Cytokine Withdrawal Research Articles

Lysine dimethylation in the BH4 domain of Bcl-2 and functional significance (P1306)

Abstract Bcl-2 is an anti-apoptotic protein that can protect developing and mature T cells against a variety of apoptotic signals. The Bcl-2 family of proteins, compromised of anti-apoptotic as well as pro-apoptotic members, plays key roles in apoptosis; the balance of these members determines cell survival. Anti-apoptotic Bcl-2 contains four conserved Bcl-2 homology (BH) domains and the BH4 domain is indispensable for its ability to suppress cell death. Using mass spectroscopy to analyze a murine T cell line, we identified dimethylation of two lysine residues in the BH4 domain: K17 and K22. Mutation of K22 (but not K17) to alanine abrogated Bcl-2 binding to pro-apoptotic proteins Bax, Bak, Bad, Bim and Puma and greatly reduced Bcl-2 anti-apoptotic function in cytokine withdrawal. This assigns functional importance to K22 (which is conserved across species and other anti-apoptotic members) and suggests that dimethylation could affect its function. K22, based on structural models, appears to be involved in stabilizing peptide secondary structure, rather than directly interacting with pro-apoptotic proteins. We developed a monoclonal antibody that recognizes dimethylated K22 in the Bcl-2 sequence and detected expression in murine B cells (but not thymocytes). This suggests that Bcl-2 dimethylation may regulate B cell survival.

FOXO3A directs a protective autophagy program in haematopoietic stem cells

Blood production is ensured by rare self-renewing hematopoietic stem cells (HSCs). How HSCs accommodate the diverse cellular stresses associated with their life-long activity remains elusive. Here, we identify autophagy as an essential mechanism protecting HSCs from metabolic stress. We show that HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy following ex vivo cytokine withdrawal and in vivo caloric restriction. We demonstrate that FoxO3a is critical to maintain a gene expression program that poise HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FoxO3a-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.

MiR-24 Promotes the Survival of Hematopoietic Cells

The microRNA, miR-24, inhibits B cell development and promotes myeloid development of hematopoietic progenitors. Differential regulation of cell survival in myeloid and lymphoid cells by miR-24 may explain how miR-24′s affects hematopoietic progenitors. MiR-24 is reported to regulate apoptosis, either positively or negatively depending on cell context. However, no role for miR-24 in regulating cell death has been previously described in blood cells. To examine miR-24′s effect on survival, we expressed miR-24 via retrovirus in hematopoietic cells and induced cell death with cytokine or serum withdrawal. We observed that miR-24 enhanced survival of myeloid and B cell lines as well as primary hematopoietic cells. Additionally, antagonizing miR-24 with shRNA in hematopoietic cells made them more sensitive to apoptotic stimuli, suggesting miR-24 functions normally to promote blood cell survival. Since we did not observe preferential protection of myeloid over B cells, miR-24′s pro-survival effect does not explain its promotion of myelopoiesis. Moreover, expression of pro-survival protein, Bcl-xL, did not mimic miR-24′s impact on cellular differentiation, further supporting this conclusion. Our results indicate that miR-24 is a critical regulator of hematopoietic cell survival. This observation has implications for leukemogenesis. Several miRNAs that regulate apoptosis have been shown to function as either tumor suppressors or oncogenes during leukemogenesis. MiR-24 is expressed highly in primary acute myelogenous leukemia, suggesting that its pro-survival activity could contribute to the transformation of hematopoietic cells.

Regulation of Antiviral CD8 T-Cell Responses

A balanced immune response to a viral pathogen leads to clearance of the virus while limiting immune mediated pathology. Control of this process occurs at all stages of the immune response, including during the induction of an antiviral response, clearance of virally infected cells, and the resolution of this response. Regulation of antiviral immune response is further modified when the immune system fails to clear the pathogen and by the nature of chronic infection itself. A number of processes have been implicated in the regulation of antiviral immune responses, such as the limitation of viral antigen load by interferons, apoptosis through cytokine withdrawal or Fas-mediated killing, and control of these responses by regulatory T cells. This review addresses several of these mechanisms.

