Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection

During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8⁺ T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcμR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso^–/–) mice reduced CD8⁺ T-cell function in the liver and resulted in virus persistence. Furthermore, Toso^–/– DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection.More than 500 million people worldwide suffer from chronic infections with hepatitis B or hepatitis C viruses.¹ Although both viruses are poorly cytopathic, persistence of either virus can lead to chronic liver inflammation and potentially cause liversteatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific CD8⁺ T cells are a major determinant governing the outcome of viral hepatitis due to their antiviral activity against virus-infected hepatocytes.^{2, 3, 4, 5} However, during prolonged infection, virus-specific CD8⁺ T cells are exhausted, resulting in their loss of function and consequently virus persistence.^{1, 6} Regulators influencing CD8⁺ T-cell function during chronic virus infection still remain ill defined.Inflammatory dendritic cells (iDCs) can develop from a subset of monocytes recruited to the site of inflammation.^{7, 8} This monocyte subset is characterized by the expression of CD115⁺/Ly6C^hi/CCR2⁺.⁷ iDCs express CD11c, CD11b, and Ly6C.^{9, 10, 11} IDCs that exhibit tumor necrosis factor (TNF)-α production and inducible nitric oxide synthase (iNOS) were named TNF-α and iNOS producing DCs (Tip-DCs). iDCs contribute to the elimination of pathogens following bacterial infection.^{12, 13, 14} During infection with influenza virus, iDCs enhance CD8⁺ T-cell immunopathology, but have limited impact on viral replication.^{11, 15} According to recent observations, chronic activation of toll-like receptor 9 leads to intrahepatic myeloid-cell aggregates (iMATE).¹⁶ These aggregates, which contain iDCs, are essential for T-cell activation and therefore participate in virus control.¹⁶ Co-stimulatory signals from either direct cell contact or from cytokines in combination with continued antigen contact in iMATEs lead to proliferation and activation of virus-specific T cells.¹⁶ These observations suggest that infiltration of professional antigen-presenting cells into target organs is important for the maintenance of strong antiviral cytotoxic CD8⁺ T-cell activity. Factors regulating iDC infiltration into the liver remain poorly understood.Toso is a membrane protein whose extracellular domain has homology to the immunoglobulin variable (IgV) domains. The cytoplasmic region has partial homology to the FAST kinase (Fas-activated serine/threonine kinase).¹⁷ Toso is expressed on B cells and activated T cells¹⁷ and is overexpressed in B-cell lymphomas.^{18, 19} Expression of Toso can influence survival of macrophages.²⁰ Originally, Toso was described as an inhibitor of FAS signaling.^{17, 21} More recently, a role of Toso in IgM binding and TNFR signaling was also demonstrated^{22, 23, 24} and consistently, Toso-deficient animals are protected from lipopolysaccharide (LPS)-induced septic shock.^{24, 25} Recently, we identified a role of Toso in the activation of granulocytes, monocytes, and DCs.^{26, 27, 28} During infection with Listeria, the expression of Toso regulated granulocyte function.^{26, 27} The role of Toso in the function of monocytes and other myeloid cells still remains to be further elucidated.In this study, we investigated the role of Toso during chronic viral infection by using the murine lymphocytic choriomeningitis virus (LCMV). We report that Toso promotes the differentiation and maturation of iDCs at virus-infected sites, which were essential for effector CD8⁺ T-cell function and in accelerating the control of the virus. We further tested the role of Toso in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model and found that Toso was required to trigger diabetes in RIP-GP mice. Taken together, we have identified an essential role of Toso in the differentiation and maturation of iDCs, which is essential for the control of persistence-prone virus infection and triggering of autoimmune disease.     

Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses

Acid sphingomyelinase (ASM), a lipid hydrolase enzyme, has the potential to modulate various cellular activation responses via the generation of ceramide and by interaction with cellular receptors. We have hypothesized that ASM modulates CD4⁺ T-cell receptor activation and impacts immune responses. We first observed interactions of ASM with the intracellular domains of both CD3 and CD28. ASM further mediates T-cell proliferation after anti-CD3/CD28 antibody stimulation and alters CD4⁺ T-cell activation signals by generating ceramide. We noted that various pharmacological inhibitors of ASM or knockdown of ASM using small hairpin RNA inhibit CD3/CD28-mediated CD4⁺ T-cell proliferation and activation. Furthermore, such blockade of ASM bioactivity by biochemical inhibitors and/or molecular-targeted knockdown of ASM broadly abrogate T-helper cell responses. In conclusion, we detail immune, pivotal roles of ASM in adaptive immune T-cell responses, and propose that these pathways might provide novel targets for the therapy of autoimmune and inflammatory diseases.Acid sphingomyelinase (ASM), a lipid hydrolase enzyme localized to lysosomes and cell membranes, converts sphingomyelin to ceramide,¹ an important lipid messenger mediating cell signaling.^{2, 3} Through the generation of ceramide, ASM appears to have an important role in regulating cell differentiation, proliferation, and apoptosis.^{1, 4} Abnormalities in ASM bioactivity result in multiple system disorders. As an example, patients with Niemann–Pick disease, who have mutations in the ASM gene, exhibit neurological symptoms at early age, and develop visceral organ abnormalities in later life.⁴ Patients with Niemann–Pick disease are at risk of infections,⁵ as can be modeled in ASM-deficient mice.^{6, 7} This phenotype has been attributed to phagocyte dysfunction.⁸ Recently, however, ASM function has also been described and noted in various other non-phagocytic immune cells, for example, regulating cytotoxic granule secretion by CD8⁺ T cells.⁹ASM has been reported to modulate T-cell receptor (TCR) signaling initiated by TNF,¹⁰ mediate CD28 signals,¹¹ and induce or rescue CD4⁺ T cells from apoptosis under certain circumstances.^{12, 13} By generating ceramide, ASM serves as a regulator of intracellular downstream signaling. However, the exact manner whereby ASM participates in TCR/CD3 or/and CD28 signaling remains controversial.^{10, 11, 14} Furthermore, the molecular mechanisms as to how ASM regulates CD4⁺ T-cell activation are still largely unexplored.Adaptive immune responses are important in the maintenance of human immune homeostasis. Imbalances in T-helper cell (Th) responses associated with aberrant CD4⁺ T-cell activation contribute to the development of inflammation as in human autoimmune diseases.^{15, 16} It remains unclear whether or how ASM might dictate Th responses during the progression of inflammatory diseases.In the present study, we confirm that ASM interacts with CD3 and CD28, and mediates intracellular signals that control CD4⁺ T-cell activation. ASM inhibition either by pharmacological inhibitors of ASM or knockdown of ASM results in decreased ceramide production. This leads to non-responsiveness of CD4⁺ T-cell to CD3/CD28 engagement, and causes globally diminished Th responses. These data suggest the pivotal role of ASM in CD3/CD28 intracellular signaling and adaptive immune responses, and also provide a potential target for the therapy of immune disease.     

Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L

Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer.Clinical and experimental studies have established that several types of solid tumors are characterized by infiltration of both innate and adaptive immune cells. Indeed, it has been reported that tumors can be infiltrated by different cell populations such as B cells, NK cells, Th1 and Th2 cells, regulatory T cells (Tregs), senescent T cells and macrophages, among others.^{1, 2, 3} Investigating the nature and effector function of these tumor-infiltrating subsets is highly relevant as accumulating evidence indicates that a dynamic cross-talk between tumors and immune system cells can regulate tumor growth and metastasis.^{4, 5}Macrophages constitute a major component of the leukocytes that infiltrate tumors. Tumor-associated macrophages (TAMs) derive almost entirely from circulating monocytes, which acquire distinct phenotypic characteristics and diverse functions according to the tumor microenvironment. Prototypically, two different types of activated macrophages have been recognized: the classically activated (M1) or pro-inflammatory macrophages and the alternatively activated (M2) macrophages. Thus, in response to diverse signals like cytokines or membrane receptor ligation, macrophages undergo M1 or M2 polarization states characterized by particular profiles of cytokine and chemokine production. M1 macrophages express high levels of pro-inflammatory cytokines, major histocompatibility complex (MHC) molecules and inducible nitric oxide (NO) sintetase. By contrast, M2 macrophages downregulate MHC class II and show increased expression of the anti-inflammatory cytokine IL-10 and mannose receptor. In addition, macrophages can also be polarized into a M2-like state, which shares some but not all the signature features of M2 cells.¹ Macrophages with intermediate or overlapping phenotypes have been observed in many pathological conditions in vivo, probably as a result of the effect of diverse signals that occur along different times of the immune response. In fact, many studies emphasize the heterogeneity and plasticity of macrophages and indicate that typical M1 and M2 phenotypes are extremes of a wide spectrum of functional states.^{6, 7, 8}Within the tumor microenvironment, the macrophages interact with or receive signals from different tumor-infiltrating immune cells such as Tregs, myeloid-derived suppressor cells, Th1 and Th2 cells, among others.^{1, 9} This interaction may regulate the profile of macrophage activation and consequently impact on tumor progression. It has been described that the excessive activity of either M1 or M2 subsets may be detrimental to the host by preventing the development of an efficient anti-tumor immune response.¹⁰ Understanding the cellular interactions that lead to the control of monocyte/macrophage (Mo/Ma) activation is, therefore, of fundamental importance to the field of tumor immunology.Senescent T cells are reported to be increased during chronic infections and some tumor processes.^{11, 12} In fact, senescent T cells circulate in the peripheral blood of most cancer patients and infiltrate tumors.² Although the hallmark of human senescent T cells is the loss of CD27 and CD28 expression, other features of these cells include shortened telomeres, reduced proliferative capacity and cytokine production as well as suppressor activity.^{13, 14} We previously reported that, after a brief contact with tumor cells, CD4+ as well as CD8+ T lymphocytes from healthy donors acquire a senescent phenotype. These CD4+ and CD8+ tumor-induced senescent (TIS)-T cells are characterized by the loss of CD27 and CD28 expression, lack of proliferative capacity, telomere shortening and increment in the expression of senescence-associated molecules such as p53, p21 and p16. Remarkably, these CD4+ and CD8+ TIS-T populations also show a potent suppressive ability.¹⁵ We also demonstrated that tumor-induced senescence of T cells is triggered by soluble factors secreted by tumor cells and that this process can be prevented by IL-7.¹⁶ These data support the hypothesis that, within the tumor microenvironment, tumor-infiltrating T lymphocytes encounter tumor cells that promote their senescence and dysfunction. These TIS-T cells would be able to suppress the lympho-proliferative response and potentially modulate other immune cells. Thus, they may serve as an intercellular cross-talk in the tumor microenvironment and impact on tumor progression.Although macrophages and TIS-T lymphocytes are both highly represented in tumors, the biological consequences of a TIS-T cell and macrophage interaction have not been studied so far. Here, we demonstrate that monocytes co-cultured with TIS-T cells in inflammatory conditions increase their production of inflammatory cytokines and angiogenic factors. In addition, we determined that Mo/Ma modulation mediated by TIS-T lymphocytes requires cell-to-cell contact and identified T-cell immunoglobulin and mucin protein 3 (Tim-3) and CD40 ligand (CD40L) molecules as mediators of this previously uncharacterized modulatory function of senescent T cells.     

CD300b regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition

The CD300 receptor family members are a group of molecules that modulate a variety of immune cell processes. We show that mouse CD300b (CLM7/LMIR5), expressed on myeloid cells, recognizes outer membrane-exposed phosphatidylserine (PS) and does not, as previously reported, directly recognize TIM1 or TIM4. CD300b accumulates in phagocytic cups along with F-actin at apoptotic cell contacts, thereby facilitating their engulfment. The CD300b-mediated activation signal is conveyed through CD300b association with the adaptor molecule DAP12, and requires a functional DAP12 ITAM motif. Binding of apoptotic cells promotes the activation of the PI3K-Akt kinase pathway in macrophages, while silencing of CD300b expression diminishes PI3K-Akt kinase activation and impairs efferocytosis. Collectively, our data show that CD300b recognizes PS as a ligand, and regulates the phagocytosis of apoptotic cells via the DAP12 signaling pathway.In both developing and mature multicellular organisms, large numbers of apoptotic cells are continually generated and must be cleared by neighboring cells or ‘professional'' phagocytes.^{1, 2, 3, 4} If not properly cleared, they become necrotic, pro-inflammatory and immunogenic, potentially leading to the development of autoimmune diseases, such as systemic lupus erythematous (SLE).^{5, 6, 7, 8} Therefore, phagocytes possess sensing systems to facilitate the clearance of apoptotic cells.^{1, 2, 3} Once guided to their location by diffusible ‘find me'' signals, phagocytes recognize apoptotic cells through their display of characteristic cell surface molecules (‘eat me'' signals).^{4, 7} The most common signal promoting phagocytosis is the recognition of phosphatidylserine (PS), which when exposed on the outer leaflet of the plasma membrane signals phagocytes to engulf apoptotic cells.² Multiple receptors for PS exist on phagocytic cells, although not necessarily simultaneously; these include stabilins,^{9, 10} T cell Ig mucin (TIM) 1 and TIM4,^{11, 12} BAI1,¹³ MFGE8, which bridges PS to integrin αvβ3,¹⁴ and Protein S and Gas6, which bridge PS to TAM receptors.¹⁵ Recently, we and others demonstrated that the CD300 family members, human and mouse CD300a,^{16, 17} and mouse CD300f,^{18, 19} also bind PS, and their expression regulates apoptotic cell phagocytosis.The CD300 family contains both activating and inhibitory receptor members.²⁰ CD300b has a short intracellular tail and gains activation potential by association with DNAX activating protein of 12 kDa (DAP12) or DAP10 adaptor molecules.²¹ CD300b is predominantly expressed on myeloid cells, including neutrophils, macrophages and mast cells. Antibody cross-linking of human and mouse CD300b has been shown to induce the release of inflammatory cytokines from mast cells.²¹ The ligand for CD300b remains a matter of debate. A recent study found that a soluble form of CD300b, released in response to Toll-like receptor ligation, recognizes unknown ligands on the surface of macrophages, resulting in the release of inflammatory cytokines.²² Others have identified the PS-binding receptors TIM1 and TIM4 as endogenous ligands for CD300b, but not PS itself.²³Here, we show that CD300b binds to PS, and recognizes PS on TIM1 or TIM4 expressing cells rather than TIM1 or TIM4 alone. We found that CD300b promotes PS-dependent apoptotic cell phagocytosis upon ectopic expression in cell lines, without the need for additional PS receptors. In addition, CD300b-mediated phagocytosis requires the association of the adaptor protein DAP12 for effective signaling. Inhibition of CD300b function by either anti-CD300b antibody treatment or siRNA transfection significantly decreases macrophage-dependent phagocytosis of apoptotic cells. Furthermore, CD300b silencing in macrophages severely impairs the apoptotic cell-induced phosphorylation of PI3K, Akt and Syk, but not Erk. Thus, our data show that CD300b is an activating receptor that has an important role in macrophage-mediated clearance of apoptotic cells.     

Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury

The loss of stem cells residing in the base of the intestinal crypt has a key role in radiation-induced intestinal injury. In particular, Lgr5⁺ intestinal stem cells (ISCs) are indispensable for intestinal regeneration following exposure to radiation. Mesenchymal stem cells (MSCs) have previously been shown to improve intestinal epithelial repair in a mouse model of radiation injury, and, therefore, it was hypothesized that this protective effect is related to Lgr5⁺ ISCs. In this study, it was found that, following exposure to radiation, transplantation of MSCs improved the survival of the mice, ameliorated intestinal injury and increased the number of regenerating crypts. Furthermore, there was a significant increase in Lgr5⁺ ISCs and their daughter cells, including Ki67⁺ transient amplifying cells, Vil1⁺ enterocytes and lysozyme⁺ Paneth cells, in response to treatment with MSCs. Crypts isolated from mice treated with MSCs formed a higher number of and larger enteroids than those from the PBS group. MSC transplantation also reduced the number of apoptotic cells within the small intestine at 6 h post-radiation. Interestingly, Wnt3a and active β-catenin protein levels were increased in the small intestines of MSC-treated mice. In addition, intravenous delivery of recombinant mouse Wnt3a after radiation reduced damage in the small intestine and was radioprotective, although not to the same degree as MSC treatment. Our results show that MSCs support the growth of endogenous Lgr5⁺ ISCs, thus promoting repair of the small intestine following exposure to radiation. The molecular mechanism of action mediating this was found to be related to increased activation of the Wnt/β-catenin signaling pathway.The epithelium of the small intestine contains crypts and villi. Intestinal stem cells (ISCs) reside in the base of the crypts and are responsible for maintaining intestinal epithelial homeostasis and regeneration following injury.^{1, 2} Recent studies have identified two populations of stem cells in the small intestine of mice called Lgr5⁺ and Bmi1⁺ ISCs.^{3, 4, 5, 6, 7, 8, 9, 10, 11} Lgr5⁺ ISCs, also known as crypt base columnar cells (CBCs), are interspersed among the Paneth cells and are active rapidly cycling stem cells.¹² A single Lgr5⁺ ISC can grow to form ‘enteroids'' in vitro that develop into all the differentiated cell types found in the intestinal crypt.¹³ Conversely, Bmi1⁺ cells are a population of ISCs located at position +4 relative to the base of the crypt, and are quiescent, slowly cycling stem cells.¹⁴ The loss of ISCs has a critical role in radiation-induced intestinal injury (RIII).^{15, 16, 17, 18} Apoptosis of stem cells because of exposure to radiation prevents normal re-epithelialization of the intestines. Therefore, enhancing the survival of ISCs following radiation is a potential effective treatment for RIII.Mesenchymal stem cells (MSCs) possess significant potential as a therapeutic for tissue damage because of their ability to regulate inflammation, inhibit apoptosis, promote angiogenesis, and support the growth and differentiation of local stem and progenitor cells.^{19, 20} However, the mechanisms by which MSCs mediate these beneficial effects remain unclear, although it has been suggested that MSCs may actively secrete a broad range of bioactive molecules with immunomodulatory (PGE2, IDO, NO, HLA-G5, TSG-6, IL-6, IL-10 and IL-1RA), mitogenic (TGFα/β, HGF, IGF-1, bFGF and EGF), angiogenic (VEGF and TGF-β1) and/or anti-apoptotic (STC-1 and SFRP2) properties that function to modulate the regenerative environment at the site of injury.²¹ Upon re-establishment of the microenvironment following damage, the surviving endogenous stem and progenitor cells can then regenerate the injured tissue completely.Our previous study, as well as other published studies, has found that systemic administration of MSCs improves intestinal epithelial repair in an animal model of radiation injury.^{22, 23, 24, 25} Following MSC treatment, radiation-induced lesions in mice were significantly smaller than those in the control group. However, the mechanism behind this protective effect is not fully understood. Lgr5⁺ ISCs have been previously shown to be indispensable for radiation-induced intestinal regeneration.²⁶ Therefore, in this study, we tested whether the therapeutic effects of MSCs in response to RIII are related to the Lgr5⁺ population of resident ISCs.     

PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy

CD8⁺ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3⁺PD-1⁺CD8⁺ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8⁺ T cells upregulated PD-1⁺ and/or Tim-3⁺ immune cells. Furthermore, the population of CD8⁺ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8⁺ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8⁺ T-cell dysfunction. Importantly, the number and function of Tim-3⁺PD-1⁺CD8⁺ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8⁺ T-cell function and maintaining normal pregnancy.Successful pregnancy requires the maternal immune system to tolerate the semi-allogeneic fetus. A failure in immune tolerance may result in abnormal pregnancies, such as recurrent spontaneous abortion. For many years, the model of immune regulation during pregnancy has been based on a shift in the maternal immune response towards a Th2 bias. The shift from producing inflammatory Th1-type cytokines toward Th2-type cytokines promotes maternal–fetal tolerance.^{1, 2} In addition, maternal administration of the Th2-type cytokine interleukin (IL)-10 or blockade of the Th1-type cytokine tumor necrosis factor (TNF)-α is known to prevent pregnancy loss induced by lipopolysaccharide.^{3, 4}Compared with CD4⁺ T cells, our understanding of the role of CD8⁺ T cells during pregnancy remains poorly understood. CD8⁺ T cells, which directly recognize allogeneic major histocompatibility complex (MHC) class I molecules, have important roles in defense against viral infections. Studies on several murine models have demonstrated the existence of CD8⁺ T cells at the maternal–fetal interface.⁵ During normal pregnancy, the major antigen present is the embryo-derived paternal antigen expressed on extravillous trophoblast (EVT) cells. These cells do not express MHC class I human leukocyte antigens (HLA)-A and HLA-B,⁶ which are the main causes of CD8⁺ T cell-mediated rejection. However, HLA-C and HLA-G, highly expressed on EVT cells,⁶ can elicit a direct cytotoxic response by CD8⁺ T cells during hematopoietic stem cell and allogeneic organ transplantation.^{7, 8} Therefore, whether suppressor or regulatory CD8⁺ T cells are present at the maternal–fetal interface, and how they function to maintain normal pregnancy, remain to be explored.Inhibitory co-stimulatory signals have crucial roles in regulating CD8⁺ T-cell activation or tolerance. It has been shown that exhausted T cells express up to seven different inhibitory molecules,⁹ including PD-1 and Tim-3. PD-1 has been identified as a marker for dysfunctional T cells, and blockade of PD-1 signals has been shown to revert the dysfunctional state of exhausted CD8⁺ T cells in most cases.^{10, 11} Tim-3 has been similarly associated with CD8⁺ T-cell exhaustion as Tim-3 blockade restores proliferation and cytokine production.^{12, 13} Tim-3 and PD-1 co-expression on T cells characterizes the most severely exhausted CD8⁺ T-cell subset, and combined blockade of Tim-3 and PD-1 restores the function of exhausted CD8⁺ T cells.^{14, 15, 16} However, much less is known about the functional regulation of Tim-3 and PD-1 on CD8⁺ T cells during pregnancy.In this study, we investigated Tim-3 and PD-1 expression on CD8⁺ T cells from decidua and peripheral blood in normal pregnant women and those who underwent miscarriage. In particular, we used surface and intracellular phenotype analysis, as well as multifunctional assays, to study the role of Tim-3 and PD-1 signaling pathways in regulating decidual CD8⁺ (dCD8⁺) T-cell function and maintenance of pregnancy. Our data indicate that Tim-3 and PD-1 co-expression on CD8⁺ T cells might be important in maintaining maternal–fetal immune tolerance and successful pregnancy. These results could provide a strategy for developing novel therapies that enhance Tim-3 and PD-1 signals to promote maternal–fetal tolerance and prevent pregnancy loss.     

Transforming growth factor-beta signaling network regulates plasticity and lineage commitment of lung cancer cells

Identification of target cells in lung tumorigenesis and characterization of the signals that control their behavior is an important step toward improving early cancer diagnosis and predicting tumor behavior. We identified a population of cells in the adult lung that bear the EpCAM+CD104+CD49f+CD44+CD24loSCA1+ phenotype and can be clonally expanded in culture, consistent with the properties of early progenitor cells. We show that these cells, rather than being restricted to one tumor type, can give rise to several different types of cancer, including adenocarcinoma and squamous cell carcinoma. We further demonstrate that these cells can be converted from one cancer type to the other, and this plasticity is determined by their responsiveness to transforming growth factor (TGF)-beta signaling. Our data establish a mechanistic link between TGF-beta signaling and SOX2 expression, and identify the TGF-beta/SMAD/SOX2 signaling network as a key regulator of lineage commitment and differentiation of lung cancer cells.Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. Lung cancers are divided into two major categories: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. NSCLC accounts for ∼80% of all lung cancers and is divided further into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large-cell lung carcinoma. Of the four major types of lung cancer, Kras mutations are present in about 30–50% of ADC, a smaller percentage of SCC (5–7%) and <1% of SCLC.^{1, 2} Mutations of the p53 gene are common in all types of lung cancer and range from ∼30% in ADC to more than 70% in SCC and SCLC.³ Other alterations occur at lower frequencies in NSCLC, including mutations in EGFR (15%), EML4-ALK (4%), ERBB2 (2%), AKT1, BRAF, MAP2K1 and MET.^{2, 4} Previous efforts in comprehensive characterization of lung cancer include copy number and gene expression profiling, targeted sequencing of candidate genes and large-scale genome sequencing of tumor samples.^{5, 6, 7, 8, 9} Significant progress has also been made in developing mouse models of lung carcinogenesis.^{10, 11} The unifying theme underlying these studies is that there exists a permissive cellular context for each specific oncogenic lesion, and that only certain types of cells are capable of cancer initiation.^{12, 13, 14}The lung consists of three anatomically distinct regions such as trachea, bronchioles and alveoli, each maintained by a distinct population of progenitor cells, that is, basal, Clara and alveolar type 2 (AT2) cells, respectively.^{15, 16} Previous work has focused upon AT2 cells, Clara cells (or variant Clara cells with low CC10 expression) and the putative bronchioalveolar stem cells (BASCs) as potential cells of origin for lung ADC.^{12, 14, 17} However, to date, only AT2 cells have been conclusively identified as having the potential to be the cells of origin for lung ADC.^{14, 17} This raises the question of whether Clara cells, their restricted subpopulations or the newly identified candidate stem cells, termed distal airway stem cells,¹⁸ alveolar epithelial progenitor cells (AECs)^{19, 20} and BASCs,¹² also have the capacity to give rise to ADC. Current knowledge on the cellular origins of SCC, the second most common type of lung cancer, lags behind that of ADC, partly owing to the fact that squamous cells are not normally present in the respiratory epithelium, and therefore arise through either metaplasia (conversions between stem cell states) or trans-differentiation (conversions between differentiated cells).^{21, 22} Whether the mechanisms of SCC causation vary by cell type, their responses to various cells signaling cascades (e.g., transforming growth factor (TGF)-beta, WNT, etc.), or other tumor characteristics is unknown at present.To address the questions of cell type of origin and signal cascades that control their behavior, we developed in vitro culture conditions that favor the growth of lung epithelial cells with stem cell-like properties. We describe a population of cells isolated from the adult lung that, rather than being restricted to one tumor type, can give rise to several different types of cancer, including ADC and SCC. We also show that these cells can be converted from one cancer type to the other, and this plasticity is largely, if not solely, determined by TGF-beta signaling.     

RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8+ T cells

Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α⁺ DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8⁺ T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity.Patients diagnosed with pancreatic cancer face a poor prognosis due to early metastasis and therapy resistance, resulting in a 5-year survival rate of only 6%.¹ Treatment options for inoperable tumors are limited and offer little benefit for the patients. But even after tumor resection most patients relapse and succumb to their disease, as evidenced by a 5-year survival rate of 20%.² Novel treatment strategies such as immunotherapy are being investigated.³ Pancreatic cancer is characterized by an immunosuppressive microenvironment, which is mediated by cytokines such as TGF-β, modulation of antigen-presenting cells, impaired T-cell effector function as well as recruitment of regulatory T cells and myeloid-derived suppressor cells.⁴ Immunosuppressive factors correlate with a poor prognosis for patients with pancreatic cancer.^{5, 6, 7, 8} On the other hand, T-cell infiltrates of the tumor were found to be a positive prognostic factor.⁹ The major challenge for immunotherapy will be to counteract immunosuppressive mechanisms for tipping the balance toward productive immune responses against the tumor.Tumor cell death occurs spontaneously in fast growing tumors or is induced by specific therapies, such as cytotoxic agents or irradiation. Several forms of cell death, such as apoptosis, necrosis, autophagy, mitotic catastrophe and senescence can be discriminated. It appears that the conditions leading to tumor cell death dictate immunological consequences.^{10, 11} In most circumstances, cell death is immunologically silent, leading to tolerance rather than immunity. In specific situations, dying cells release immunogenic signals to the cell surface or the extracellular space leading to the activation of antigen-presenting cells, such as DCs, and facilitating antigen uptake and presentation. These signals are collectively called danger-associated molecular patterns (DAMPs) and include calreticulin exposure on the outer cell membrane, release of heat shock proteins, HMGB1, DNA, RNA, ATP and uric acid crystals, or the secretion of proinflammatory cytokines, such as IL-1 and IL-6.¹² Evidence has accumulated that certain chemotherapeutic drugs, which were traditionally considered to mediate antitumor effects via their antiproliferative properties, induce an immunogenic form of cell death leading to tumor-directed immunity.^{11, 13}Immune responses against viruses share many features with those against tumors. Mimicking a viral infection can be exploited for tumor immunotherapy. Double-stranded viral RNA is recognized by cytosolic pattern recognition receptors called RIG-I-like helicases (RLH), including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated antigen 5 (MDA5).^{14, 15, 16} Synthetic RLH ligands include 5′-triphosphate RNA (ppp-RNA) for RIG-I, and polyinosinic:polycytidylic acid (poly(I:C)) for MDA5. RLH initiate a signaling cascade mediated by IFN regulatory factor 3 (IRF-3), IRF-7 and NF-κB, leading to an antiviral response program characterized by the production of type I IFN and other innate immune response genes.^{17, 18} In addition, RLH signaling induces intrinsic apoptosis in tumor cells, which are highly susceptible, as compared with nonmalignant cells.^{19, 20} RLH ligands have been evaluated as therapeutic agents in preclinical tumor models for melanoma, ovarian cancer and pancreatic cancer.^{19, 21, 22, 23, 24} Therapeutic efficacy was enhanced by combining RNAi-mediated gene silencing with RIG-I activation in a single RNA molecule.^{21, 24} A ppp-siRNA targeting the anti-apoptotic protein Bcl-2 to promote tumor apoptosis showed therapeutic efficacy in experimental melanoma.²⁴ In this model, the antitumor effect was dependent on NK cells. To counteract tumor-induced immunosuppression, our group generated a ppp-siRNA silencing TGF-β1, which showed therapeutic efficacy in an orthotopic model of pancreatic cancer.²¹ Interestingly, with this approach CD8⁺ T cells mediated antitumor efficacy. Others reported that treatment of human ovarian cancer cells with RLH ligands resulted in phagocytosis of apoptotic tumor cells by monocyte-derived DCs and DC activation.^{22, 23} Together, these findings indicate that RLH-induced tumor cell death may promote adaptive immunity against tumors. However, mechanisms leading to DC activation and the impact on tumor antigen cross-presentation by DCs, which defines immunogenic cell death, have not been explored.In this study, we investigated the effects of RLH-induced tumor cell death on DC activation, antigen uptake and cross-presentation of tumor antigen by primary murine DC populations. We also studied mechanisms leading to DC activation using mice deficient in pathways of TLR, RAGE, inflammasome and type I IFN signaling. In addition, we assessed the immunogenicity and therapeutic efficacy of RLH-based immunotherapy in two different mouse models for pancreatic cancer.     

Severe influenza A(H1N1)pdm09 infection induces thymic atrophy through activating innate CD8+CD44hi T cells by upregulating IFN-γ

Thymic atrophy has been described as a consequence of infection by several pathogens including highly pathogenic avian influenza virus and is induced through diverse mechanisms. However, whether influenza A(H1N1)pdm09 infection induces thymic atrophy and the mechanisms underlying this process have not been completely elucidated. Our results show that severe infection of influenza A(H1N1)pdm09 led to progressive thymic atrophy and CD4⁺CD8⁺ double-positive (DP) T-cells depletion due to apoptosis. The viruses were present in thymus, where they activated thymic innate CD8⁺CD44^hi single-positive (SP) thymocytes to secrete a large amount of IFN-γ. Milder thymic atrophy was observed in innate CD8⁺ T-cell-deficient mice (C57BL/6J). Neutralization of IFN-γ could significantly rescue the atrophy, but peramivir treatment did not significantly alleviate thymic atrophy. In this study, we demonstrated that thymic innate CD8⁺CD44^hi SP T-cells have critical roles in influenza A(H1N1)pdm09 infection-induced thymic atrophy through secreting IFN-γ. This exceptional mechanism might serve as a target for the prevention and treatment of thymic atrophy induced by influenza A(H1N1)pdm09.Influenza A virus can cause recurrent epidemics and is the cause of one of the most important diseases, resulting in substantial human morbidity and mortality. The recent swine-origin 2009 pandemic influenza A H1N1 virus (influenza A(H1N1)pdm09) lead more than 60 million laboratory-confirmed cases in 214 countries and over 18 449 deaths until August 2010.¹ However, the basis for the increased pathogenesis of the virus remains not fully clear.Although influenza A(H1N1)pdm09 did not cause high mortality, there was an unusually high frequency of fatal cases in healthy young and middle-aged patients.^{2, 3, 4} More than 60% of the confirmed cases occurred in individuals between 5 and 29 years of age.⁵ In addition to severe pathological pneumonia and hypercytokinemia in the lungs and serum,^{2, 6} we also previously found another hallmark of H5N1 or H1N1 virus infection in humans, which was strong reduction in T lymphocytes, also known as lymphopenia.^{7, 8, 9, 10} Peripheral lymphopenia occurs in parallel with thymic atrophy. Several microorganisms can infect the thymus and perturb the systemic T-cell pool.¹¹ Lymphopenia in fatal influenza A(H1N1)pdm09 cases in the young population may also be related to thymic atrophy.¹² Several mechanisms have been implicated in infection-induced thymic atrophy, and vary depending on the microorganism. Thymic atrophy in HPAIV infection has been reported to interfere with T-lymphocyte development through negative selection and glucocorticoids (GCs).^{13, 14} However, the mechanisms of influenza A(H1N1)pdm09-induced thymic atrophy have not been completely elucidated.Unlike conventional T cells, which acquire effector function in the periphery following interaction with Ag,^{15, 16} some innate CD8⁺ thymocytes in thymus display an effector-memory phenotype and effector function ‘from birth'' by rapidly producing cytokines upon stimulation.^{16, 17} A large proportion of innate CD8⁺ thymocyte were found and developed in the thymus of Itk^−/−/RLK^−/−, KLF2^−/−or Id3^−/− mice.^{17, 18} Subsequently, it was found that ∼10% of TCRαβ⁺ CD4⁻CD8⁺ thymocytes were innate polyclonal T cells (CD8⁺CD44^hi) in normal mice.¹⁹ Whether innate CD8⁺ thymocytes are involved in the pathogenesis of influenza A(H1N1)pdm09-induced thymic atrophy should be further evaluated.In this study, we demonstrated that severe influenza A(H1N1)pdm09 infection induced strong thymic atrophy. The viruses could infect the thymus, and further primed the innate CD8⁺CD44^hi T cells. Innate CD8⁺ T cells induced apoptosis of thymocytes by upregulating IFN-γ. Our results indicated that the pathogenesis of influenza A(H1N1)pdm09 infection was not only due to severe lung damage but also due to innate CD8⁺ T-cell-induced thymic atrophy.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

