Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Glucocorticoid Receptor-α and MKP-1 as Candidate Biomarkers for Treatment Response and Disease Activity in Vogt-Koyanagi-Harada Disease.

PURPOSE: To investigate the potential of utilizing the expression of genes for glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase-1 (MKP-1) as biomarkers of corticosteroid (CS) refractoriness and disease activity in patients with Vogt-Koyanagi-Harada (VKH) disease. DESIGN: Prospective cohort study. METHODS: Twenty VKH patients receiving their first cycle of CS treatment in the absence of additional systemic immunosuppressive therapy and a control group of fifteen healthy volunteers were recruited from the University of Chile (Santiago, Chile) and US National Institutes of Health (Bethesda, United States). Intraocular inflammation was clinically quantified at enrolment and all follow-up visits. CS refractoriness was defined as an ocular reactivation of VKH upon CS withdrawal at a daily oral prednisone dose of 10 mg or more. Quantitative Reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the mRNA levels of the alpha (α) and beta (ß) isoforms of GR and MKP-1 in peripheral blood mononuclear cells (PBMC) after in vitro stimulation with either anti-CD3/anti-CD28 antibodies, lipopolysaccharide (LPS), or phytohemagglutinin (PHA), in the presence or absence of dexamethasone (Dex). RESULTS: After 6 hours of stimulation in the presence of Dex, PBMC from CS-refractory VKH patients had an impaired elevation in GRα expression (P = .03). Furthermore, inactive patients showed a significant Dex-induced upregulation of MKP-1 (P = .005). CONCLUSIONS: In this pilot study, the expression of GR isoforms and MKP-1 corresponded with patients' clinical response to systemic CS treatment and disease activity, respectively. Hence, these candidate biomarkers have potential clinical utility in the early identification of CS refractoriness and subclinical inflammation in patients with VKH disease.

CytoBinning: Immunological insights from multi-dimensional data.

New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses.

STAT5B: A Differential Regulator of the Life and Death of CD4+ Effector Memory T Cells.

Understanding the control of Ag restimulation-induced T cell death (RICD), especially in cancer immunotherapy, where highly proliferating T cells will encounter potentially large amounts of tumor Ags, is important now more than ever. It has been known that growth cytokines make T cells susceptible to RICD, but the precise molecular mediators that govern this in T cell subsets is unknown until now. STAT proteins are a family of transcription factors that regulate gene expression programs underlying key immunological processes. In particular, STAT5 is known to favor the generation and survival of memory T cells. In this study, we report an unexpected role for STAT5 signaling in the death of effector memory T (TEM) cells in mice and humans. TEM cell death was prevented with neutralizing anti-IL-2 Ab or STAT5/JAK3 inhibitors, indicating that STAT5 signaling drives RICD in TEM cells. Moreover, we identified a unique patient with a heterozygous missense mutation in the coiled-coil domain of STAT5B that presented with autoimmune lymphoproliferative syndrome-like features. Similar to Stat5b-/- mice, this patient exhibited increased CD4+ TEM cells in the peripheral blood. The mutant STAT5B protein dominantly interfered with STAT5-driven transcriptional activity, leading to global downregulation of STAT5-regulated genes in patient T cells upon IL-2 stimulation. Notably, CD4+ TEM cells from the patient were strikingly resistant to cell death by in vitro TCR restimulation, a finding that was recapitulated in Stat5b-/- mice. Hence, STAT5B is a crucial regulator of RICD in memory T cells in mice and humans.

B Cell Anomalies in Autoimmune Retinopathy (AIR).

Purpose: Autoimmune retinopathy (AIR) is a retinopathy associated with unexplained vision loss presumably linked to circulating antiretinal antibodies; currently, however, there are no standardized criteria regarding the diagnosis, treatment strategy, or pathogenesis of this disease. The importance of B-lymphocyte immunophenotyping in the classification of AIR is unknown. Methods: We utilized 15-color multiparametric flow cytometry to identify aberrations in B cell subsets that may contribute to the pathophysiology of AIR. Luminex cytokine analysis was also performed on plasma samples from AIR patients. Results: Significant differences in AIR patients compared to individuals with other inflammatory conditions or healthy donors were found in the B cell memory compartment, including an increase in naïve B cells and a decrease in switched and unswitched memory B cells, which correlated with alterations in immunoglobulin secretion. Conclusions: These findings suggest that the maturation process of B cells may be impaired and that B cell immunophenotyping may help in understanding disease process in AIR.

Modulation of Immune Responses by Extracellular Vesicles From Retinal Pigment Epithelium.

