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.

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.

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.

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.

Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis.

The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.

The necrotic signal induced by mycophenolic acid overcomes apoptosis-resistance in tumor cells.

BACKGROUND: The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA-mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL-overexpressing cells). All tested cells remained sensitive to MPA-mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers. CONCLUSIONS/SIGNIFICANCE: These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

An atypical necrotic signal induced by immunosuppressive and anti-viral agents.

We recently demonstrated that the anti-viral agent ribavirin and the immunosuppressor mycophenolic acid (MPA) are both potent inducers of a necrotic signal. These two chemicals deplete the intracellular pool of guanosine triphosphate through the inhibition of inosine monophosphate dehydrogenase (IMPDH) activity. The cellular stress resulting from the GTP/GDP depletion leads to the activation of the small GTPase Cdc42 and the remodeling of actin, which are crucial events in the transmission of the MPA-mediated necrotic signal. Nevertheless, we observe for each tested cell (leukemic T and B-cell lines, activated PBLs) that a minor part of the cell population is killed through a caspase-dependent apoptotic signal. Blockade of the caspase activity eliminates the apoptotic cells, which are replaced by cells exhibiting autophagic features. In light of our findings, we assume that the newly characterized atypical cell death induced by MPA may account for the decreased risk of cancer occurrence observed in transplant recipients treated with mycophenolate mofetil (MMF) versus a non-MMF regimen.

The immunosuppressor mycophenolic acid kills activated lymphocytes by inducing a nonclassical actin-dependent necrotic signal.

Mycophenolate mofetil (MMF) is an immunosuppressive agent used in transplantation. Over the last decade, MMF has also emerged as an alternative therapeutic regimen for autoimmune diseases, mainly for patients refractory to other therapies. The active compound of MMF, mycophenolic acid (MPA), depletes the intracellular pool of guanosine tri-phosphate through inosine monophosphate dehydrogenase blockade. The molecular mechanism involved in the elimination of T and B lymphocytes upon inhibition of inosine monophosphate dehydrogenase remains elusive. In this study, we showed that in contrast to the immunosuppressors azathioprine, cyclosporin A, and tacrolimus, MPA killed lymphocytes through the activation of a caspase-independent necrotic signal. Furthermore, the MPA-mediated necrotic signal relied on the transmission of a novel intracellular signal involving Rho-GTPase Cdc42 activity and actin polymerization. In addition to its medical interest, this study sheds light on a novel and atypical molecular mechanism leading to necrotic cell death.

Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.

Rewinding the DISC.

Fas (CD95/APO-1) belongs to the tumor necrosis factor receptor family and its signaling pathway has been extensively studied over the past 15 years. Blockade of the Fas-mediated apoptotic signal leads to abusive lymphoproliferation, auto-immunity, and an increased risk of developing lymphoma and leukemia. Fas engagement drives the formation of a complex termed DISC (death-inducing signaling complex), which contains the adaptor molecule Fas-associated protein, two members of the caspase family caspase-8 and -10, and a pseudo-caspase termed c-FLIP. According to different authors, DISC formation relies either on the redistribution of Fas into the lipid rafts or the recruitment of the actin cytoskeleton and receptor endocytosis or the production of ceramide. However, the accurate molecular ordering upstream from the formation of DISC remains very puzzling and is highly debated. Herein we review some of the factors that would potentially facilitate or limit the formation of the DISC.

RhoGTPases and p53 are involved in the morphological appearance and interferon-alpha response of hairy cells.

Hairy cell leukemia is an uncommon B-cell lymphoproliferative disease of unknown etiology in which tumor cells display characteristic microfilamentous membrane projections. Another striking feature of the disease is its exquisite sensitivity to interferon (IFN)-alpha. So far, none of the known IFN-alpha regulatory properties have explained IFN-alpha responsiveness nor have they taken into account the morphological characteristics of hairy cells. IFN-alpha profoundly alters cytoskeletal organization of hairy cells and causes reversion of the hairy appearance into a rounded morphology. Because cytoskeletal rearrangements are controlled by the Rho family of GTPases, we investigated the GTPase activation status in hairy cells and their regulation by IFN-alpha. Using immunolocalization techniques and biochemical assays, we demonstrate that hairy cells display high levels of active Cdc42 and Rac1 and that IFN-alpha down-regulates these activities. In sharp contrast, RhoA activity was low in hairy cells but was increased by IFN-alpha treatment. Finally, IFN-alpha-mediated morphological changes also implicated a p53-induced response. These observations shed light on the mechanism of action of IFN-alpha in hairy cell leukemia and are of potential relevance for the therapeutical applications of this cytokine.

Soluble mannose 6-phosphate/insulin-like growth factor II (IGF-II) receptor inhibits interleukin-6-type cytokine-dependent proliferation by neutralization of IGF-II.

The calcium-independent mannose 6-phosphate receptor (CIMPR) is a receptor for multiple ligands, including leukemia inhibitory factor (LIF), an IL-6 type cytokine, and IGF-II. CIMPR targets newly synthesized ligands to lysosomes and induces internalization/degradation of secreted ligands. A natural soluble form of CIMPR (sCIMPR) neutralizes IGF-II mitogenic potency on hepatocytes and fibroblasts. Herein we show that sCIMPR also inhibits LIF-driven proliferation of myeloid and lymphoid cell lines. Similar inhibition was observed with IL-6 and IL-11, two other IL-6-type cytokines that do not interact with CIMPR. Neutralizing anti-IGF-II antibodies inhibited IL-6-, IL-11-, and LIF-driven cell proliferation to the same extent as sCIMPR, suggesting that neutralization of serum IGF-II by sCIMPR plays a major role in IL-6-type cytokine-dependent cell proliferation. Confirming this idea, ERK1/2 and AKT/protein kinase B, the kinases necessary for cell proliferation and survival, were activated by IGF-II alone or by the association of IL-6-type cytokines and IGF-II. IL-6-type cytokines alone (up to 10 ng/ml) did not activate ERK1/2 or AKT, but did activate STAT3 (signal transducer and activator of transcription 3), a transcription factor necessary for the G1 to S phase cell cycle transition. Activation of ERK1/2 and AKT by IGF-II thus appears essential to sustain cellular expansion driven by IL-6-type cytokines.