Apoptotic mechanisms during competition of ribosomal protein mutant cells: roles of the initiator caspases Dronc and Dream/Strica

Heterozygosity for mutations in ribosomal protein genes frequently leads to a dominant phenotype of retarded growth and small adult bristles in Drosophila (the Minute phenotype). Cells with Minute genotypes are subject to cell competition, characterized by their selective apoptosis and removal in mosaic tissues that contain wild-type cells. Competitive apoptosis was found to depend on the pro-apoptotic reaper, grim and head involution defective genes but was independent of p53. Rp/+ cells are protected by anti-apoptotic baculovirus p35 expression but lacked the usual hallmarks of ‘undead'' cells. They lacked Dronc activity, and neither expression of dominant-negative Dronc nor dronc knockdown by dsRNA prevented competitive apoptosis, which also continued in dronc null mutant cells or in the absence of the initiator caspases dredd and dream/strica. Only simultaneous knockdown of dronc and dream/strica by dsRNA was sufficient to protect Rp/+ cells from competition. By contrast, Rp/Rp cells were also protected by baculovirus p35, but Rp/Rp death was dronc-dependent, and undead Rp/Rp cells exhibited typical dronc-dependent expression of Wingless. Independence of p53 and unusual dependence on Dream/Strica distinguish competitive cell death from noncompetitive apoptosis of Rp/Rp cells and from many other examples of cell death.In Drosophila, heterozygous mutation of many ribosomal protein gene loci leads to the dominant ‘Minute'' phenotype, named for its small thin bristles.^{1, 2} Minute animals show a dominant developmental delay. In addition, Minute (that is, Rp/+) cells tend to be lost from mosaics that contain wild-type cells, making it difficult for clones of Rp/+ genotypes to survive and contribute to the adult.^{3, 4, 5, 6, 7} Such conditional cell viability that depends on a heterotypic cellular environment is termed ‘cell competition''.⁴Competition of Rp/+ clones is suppressed by equalizing growth rates through starvation⁸ or nonmosaic mutation of a second Rp locus.⁴ Hyperplastic clones that express higher levels of myc^{9, 10} or lower levels of the Salvador-Hippo-Warts pathway tumor suppressors out-compete nearby wild-type cells, that is, they are ‘super-competitors''.^{7, 11} Competition based on c-myc also occurs in mouse embryogenesis.¹² Differential growth is not always sufficient to cause cell competition, as cells growing rapidly due to elevated CyclinD/Cdk4 activity or higher activity of the insulin/IGF pathway are not super-competitive.⁹ Differences in Jak/Stat signaling, Wg signaling and cell adhesion are also reported to generate cell competition.^{13, 14, 15} These findings suggest that cell competition arises from specific interactions between cells, rather than as a general consequence of differential growth.Apoptotic cell death is a fundamental part of cell competition. Elimination of Rp/+ clones is delayed by expression of the caspase inhibitor baculovirus p35.⁵ Apoptosis of Rp/+ cells also occurs when clones of wild-type cells arise in Rp/+ backgrounds, predominantly among Rp/+ cells nearby wild-type cells.^{6, 16} As expected, such apoptosis is prevented by expression of baculovirus p35 or DIAP1.^{6, 16, 17}Cell competition has been hypothesized to contribute to human cancer, because most tumors have an altered genotype, and because many genes implicated in cell competition are homologs of oncogenes and tumor suppressors.^{18, 19, 20, 21} Cell competition may contribute to homeostasis of organ growth^{4, 9} and to antitumor surveillance.^{22, 23, 24, 25, 26}Cell competition may be a means to eliminate certain categories of aneuploid cells.^{27, 28} Seventy-nine ribosomal protein genes, sixty-six of which are haploinsufficient Minute loci, are distributed throughout the Drosophila genome.² Copy number changes to parts of the genome are likely to perturb relative dose of Rp/+ genes, and those that reduce Rp gene dose could be subject to cell competition. This suggests cell competition can eliminate some aneuploid cells even after DNA damage responses have ceased.^{27, 28, 29}In humans, heterozygosity for multiple different Rp mutations causes Diamond Blackfan Anemia.³⁰ Accumulation of ribosomal assembly intermediates or of unassembled ribosomal proteins in these genotypes activates p53, for example through the binding of the p53 ubiquitin ligase Mdm2 by RpL11 or RpL5.³¹ The p53 pathway leads to cell cycle arrest and/or apoptosis,³² and loss of hematopoietic stem cells causes anemia. Diamond Blackfan Anemia is a condition of nonmosaic individuals, so its relationship to cell competition is unclear.The uncertain nature of the cell interactions that trigger competition might be illuminated if the initiation of competitive apoptosis was understood. The Drosophila genome encodes three potential initiator caspases that might be activated through long prodomains, and four effector caspase zymogens lacking prodomains that are activated by initiator caspases and by one another.³³ Here, the p53 and initiator caspase requirements for competitive cell death of Rp/+ cells were determined. Whereas Dronc is the initiator caspase for most apoptosis in Drosophila,^{34, 35, 36, 37} we found that competitive cell death could occur without dronc or p53. Experiments that eliminated multiple initiator caspases simultaneously demonstrated that competitive apoptosis of Rp/+ cells required Dronc and Dream/Strica redundantly, a difference from most other apoptotic genotypes in Drosophila, for example, Rp/Rp cells generated in these experiments died in a Dronc-dependent manner.     

TRPM2 channel deficiency prevents delayed cytosolic Zn2+ accumulation and CA1 pyramidal neuronal death after transient global ischemia

Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn²⁺ level ([Zn²⁺]_c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia–reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn²⁺]_c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn²⁺]_c but abolished the cytosolic Zn²⁺ accumulation during reperfusion as well as ROS-elicited increases in the [Zn²⁺]_c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn²⁺]_c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury.Transient ischemia is a major cause of chronic neurological disabilities including memory impairment and cognitive dysfunctions in stroke survivors.^{1, 2} The underlying mechanisms are complicated and multiple, and remain not fully understood.³ It is well documented in rodents, non-human primates and humans that pyramidal neurons in the CA1 region of the hippocampus are particularly vulnerable and these neurons are demised after transient ischemia, commonly referred to as the delayed neuronal death.⁴ Studies using in vitro and in vivo models of transient ischemia have demonstrated that an increase in the [Zn²⁺]_c or cytosolic Zn²⁺ accumulation is a critical factor.^{5, 6, 7, 8, 9, 10, 11} There is evidence supporting a role for ischemia-evoked release of vesicular Zn²⁺ at glutamatergic presynaptic terminals and subsequent entry into postsynaptic neurons via GluA2-lacking AMPA subtype glutamate receptors (AMPARs) to raise the [Zn²⁺]_c.^{12, 13, 14, 15, 16} Upon reperfusion, while glutamate release returns to the preischemia level,¹⁷ Zn²⁺ can activate diverse ROS-generating machineries to generate excessive ROS as oxygen becomes available, which in turn elicits further Zn²⁺ accumulation during reperfusion.^{18, 19} ROS generation and cytosolic Zn²⁺ accumulation have a critical role in driving delayed CA1 pyramidal neuronal death,^{7, 12, 20, 21, 22} but the molecular mechanisms underlying such a vicious positive feedback during reperfusion remain poorly understood.Transient receptor potential melastatin-related 2 (TRPM2) forms non-selective cationic channels; their sensitivity to activation by ROS via a mechanism generating the channel activator ADP-ribose (ADPR) confers diverse cell types including hippocampal neurons with susceptibility to ROS-induced cell death, and thus TRPM2 acts as an important signaling molecule mediating ROS-induced adversities such as neurodegeneration.^{23, 24, 25, 26} Emergent evidence indeed supports the involvement of TRPM2 in transient ischemia-induced CA1 pyramidal neuronal death.^{27, 28, 29, 30} This has been attributed to the modulation of NMDA receptor-mediated signaling; despite that ROS-induced activation of the TRPM2 channels results in no change in the excitability of neurons from the wild-type (WT) mice, TRPM2 deficiency appeared to favor prosurvival synaptic Glu2A expression and inhibit prodeath extrasynaptic GluN2B expression.³⁰ A recent study suggests that TRPM2 activation results in extracellular Zn²⁺ influx to elevate the [Zn²⁺]_c.³¹ The present study, using TRPM2-deficient mice in conjunction with in vivo and in vitro models of transient global ischemia, provides compelling evidence to show ROS-induced TRPM2 activation during reperfusion as a crucial mechanism determining the delayed cytosolic Zn²⁺ accumulation, CA1 neuronal death and postischemic memory impairment.     

Differential effects of P2Y1 deletion on glial activation and survival of photoreceptors and amacrine cells in the ischemic mouse retina

Gliosis of retinal Müller glial cells may have both beneficial and detrimental effects on neurons. To investigate the role of purinergic signaling in ischemia-induced reactive gliosis, transient retinal ischemia was evoked by elevation of the intraocular pressure in wild-type (Wt) mice and in mice deficient in the glia-specific nucleotide receptor P2Y₁ (P2Y₁ receptor-deficient (P2Y1R-KO)). While control retinae of P2Y1R-KO mice displayed reduced cell numbers in the ganglion cell and inner nuclear layers, ischemia induced apoptotic death of cells in all retinal layers in both, Wt and P2Y1R-KO mice, but the damage especially on photoreceptors was more pronounced in retinae of P2Y1R-KO mice. In contrast, gene expression profiling and histological data suggest an increased survival of amacrine cells in the postischemic retina of P2Y1R-KO mice. Interestingly, measuring the ischemia-induced downregulation of inwardly rectifying potassium channel (Kir)-mediated K⁺ currents as an indicator, reactive Müller cell gliosis was found to be weaker in P2Y1R-KO (current amplitude decreased by 18%) than in Wt mice (decrease by 68%). The inner retina harbors those neurons generating action potentials, which strongly rely on an intact ion homeostasis. This may explain why especially these cells appear to benefit from the preserved Kir4.1 expression in Müller cells, which should allow them to keep up their function in the context of spatial buffering of potassium. Especially under ischemic conditions, maintenance of this Müller cell function may dampen cytotoxic neuronal hyperexcitation and subsequent neuronal cell loss. In sum, we found that purinergic signaling modulates the gliotic activation pattern of Müller glia and lack of P2Y₁ has janus-faced effects. In the end, the differential effects of a disrupted P2Y₁ signaling onto neuronal survival in the ischemic retina call the putative therapeutical use of P2Y₁-antagonists into question.Glial cells are crucially involved in the maintenance of neuronal activity in nervous tissues.¹ The homeostasis of the extracellular space is regulated by various glial functions including spatial K⁺ buffering, cell volume regulation and uptake of neurotransmitters.^{2, 3, 4} Activation of membrane receptors and ion channels is critically implicated in mediating the neuron-supportive glial functions. The dominant K⁺ conductance of glial cells mediates spatial K⁺ buffering and is important for the very negative membrane potential of these cells, thereby supporting electrogenic membrane transporters.⁵ Alterations in glial function are characteristic for pathological processes of the nervous system.⁶ Reactive gliosis may have beneficial and detrimental effects and is considered as an attempt to maintain neuronal function, protecting the tissue from further destruction, and to initiate tissue regeneration.^{7, 8} However, reactive gliosis may cause secondary neuronal damage as major neuron-supportive functions of glial cells get lost.⁶Gliotic alterations of Müller cells, the dominant macroglia of the vertebrate retina, have been observed in various models of retinal diseases.^{9, 10} A prominent feature of Müller cell gliosis is the downregulation of the inwardly rectifying K⁺ conductance mediated by inwardly rectifying K⁺ (Kir) channels.⁹ It has been demonstrated in astrocytes that downregulation or conditional knockout of Kir4.1 results in an impairment of glial glutamate (Glu) uptake.^{11, 12} In addition, it has been suggested that autocrine/paracrine purinergic signaling may have a causative role in the development of reactive gliosis in brain and retina.^{13, 14} Müller cells express different subtypes of P2 nucleotide receptors including P2Y₁ and P2Y₄.^{15, 16} P2Y₁ receptors have been demonstrated to be functionally expressed by Müller cells and microglial cells, rather than by neurons.^{15, 16, 17, 18}Retinal ischemia, a characteristic of various important human blinding diseases including diabetic retinopathy, results in neuronal degeneration and reactive gliosis.^{19, 20} The reduced K⁺ permeability of Müller cell membranes is associated with an impaired cell volume regulation under hypoosmotic stress after high intraocular pressure (HIOP)-induced ischemia.²¹ It has been observed that tandem-pore domain K⁺ channels may fulfill certain functions under conditions where Kir channels are downregulated or lacking.^{22, 23} A malfunctional Müller cell volume regulation was also found after deletion of P2Y₁ in the mouse retina.¹⁶ It has been suggested that impaired glial K⁺ buffering and cell volume regulation may contribute to neuronal degeneration in the ischemic retina by inducing neuronal hyperexcitation and Glu-induced cell death.¹⁴ In order to determine whether endogenous purinergic signaling is implicated in mediating and/or protecting from neuronal degeneration, we investigated the effects of HIOP-induced ischemia in the retinae of P2Y₁-deficient mice.     

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as ''lethal exosomes'' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types.¹ ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process.^{2, 3} Two main pathways are involved in MVB maturation.^{4, 5} In addition to the ESCRT (endosomal complex required for traffic) proteins,⁶ there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA),⁷ ceramides⁸ and diacylglycerol (DAG)⁹ contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activation-induced cell death (AICD), antigen presentation and intercellular miRNA exchange.^{10, 11, 12, 13, 14, 15} The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins.^{14, 16, 17, 18, 19, 20, 21} These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes.²² DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG.^{23, 24}The secretory vesicle pathway involves several DAG-controlled checkpoints at which DGKα may act; these include vesicle formation and fission at the trans-Golgi network (TGN), MVB maturation, as well as their transport, docking and fusion to the plasma membrane.^{9, 16, 17, 18, 19, 20} The molecular components that regulate some of these trafficking processes include protein kinase D (PKD) family members.²¹ PKD1 activity, for instance, regulates fission of transport vesicles from TGN via direct interaction with the pre-existing DAG pool at this site.¹⁹ The cytosolic serine/threonine kinases PKD1, PKD2 and PKD3^{(ref. 21)} are expressed in a wide range of cells, with PKD2 the most abundant isotype in T lymphocytes.^{25, 26} PKD have two DAG-binding domains (C1a and C1b) at the N terminus,²¹ which mediate PKD recruitment to cell membranes. Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation.²⁷Based on our previous studies showing DGKα regulation of DAG in MVB formation and exosome secretion,^{9, 14, 28} and the identification of PKD1/2 association to MVB,¹⁴ we hypothesized that DGKα control of DAG mediates these events, at least in part, through PKD. Here we explored whether, in addition to its role in vesicle fission from TGN,¹⁹ PKD regulates other steps in the DAG-controlled secretory traffic pathway. Using PKD-deficient cell models, we analyzed the role of PKD1/2 in MVB formation and function, and demonstrate their implication in exosome secretory traffic.     

Numb-dependent integration of pre-TCR and p53 function in T-cell precursor development

Numb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion. Notably, nuclear localization of Numb in early thymocyte precursors favors p53 nuclear stabilization, whereas pre-TCR-dependent Numb nuclear exclusion promotes the p53 downmodulation essential for further differentiation. Accordingly, the persistence of Numb in the nucleus impairs the differentiation and promotes precursor cell death. This study reveals a novel regulatory mechanism for Numb function based on its nucleus–cytosol shuttling, coupling the different roles of Numb with different stages of T-cell development.Cell fate decision of dividing progenitor-derived cells is a crucial event in development and diseases. Cell fate is often regulated by asymmetric cell division, which is a process by which progenitors asymmetrically segregate certain cell fate determinants during division, to generate two functionally different cells.^1,2 The adaptor protein Numb was initially identified in Drosophila as a critical cell fate determinant,³ where loss of Numb and its homolog Numb-like results in the loss of neural progenitors, indicating that the presence of Numb is essential for maintaining the progenitors during the initial progenitor versus neural fate decision.^4,5 However, re-expression of Numb is also required for further neural differentiation,^6,7 indicating that the role of Numb in the same tissue may change over time.Numb function in the immune system has been partially explored.^8,9 Numb is involved in asymmetric division in hematopoietic stem cells,¹⁰ thymocytes¹¹ and mature T lymphocytes.^12,13 T cells develop from intrathymic CD4⁻CD8⁻ double-negative (DN) precursors that, after progression through DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺) and DN4 (CD44⁻CD25⁻), have to decide between proliferation, to increase the total number of precursors, or differentiation into CD4⁺CD8⁺ double-positive (DP) cells. This decision is made during DN3 stage and appears to be dependent on asymmetric segregation of Numb.¹¹As Numb is a well-characterized inhibitor of Notch-1 receptor signaling pathway,¹⁴ the ability of Numb to regulate cell fate decisions during development has been associated with this Numb function.¹⁵ However, the role of Numb during development could not be restricted to the control of Notch-1 signaling, as Numb has been implicated in the regulation of a variety of biochemical pathways, including the tumor suppressor p53.¹⁶ Increasing evidence suggests that p53 regulates cell differentiation in addition to cell proliferation, apoptosis and senescence.^17,18Notably, T-cell development is regulated by both Notch-1 and p53. Notch-1 signals appear to be critical for the very early steps of T-cell development (i.e. T-cell commitment).¹⁹ The involvement of p53 has been instead reported in the transition from the DN to the DP stage. However, while the overexpression of p53 during DN3 stage promotes a block in the differentiation and proliferation, resulting in a small thymus size,^20,21 loss of p53 apparently does not affect thymocyte development, even though the vast majority of spontaneous malignancies in p53^−/− mice are lymphomas.²² Thus, the double function of Numb could be dependent on two different pathways, which may be differentially triggered during selected differentiation stages.Recent data describe the presence of Numb in the nuclear compartment,²³ besides its known cytoplasmic localization, raising the possibility that different Numb functions could be regulated by its differential subcellular localization. However, whether Numb may have different subcellular localizations in precursors or more differentiated T cell, how Numb import is regulated or how the nuclear localization affects its function during T-cell development remain unexplored. Here we show that Numb is an important regulator of p53 pathway during T-cell development, and we describe a novel molecular mechanism involved in the differential regulation of Numb–p53 axis based on the regulation of Numb nuclear import, emerging an interesting scenario where Numb can act as a regulator of two fundamental pathways during T-cell development.