Clinical Spectrum of RAS-Associated Autoimmune Leukoproliferative Disorder (RALD): A Distinct Clinical Entity Mimicking Juvenile Myelomonocytioc Leukemia (JMML) or Chronic Myelomonocytic Leukemia (CMML)

AbstractAbstract 1033Chronic, nonmalignant, noninfectious lymphadenopathy associated with splenomegaly and elevated circulating TCRα β+, CD4-/CD8- Double Negative T (DNT) cells are the hallmarks of the apoptosis disorder called autoimmune lymphpoproliferative syndrome (ALPS). ALPS is most frequently associated with defects in the FAS gene affecting the extrinsic pathway of apoptosis. Patients with ALPS-like syndromes caused by somatic mutations in NRAS and KRAS affecting the intrinsic (mitochondrial) pathway of apoptosis are recently recognized and currently classified separately as ALPS-related apoptosis disorders. Reduced peripheral blood lymphocyte apoptosis after withdrawal of IL-2 is a key feature of their diagnosis. They present with autoimmune phenomena, massive splenomegaly, modest lymphadenopathy, and normal or only marginally elevated DNT cells. Concomitant abnormalities of the myeloid compartment, with an absolute monocytosis with or without concurrent leukocytosis and neutrophilia, mimic juvenile or chronic myelomonocytic leukemia in otherwise asymptomatic younger patients. These patients are classified as RAS-associated autoimmune leukoproliferative disorder (RALD) (Oliveira et al, Blood 2010 and Niemela et al, Blood 2011). [Display omitted] Here we summarize the clinical features in our cohort of 10 patients with RALD. They are currently 4–54 years old (M:F ratio 5:5) and all are alive except one patient who died at age 13. Their median age of disease onset was 2 years; while 50% presented with symptoms at or before age of 1year; one patient (380.1) presented with symptoms as an adult (age 36) with hypersplenism and leukocytosis. All presented with significantly enlarged spleens and 3/10 have undergone splenectomy. All 5 patients with NRAS mutation are male in our cohort, whereas all 5 patients with KRAS mutation are female and three of them presented with ecchymotic periarthritis. As a group these patients have an absolute monocytosis (median 1.7 K/uL, range 0.8–49.7 K/uL), increased circulating B lymphocytes (median 743, range 497–5970) and elevated serum IgG (median 1960 mg/dL, range 1310–2450 mg/dL). Monocytes appear to express unique activation markers in the two patients studied to date. Monocytes from patients 380.1 and 381.1 showed atypical expression of CD56 and 40% of them from patient 381.1 were atypical with an immunophenotype consistent with dendritic cells (non-classical monocytes) that expressed CD16, bright CD45, bright HLA-DR, dim CD14, and dim CD36.Bone marrow biopsies performed on a subset of patients show hypercellular marrow with monocytosis, granulocytic hyperplasia, and no increase in blasts. Atypical megakaryocytes and myeloid maturation asynchrony have been noted in some, without overt dysplasia. Autoantibodies noted in these patients include DAT, lupus anticoagulant, ACA IgG, ACA IgM, and ANA. Importantly cytokine (IL-2) withdrawal assay showed diminished apoptosis in each of these patients while GM-CSF hypersensitivity assay was positive in the 3/5 patients tested. Only one patient in our cohort (Patient 58.1) with an NRAS mutation has developed B cell lymphoma at age 44 and is alive 10 years after receiving chemotherapy. RALD should be considered in patients with unexplained splenomegaly, autoimmune phenomena and persistent monocytosis. Diligent clinical and laboratory assessments and long-term follow up will help us understand the natural history of this rare leukoproliferative disorder and distinguish it from myelodysplastic conditions like JMML and CMML. Disclosures:No relevant conflicts of interest to declare.

