Search

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I–low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A + NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A + NK cells by the MHC-I molecule H2D d led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6 , which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2D d -educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.

Signaling via inhibitory KIR/Ly49 receptors preserves natural killer (NK) cell self-tolerance but also conveys NK cell reactivity towards MHC class-I low target cells in an education process. Here, we demonstrate that mouse NK cell education by H-2Dd regulates transcription of several genes in Ly49A+ NK cells including Ptpn6, encoding the phosphatase SHP-1. SHP-1 was highly expressed in uneducated NK cells, in which knock-out of Ptpn6 increased responsiveness. Following NKp46 triggering of uneducated NK cells, a higher synaptic abundance of phosphorylated SHP-1 was found relative to educated NK cells, concomitant with reduced phosphorylation of several signaling molecules, including PLC-g2, SLP-76, ZAP70/Syk and ERK1/2. SHP-1 overlapped extensively with F-actin and SLP-76 in the uneducated activating synapse of Ly49A+ NK cells, whereas a greater association between Ly49A and SHP-1 was observed in educated NK cells. Thus, our results indicate that in addition to transcriptional regulation, a distinct SHP-1 patterning in NK cell activating synapses can determine their tolerance.

BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:149 issue:3 pages:1069-1084 ispartof: location:United States status: published

Actin cytoskeletal dynamics drive cellular shape changes, linking numerous cell functions to physiological and pathological cues. Mutations in actin regulators that are differentially expressed or enriched in immune cells cause severe human diseases known as primary immunodeficiencies underscoring the importance of efficienct actin remodeling in immune cell homeostasis. Here we discuss recent findings on how immune cells sense the mechanical properties of their environement. Moreover, while the organization and biochemical regulation of cytoplasmic actin have been extensively studied, nuclear actin reorganization is a rapidly emerging field that has only begun to be explored in immune cells. Based on the critical and multifaceted contributions of cytoplasmic actin in immune cell functionality, nuclear actin regulation is anticipated to have a large impact on our understanding of immune cell development and functionality.

X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood; however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from 2 patients with XLN harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased granzyme B content and elevated degranulation and IFN-γ production when compared with healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28-coated beads, respectively. WASpL272P mouse T cells had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8+ T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to lymphocyte function associated antigen-1 engagement. When compared with WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggest that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells. ispartof: JCI INSIGHT vol:6 issue:6 ispartof: location:United States status: published

Congenital defects of the immune system called primary immunodeficiency disorders (PID) describe a group of diseases characterized by a decrease, an absence, or a malfunction of at least one part of the immune system. As a result, PID patients are more prone to develop life-threatening complications, including cancer. PID currently include over 400 different disorders, however, the variety of PID-related cancers is narrow. We discuss here reasons for this clinical phenotype. Namely, PID can lead to cell intrinsic failure to control cell transformation, failure to activate tumor surveillance by cytotoxic cells or both. As the most frequent tumors seen among PID patients stem from faulty lymphocyte development leading to leukemia and lymphoma, we focus on the extensive genomic alterations needed to create the vast diversity of B and T lymphocytes with potential to recognize any pathogen and why defects in these processes lead to malignancies in the immunodeficient environment of PID patients. In the second part of the review, we discuss PID affecting tumor surveillance and especially membrane trafficking defects caused by altered exocytosis and regulation of the actin cytoskeleton. As an impairment of these membrane trafficking pathways often results in dysfunctional effector immune cells, tumor cell immune evasion is elevated in PID. By considering new anti-cancer treatment concepts, such as transfer of genetically engineered immune cells, restoration of anti-tumor immunity in PID patients could be an approach to complement standard therapies.

Actin cytoskeleton remodeling facilitates and fine-tunes diverse cellular processes. Cells have evolved to use the same building blocks of actin monomers to form filaments through the sequential and synchronous use of actin filament regulators. This is best illustrated in immune cells which rely on a highly dynamic cytoskeleton to patrol the body and recognize and respond to cancer cells. Here, we highlight key actin regulators that are differentially expressed in immune cells and the immune cell biology learned from disease-causing mutations in these actin regulators. Moreover, we discuss two important aspects of the actin cytoskeleton in controlling cancer: the engagement in multiple phases of immune cell activation and effector function as well as the role in cellular transformation. We conclude by reflecting on how these two aspects can be balanced in developing novel chemotherapies.

Actin cytoskeleton remodeling facilitates and fine-tunes diverse cellular processes. Cells have evolved to use the same building blocks of actin monomers to form filaments through the sequential and synchronous use of actin filament regulators. This is best illustrated in immune cells which rely on a highly dynamic cytoskeleton to patrol the body and recognize and respond to cancer cells. Here, we highlight key actin regulators that are differentially expressed in immune cells and the immune cell biology learned from disease-causing mutations in these actin regulators. Moreover, we discuss two important aspects of the actin cytoskeleton in controlling cancer: the engagement in multiple phases of immune cell activation and effector function as well as the role in cellular transformation. We conclude by reflecting on how these two aspects can be balanced in developing novel chemotherapies.

The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.

An emerging new paradigm is that immune cell activation is controlled by transient interactions between supramolecular assemblies of receptors and ligands. Current immunotherapy biologic pharmaceuticals that activate or desensitize NK cells are, however, individual molecules that do not replicate this nanoscale organization of proteins. Here, we use nanoscale graphene oxide(NGO) as a template to generate soluble nanoscale clusters of Natural Killer cell-activating antibodies. We control nanocluster size and molecular number to mimic reported values for cell surface proteins. These NGO-templated molecular nanoclusters, used to stimulate NK cells via the CD16 receptor, successfully induced cellular activation, indicated by degranulation of cytolytic granules and IFN-g secretion. Importantly, activation significantly exceeded that induced by the same antibodies applied as a solution of individual molecules. These resultsdemonstrate that future immunotherapies could be enhanced by assemblingimmunomodulatory drugs into nanoclusters, and establish NGO-templating as a candidate technology.

Natural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.

Background Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. Objective We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. Methods We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. Results Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. Conclusion The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.