and target cells. Over-expression of human NKp46 is correlated with increased accumulation of F-actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse. Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function

Seaweed Inclusion in Finishing Lamb Diet Promotes Changes in Micronutrient Content and Flavour-Related Compounds of Raw Meat and Dry-Cured Leg (Fenalår)

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Preformed CD40L Is Stored in Th1, Th2, Th17, and T Follicular Helper Cells as Well as CD4+8− Thymocytes and Invariant NKT Cells but Not in Treg Cells

CD40L is essential for the development of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, imaging studies show that the majority of cognate interactions between effector CD4+ T cells and APCs in vivo are too short to allow de novo CD40L synthesis. We previously showed that Th1 effector and memory cells store preformed CD40L (pCD40L) in lysosomal compartments and mobilize it onto the plasma membrane immediately after antigenic stimulation, suggesting that primed CD4+ T cells may use pCD40L to activate APCs during brief encounters. Indeed, our recent study showed that pCD40L is sufficient to mediate selective activation of cognate B cells and trigger DC activation in vitro. In this study, we show that pCD40L is present in Th1 and follicular helper T cells developed during infection with lymphocytic choriomeningitis virus, Th2 cells in the airway of asthmatic mice, and Th17 cells from the CNS of animals with experimental autoimmune encephalitis (EAE). pCD40L is nearly absent in both natural and induced Treg cells, even in the presence of intense inflammation such as occurs in EAE. We also found pCD40L expression in CD4 single positive thymocytes and invariant NKT cells. Together, these results suggest that pCD40L may function in T cell development as well as an unexpectedly broad spectrum of innate and adaptive immune responses, while its expression in Treg cells is repressed to avoid compromising their suppressive activity

Case Study: Mechanism for Increased Follicular Helper T Cell Development in Activated PI3K Delta Syndrome

Gain-of-function variants in p110δ, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) expressed in lymphocytes, cause activated PI3-kinase δ syndrome (APDS), a primary immunodeficiency that is characterized by recurrent infections, viremia, lymphadenopathy, and autoimmunity. The mechanism of autoimmunity in APDS has not been well-understood. Here, we show the profound skewing of peripheral CD4+ T cells to a T follicular helper (TFH) phenotype in a patient with APDS bearing a novel p110δ variant, Y524S. We also saw a diminishment of transient Foxp3 expression in activated T cells. Mechanistic studies revealed that both the new variant and a previously described, pathogenic variant (E81K) enhanced an interaction between intracellular Osteopontin and p85α. This interaction had been shown in mice to promote TFH differentiation. Our results demonstrate a new influence of PI3K on human T cell differentiation that is unrelated to its lipid-kinase activity and suggest that TFH should be monitored in APDS patients

CD28 between tolerance and autoimmunity: The side effects of animal models [version 1; referees: 2 approved]

Regulation of immune responses is critical for ensuring pathogen clearance and for preventing reaction against self-antigens. Failure or breakdown of immunological tolerance results in autoimmunity. CD28 is an important co-stimulatory receptor expressed on T cells that, upon specific ligand binding, delivers signals essential for full T-cell activation and for the development and homeostasis of suppressive regulatory T cells. Many in vivo mouse models have been used for understanding the role of CD28 in the maintenance of immune homeostasis, thus leading to the development of CD28 signaling modulators that have been approved for the treatment of some autoimmune diseases. Despite all of this progress, a deeper understanding of the differences between the mouse and human receptor is required to allow a safe translation of pre-clinical studies in efficient therapies. In this review, we discuss the role of CD28 in tolerance and autoimmunity and the clinical efficacy of drugs that block or enhance CD28 signaling, by highlighting the success and failure of pre-clinical studies, when translated to humans

T cell immunoengineering with advanced biomaterials

Recent advances in biomaterials design offer the potential to actively control immune cell activation and behaviour. Many human diseases, such as infections, cancer, and autoimmune disorders, are partly mediated by inappropriate or insufficient activation of the immune system. T cells play a central role in the host immune response to these diseases, and so constitute a promising cell type for manipulation. In vivo, T cells are stimulated by antigen presenting cells (APC), therefore to design immunoengineering biomaterials that control T cell behaviour, artificial interfaces that mimic the natural APC-T cell interaction are required. This review draws together research in the design and fabrication of such biomaterial interfaces, and highlights efforts to elucidate key parameters in T cell activation, such as substrate mechanical properties and spatial organization of receptors, illustrating how they can be manipulated by bioengineering approaches to alter T cell function

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells

Knocking at the brain’s door: intravital two-photon imaging of autoreactive T cell interactions with CNS structures

Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease