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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

Dendritic cells (DCs) devoid of the actin regulator Wiskott-Aldrich syndrome protein (WASp) show reduced directed migration and decreased formation of podosome adhesion structures. We examined DCs expressing a gain-of-function mutation in WASp, WASp L272P, identified in X-linked neutropenia patients. Analysis of WASp L272P DCs was compared to WASp-deficient DCs to examine how WASp activity influences DC migratory responses. In confined space, WASp-deficient DCs had increased migration speed whereas WASp L272P DCs had similar average speed but increased speed fluctuations, reduced displacement, and atypical rounded morphology, compared to wild-type (WT) DCs. Using an ear inflammation model and flow cytometry analysis, WT, WASp-deficient, and WASp L272P DCs were found to migrate in comparable numbers to the draining lymph nodes (LNs). However, histology analysis revealed that migratory DCs of WASp deficient and WASp L272P mice were mainly located in the collagenous capsule of the LN whereas WT DCs were located inside the LN. Analysis of ultrastructural features revealed that WASp L272P DCs had reduced cell area but formed larger podosome structures when compared to WT DCs. Together, our data suggest that WASp activity regulates DC migration and that loss-of-function and gain-of-function in WASp activity lead to different and phenotype-specific DC migratory behavior.

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

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.

Standing et al. report a novel autoinflammatory disease caused by a homozygous missense mutation in the actin-regulating protein WDR1. The disease is characterized by periodic fevers, immunodeficiency, and thrombocytopenia, with increased polymerized actin in immune cells and increased IL-18 secretion., The importance of actin dynamics in the activation of the inflammasome is becoming increasingly apparent. IL-1β, which is activated by the inflammasome, is known to be central to the pathogenesis of many monogenic autoinflammatory diseases. However, evidence from an autoinflammatory murine model indicates that IL-18, the other cytokine triggered by inflammasome activity, is important in its own right. In this model, autoinflammation was caused by mutation in the actin regulatory gene WDR1. We report a homozygous missense mutation in WDR1 in two siblings causing periodic fevers with immunodeficiency and thrombocytopenia. We found impaired actin dynamics in patient immune cells. Patients had high serum levels of IL-18, without a corresponding increase in IL-18–binding protein or IL-1β, and their cells also secreted more IL-18 but not IL-1β in culture. We found increased caspase-1 cleavage within patient monocytes indicative of increased inflammasome activity. We transfected HEK293T cells with pyrin and wild-type and mutated WDR1. Mutant protein formed aggregates that appeared to accumulate pyrin; this could potentially precipitate inflammasome assembly. We have extended the findings from the mouse model to highlight the importance of WDR1 and actin regulation in the activation of the inflammasome, and in human autoinflammation.

High levels of ecto-5'-nucleotidase (CD73) have been implicated in immune suppression and tumor progression, and have also been observed in cancer patients who progress on anti-PD-1 immunotherapy. Although regulatory T cells can express CD73 and inhibit T cell responses via the production of adenosine, less is known about CD73 expression in other immune cell populations. We found that tumor-infiltrating NK cells upregulate CD73 expression and the frequency of these CD73-positive NK cells correlated with larger tumor size in breast cancer patients. In addition, the expression of multiple alternative immune checkpoint receptors including LAG-3, VISTA, PD-1, and PD-L1 was significantly higher in CD73-positive NK cells than in CD73-negative NK cells. Mechanistically, NK cells transport CD73 in intracellular vesicles to the cell surface and the extracellular space via actin polymerization-dependent exocytosis upon engagement of 4-1BBL on tumor cells. These CD73-positive NK cells undergo transcriptional reprogramming and upregulate IL-10 production via STAT3 transcriptional activity, suppressing CD4-positive T cell proliferation and IFN-γ production. Taken together, our results support the notion that tumors can hijack NK cells as a means to escape immunity and that CD73 expression defines an inducible population of NK cells with immunoregulatory properties within the tumor microenvironment.

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:128 issue:9 pages:4115-4131 ispartof: location:United States status: published

The constitutively active mutant of the Wiskott-Aldrich Syndrome protein (CA-WASp) is the cause of X-linked neutropenia and is linked with genomic instability and myelodysplasia. CA-WASp generates abnormally high levels of cytoplasmic F-actin through dysregulated activation of the Arp2/3 complex leading to defects in cell division. As WASp has no reported role in cell division, we hypothesized that alteration of cell mechanics because of increased F-actin may indirectly disrupt dynamic events during mitosis. Inhibition of the Arp2/3 complex revealed that excess cytoplasmic F-actin caused increased cellular viscosity, slowed all phases of mitosis, and perturbed mitotic mechanics. Comparison of chromosome velocity to the cytoplasmic viscosity revealed that cells compensated for increased viscosity by up-regulating force applied to chromosomes and increased the density of microtubules at kinetochores. Mitotic abnormalities were because of overload of the aurora signaling pathway as subcritical inhibition of Aurora in CA-WASp cells caused increased cytokinesis failure, while overexpression reduced defects. These findings demonstrate that changes in cell mechanics can cause significant mitotic abnormalities leading to genomic instability, and highlight the importance of mechanical sensors such as Aurora B in maintaining the fidelity of hematopoietic cell division.

The actin cytoskeleton is crucial at many junctures of normal immune function, and consequently there are many immune specific regulators of actin dynamics. A growing number of primary immunodeficiencies are being defined as caused by mutations in the genes encoding these regulators. In addition to immunodeficiency, immune dysregulation and autoinflammation are increasingly recognised to arise from defects within this pathway.

Megakaryoblastic leukemia 1 (MKL1), also known as MAL or myocardin-related transcription factor A (MRTF-A), is a coactivator of serum response factor, which regulates transcription of actin and actin cytoskeleton–related genes. MKL1 is known to be important for megakaryocyte differentiation and function in mice, but its role in immune cells is unexplored. Here we report a patient with a homozygous nonsense mutation in the MKL1 gene resulting in immunodeficiency characterized predominantly by susceptibility to severe bacterial infection. We show that loss of MKL1 protein expression causes a dramatic loss of filamentous actin (F-actin) content in lymphoid and myeloid lineage immune cells and widespread cytoskeletal dysfunction. MKL1-deficient neutrophils displayed reduced phagocytosis and almost complete abrogation of migration in vitro. Similarly, primary dendritic cells were unable to spread normally or to form podosomes. Silencing of MKL1 in myeloid cell lines revealed that F-actin assembly was abrogated through reduction of globular actin (G-actin) levels and disturbed expression of multiple actin-regulating genes. Impaired migration of these cells was associated with failure of uropod retraction likely due to altered contractility and adhesion, evidenced by reduced expression of the myosin light chain 9 (MYL9) component of myosin II complex and overexpression of CD11b integrin. Together, our results show that MKL1 is a nonredundant regulator of cytoskeleton-associated functions in immune cells and fibroblasts and that its depletion underlies a novel human primary immunodeficiency.

The importance of the cytoskeleton in mounting a successful immune response is evident from the wide range of defects that occur in actin-related primary immunodeficiencies (PIDs). Studies of these PIDs have revealed a pivotal role for the actin cytoskeleton in almost all stages of immune system function, from hematopoiesis and immune cell development, through to recruitment, migration, intercellular and intracellular signaling, and activation of both innate and adaptive immune responses. The major focus of this review is the immune defects that result from mutations in the Wiskott-Aldrich syndrome gene (WAS), which have a broad impact on many different processes and give rise to clinically heterogeneous immunodeficiencies. We also discuss other related genetic defects and the possibility of identifying new genetic causes of cytoskeletal immunodeficiency.

Lysosome mobilization is a key cellular process in phagocytes for bactericidal activities and trans-matrix migration. The molecular mechanisms that regulate lysosome mobilization are still poorly known. Lysosomes are hard to track as they move towards phagosomes throughout the cell volume. In order to anticipate cell regions where lysosomes are recruited to, human and RAW264.7 macrophages were seeded on surfaces that were micro-patterned with immune complexes (ICs) as 4 µm-side squares. Distances between IC patterns were adapted to optimize cell spreading in order to constrain lysosome movements mostly in 2 dimensions. Fc receptors triggered local frustrated phagocytosis, frustrated phagosomes appeared as rings of F-actin dots around the IC patterns as early as 5 minutes after cells made contact with the substratum. Frustrated phagosomes recruited actin-associated proteins (vinculin, paxillin and gelsolin). The fusion of lysosomes with frustrated phagosomes was shown by the release of beta-hexosaminidase and the recruitment of Lamp-1 to frustrated phagosomes. Lysosomes of RAW264.7 macrophages were labeled with cathepsinD-mCherry to visualize their movements towards frustrated phagosomes. Lysosomes saltatory movements were markedly slowed down compared to cells layered on non-opsonized patterns. In addition, the linearity of the trajectories and the frequency and duration of contacts of lysosomes with frustrated phagosomes were measured.¬¬¬¬¬¬¬¬ Using PP2 we showed that instant velocity, pauses and frequency of lysosome/phagosome contacts were at least in part dependent on Src tyrosine kinases. This experimental set-up is the first step towards deciphering molecular mechanisms which are involved in lysosome movements in the cytoplasm (directionality, docking and fusion) using RNA interference, pharmacological inhibition or mutant expression.