PURPOSE: Extracellular vesicles (EV), such as exosomes, are important mediators of intercellular communication and have been implicated in modulation of the immune system. We investigated if EV released from retinal pigment epithelium (RPE) modulate immune responses in vitro. METHODS: Extracellular vesicles were isolated from ARPE-19 cultures stimulated or not with the inflammatory cytokines IL-1ß, IFN-γ, and TNF-α. Isolated EV were characterized by nanoparticle flow and Western blot analyses. Retinal pigment epithelium-derived EV were cultured with human peripheral blood mononuclear cells, which were assayed for T-cell proliferation by 3H-thymidine incorporation. Retinal pigment epithelium-derived EV were also independently cultured with enriched lymphocytes or monocytes. Cell phenotype and cell death were evaluated by flow cytometric analysis. Cytokine levels were assayed in culture supernatants by multiplex bead analysis. RESULTS: The concentration of ARPE-derived EV from cytokine-stimulated cultures was slightly higher than from nonstimulated cultures. The size of EV was approximately 100 nm in both groups. Extracellular vesicles from both nonstimulated and cytokine-stimulated ARPE-19 significantly inhibited T-cell proliferation without affecting T-cell viability. Culture of EV from nonstimulated ARPE-19 with undifferentiated human monocytes induced an immunoregulatory phenotype with a significantly higher percentage of CD14++CD16+ monocytes and upregulation of TGF-ß1. Culture of EV from cytokine-stimulated ARPE-19 cells with human monocytes induced upregulation of several proinflammatory cytokines and monocyte death. CONCLUSIONS: Retinal pigment epithelium cells constitutively secrete EV in the size range of exosomes, with increased release from RPE cells stimulated with inflammatory cytokines. Extracellular vesicles from both nonstimulated and cytokine-stimulated RPE inhibited T-cell stimulation. Extracellular vesicles from nonstimulated ARPE-19 cells promoted an immunoregulatory CD14++CD16+ phenotype in human monocytes, while EV from cytokine-stimulated ARPE-19 cells caused human monocyte death. These findings suggest that RPE cells use EV to induce a downregulatory immune environment under homeostatic conditions. In an inflammatory milieu, RPE-derived EV may mitigate a potentially harmful inflammatory response through killing of monocytes.

Elevated CD1c+ Myeloid Dendritic Cell Proportions Associate With Clinical Activity and Predict Disease Reactivation in Noninfectious Uveitis.

PURPOSE: To test the association between elevated proportions of CD1c+ myeloid dendritic cells (mDCs) and disease activation/reactivation in noninfectious uveitis. METHODS: Noninfectious uveitis patients (n = 89) and healthy controls (n = 111) were recruited. The proportion of CD1c+ mDCs in the total dendritic cell (DC) population of peripheral blood was measured by flow cytometry (CD1c+ mDCs gated on Lineage 1+HLADR+ DCs). Disease activity was assessed per Standardization of Uveitis Nomenclature criteria. Uveitis reactivation was ascribed to clinically quiescent patients who developed reactivation of intraocular inflammation within 6 months. RESULTS: The proportions of CD1c+ mDCs were increased in noninfectious uveitis patients, especially in active disease, compared to healthy controls. This CD1c+ mDC elevation was not associated with underlying systemic diseases, anatomic locations of uveitis, medications, or demographic factors. Longitudinal data showed that the dynamics of CD1c+ mDC levels were correlated with disease activity. The average proportion of CD1c+ mDCs in active uveitis patients was 60% so we set this as the cutoff between high and low CD1c+ mDC levels. Although 74% of quiescent patients had low proportions of CD1c+ mDCs, 26% still had high proportions. Quiescent patients with high CD1c+ mDC proportions showed increased risk of disease reactivation, compared to quiescent patients with low CD1c+ mDC proportions. CONCLUSIONS: Increased proportions of CD1c+ mDCs were associated with clinical activity, and quiescent patients with elevated CD1c+ mDCs were more likely to undergo reactivation. This suggests that CD1c+ mDC proportion may be a potential biomarker for assessing clinical activation and reactivation in noninfectious uveitis.

Identification of a novel mutation in MAGT1 and progressive multifocal leucoencephalopathy in a 58-year-old man with XMEN disease.

XMEN disease (X-linked immunodeficiency with Magnesium defect, Epstein-Barr virus infection and Neoplasia) is a novel primary immune deficiency caused by mutations in MAGT1 and characterised by chronic infection with Epstein-Barr virus (EBV), EBV-driven lymphoma, CD4 T-cell lymphopenia, and dysgammaglobulinemia [1]. Functional studies have demonstrated roles for magnesium as a second messenger in T-cell receptor signalling [1], and for NKG2D expression and consequently NK- and CD8 T-cell cytotoxicity [2]. 7 patients have been described in the literature; the oldest died at 45 years and was diagnosed posthumously [1-3]. We present the case of a 58-year-old Caucasian gentleman with a novel mutation in MAGT1 with the aim of adding to the phenotype of this newly described disease by detailing his clinical course over more than 20 years.