Casitas B-lineage lymphoma mutants activate AKT to induce transformation in cooperation with class III receptor tyrosine kinases

In addition to overexpression and the occurrence of activating mutations, receptors can be aberrantly activated by impaired downregulation. In this study, we show that an oncogenic mutant of the ubiquitin ligase casitas B-lineage lymphoma (CBL; CBLΔexon8), which is found in acute myeloid leukemia patients, predominantly cooperates with receptor tyrosine kinase (RTK) class III receptors (PDGFRA, PDGFRB, KIT, and FLT3), but not with non-class III RTKs or cytokine receptors, to induce IL-3-independent growth of Ba/F3 cells. In cells coexpressing RTK class III/CBLΔexon8, receptor internalization was delayed, and cells were protected from apoptosis after cytokine withdrawal. Ligand-stimulated Ba/F3 cells and acute myeloid leukemia cell lines coexpressing the CBL deletion mutant and FLT3 showed enhanced AKT phosphorylation. Combined pharmacologic inhibition of the PI3K/AKT pathway and FLT3 had an additive effect on cell proliferation. The transforming potential of the CBL mutant was completely abolished by the mutation of the CBL PTB domain and was decreased by the mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutively active AKT1 mutant (E17K) recapitulated the phenotype induced by the CBL deletion mutant in Ba/F3 cells. This study reveals FLT3-CBL interaction sites and the AKT pathway as critical mediators of transformation by oncogenic CBL mutants.

Notch and the Survival of Regulatory T Cells: Location Is Everything!

Signaling through the T cell receptor induces T lymphocytes to divide, differentiate, and perform numerous effector functions. Once activated, effector T cells are exquisitely sensitive to changes in available cytokines, particularly the survival cytokine interleukin-2 (IL-2). Removal of IL-2 rapidly initiates apoptosis in response to cytokine withdrawal. In contrast to effector T cells, regulatory T cells (T(regs)) are resistant to apoptosis induced by cytokine withdrawal. A study exploring the differences between these two T cell subsets reveals a role for Notch1 in protecting T(regs) from apoptosis. Protection from apoptosis induced by cytokine withdrawal correlated with Notch1 localization in the cytosol of T(regs) and its association with phosphatidylinositol 3-kinase and Rictor, a component of the mammalian target of rapamycin complex. Notch1 localization in the nucleus in effector T cells, on the other hand, was correlated with susceptibility to apoptosis induced by cytokine withdrawal. This study highlights how Notch1 can deliver opposing signals in different cellular contexts and suggests that localization of Notch1 can have a substantial influence on life-and-death decisions in T lymphocytes.

PGE2 promotes apoptosis induced by cytokine deprivation through EP3 receptor on mast cells (177.1)

Abstract Increased mast cells numbers are observed at sites of allergic inflammation. Although a critical factor in the resolution of these inflammatory responses is the restoration of normal mast cell numbers. Early studies showed that cytokines protect mast cells from programmed cells death, suggesting a simple model in which diminished cytokines levels during resolution leads to cell death. The report that prostaglandins (PGs) can contribute to the resolution of inflammation together with the demonstration that mast cells express all four PGE2 receptors raises the possibility that PGE2 act in part by augmenting mast cell apoptosis. We report here that, while exposure of mast cells to PGE2 alone does not lead to apoptosis, PGE2 through the EP3 receptor promotes cell death initiated by cytokine withdrawal. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨm), increased caspase-3 activation, and chromatin condensation with low molecular weight cleavage. PGE2 augmented cell death is dependent on increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE2 contributes to the resolution of inflammatory response in part by augmenting the removal of inflammatory cells particularly mast cells.