X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia disease: a combined immune deficiency with magnesium defect.

PURPOSE OF REVIEW: To describe the role of the magnesium transporter 1 (MAGT1) in the pathogenesis of 'X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia' (XMEN) disease and its clinical implications. RECENT FINDINGS: The magnesium transporter protein MAGT1 participates in the intracellular magnesium ion (Mg) homeostasis and facilitates a transient Mg influx induced by the activation of the T-cell receptor. Loss-of-function mutations in MAGT1 cause an immunodeficiency named 'XMEN syndrome', characterized by CD4 lymphopenia, chronic EBV infection, and EBV-related lymphoproliferative disorders. Patients with XMEN disease have impaired T-cell activation and decreased cytolytic function of natural killer (NK) and CD8 T cells because of decreased expression of the NK stimulatory receptor 'natural-killer group 2, member D' (NKG2D). Patients may have defective specific antibody responses secondary to T cell dysfunction, but B cells have not been shown to be directly affected by mutations in MAGT1. SUMMARY: XMEN disease has revealed a novel role for free intracellular magnesium in the immune system. Further understanding of the MAGT1 signaling pathway may lead to new diagnostic and therapeutic approaches.

Divalent cation signaling in immune cells.

Divalent cations of two alkaline earth metals Ca(2+) and Mg(2+) and the transition metal Zn(2+) play vital roles in the immune system, and several immune disorders are associated with disturbances of their function. Until recently only Ca(2+) was considered to serve as a second messenger. However, signaling roles for Mg(2+) and Zn(2+) have been recently described, leading to a reevaluation of their role as potential second messengers. We review here the roles of these cations as second messengers in light of recent advances in Ca(2+), Mg(2+), and Zn(2+) signaling in the immune system. Developing a better understanding of these signaling cations may lead to new therapeutic strategies for immune disorders.

XMEN disease: a new primary immunodeficiency affecting Mg2+ regulation of immunity against Epstein-Barr virus.

Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that infects and persists in 95% of adults worldwide and has the potential to cause fatal disease, especially lymphoma, in immunocompromised hosts. Primary immunodeficiencies (PIDs) that predispose to EBV-associated malignancies have provided novel insights into the molecular mechanisms of immune defense against EBV. We have recently characterized a novel PID now named "X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia" (XMEN) disease characterized by loss-of-function mutations in the gene encoding magnesium transporter 1 (MAGT1), chronic high-level EBV with increased EBV-infected B cells, and heightened susceptibility to EBV-associated lymphomas. The genetic etiology of XMEN disease has revealed an unexpected quantitative role for intracellular free magnesium in immune functions and has led to novel diagnostic and therapeutic strategies. Here, we review the clinical presentation, genetic mutation spectrum, molecular mechanisms of pathogenesis, and diagnostic and therapeutic considerations for this previously unrecognized disease.

Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D.

The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg(2+)) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg(2+) causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8(+) T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg(2+) and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg(2+) in eukaryotic cells.

Congenital B cell lymphocytosis explained by novel germline CARD11 mutations.

Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.

CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) ß2, and prevents death-inducing signaling complex formation.

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca(2+) release-activated Ca(2+) channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca(2+) influx is fundamental to recruiting the Ca(2+)-dependent protein kinase C (PKC) ß2 to the DISC. PKCß2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca(2+) and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.

Loss of MAGT1 abrogates the Mg2+ flux required for T cell signaling and leads to a novel human primary immunodeficiency.

Although Mg(2+) has a well-recognized role as an essential cofactor for all ATP-binding enzymes, its role as a signaling ion, like Ca(2+), has been controversial. A requirement for Mg(2+)for optimal T lymphocyte stimulation was demonstrated more than 30 years ago, but the mechanism of its synergistic effect with Ca(2+)in T cell activation remains elusive. Here, we summarize our recent discovery of a signaling role for Mg(2+)in the T cell antigen receptor (TCR) signaling pathway from the study of a novel primary immunodeficiency, now named X-linked immunodeficiency with Mg(2+)defect, EBV infection and neoplasia (XMEN). XMEN patients were found to have a deficiency in magnesium transporter 1 (MAGT1), an Mg(2+)-specific transporter, which leads to the absence of a TCR-stimulated Mg(2+)flux and an attenuation of T cell activation. We further showed that this Mg(2+)flux is required proximally for the temporal orchestration of phospholipase C-γ1 (PLCγ1) activation. Thus, our study not only provides a second messenger role for Mg(2+)to explain its synergism with calcium in T cell signaling, it also identifies a potential extracellular therapeutic target for T cell-specific immunomodulation.

Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death.

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr.

Second messenger role for Mg2+ revealed by human T-cell immunodeficiency.

The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.

The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²âº/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.