Antigen-Independent Induction of Tim-3 Expression on Human T Cells by the Common γ-Chain Cytokines IL-2, IL-7, IL-15, and IL-21 Is Associated with Proliferation and Is Dependent on the Phosphoinositide 3-Kinase Pathway

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8(+) and Th1 CD4(+) T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3(+) cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4(+) T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3(+) primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.

A novel mutation in MPL (Y252H) results in increased thrombopoietin sensitivity in essential thrombocythemia

Essential thrombocythemia (ET) is a rare type of myeloproliferative neoplasm characterized by clonal expansion of the megakaryocyte and platelet lineage. Here, we describe a novel mutation (Y252H) in the thrombopoietin (TPO) receptor, or MPL, in a JAK2 mutation-negative ET patient. The bone marrow examination revealed increased numbers of dysmorphic megakaryocytes with focal clustering. The x-inactivation pattern suggested clonal expansion of hematopoietic cells in the bone marrow. Furthermore, we found that the patient's bone marrow cells were hypersensitive to TPO in generating megakaryocyte colonies in vitro. More importantly, we demonstrated that this MPL Y252H mutant confers increased TPO/MPL-mediated cell growth and increased cell survival upon cytokine withdrawal in BaF3 cells, indicating it is a disease-driving mutation and may contribute to the development of ET in vivo. In summary, this is the first report describing a mutation in the extracellular domain of MPL underlying ET.

BH3-only proteins in apoptosis at a glance

Apoptosis is a genetically programmed process for the elimination of damaged or redundant cells by activation of caspases (aspartate-specific cysteine proteases). Caspases cleave vital proteins, leading the cell to fragment into vesicles that are rapidly engulfed by phagocytes (for reviews, see [

Interleukin-1β and transforming growth factor-β cooperate to induce neurosphere formation and increase tumorigenicity of adherent LN-229 glioma cells

IntroductionGlioma stem cells (GSCs) have the property of self-renewal and appear to be a driving force for the initiation and recurrence of gliomas. We recently found that the human tumorigenic LN-229 glioma cell line failed to form neurospheres in serum-free conditions and generated mostly small tumors in vivo, suggesting that either LN-229 GSCs are not active in these conditions or GSCs are absent in the LN-229 cell line.MethodsUsing self-renewal assay, soft-agar colony assay, cell proliferation assay, invasion assay, real time PCR analysis, ELISA and in vivo tumorigenic assay, we investigated the effects of interleukin (IL)-1β and transforming growth factor (TGF)-β on the development of GSCs from LN-229 cells.ResultsHere, we demonstrate that the combination of IL-1β and TGF-β can induce LN-229 cells to form neurospheres in serum-free medium. IL-1β/TGF-β-induced neurospheres display up-regulated expression of stemness factor genes (nestin, Bmi-1, Notch-2 and LIF), and increased invasiveness, drug resistance and tumor growth in vivo: hallmarks of GSCs. These results indicate that IL-1β and TGF-β cooperate to induce a GSC phenotype in the LN-229 cell line. Induction of nestin, LIF and Notch-2 by IL-1β/TGF-β can be reverted after cytokine withdrawal. Remarkably, however, up-regulated Bmi-1 levels remained unchanged after cytokine withdrawal; and the cytokine-withdrawn cells maintained strong clonogenicity, suggesting that Bmi-1 may play a crucial role in tumorigenesis.ConclusionsOur finding indicates that glioma cells without self-renewal capability in standard conditions could also contribute to glioma malignancy when cytokines, such as IL-1β and TGF-β, are present in the tumor environment. Targeting GSC-promoting cytokines that are highly expressed in glioblastomas may contribute to the development of more effective glioma therapies.

CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway

Abstract▪2447▪This icon denotes a clinically relevant abstractWe examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants.RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands.Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells.CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Novel Mutation in MPL (Y252H) Results in Increased Thrombopoietin Sensitivity and Moderate Thrombocytosis in a Pediatric Patient with Essential Thrombocytosis

Abstract▪2817▪This icon denotes a clinically relevant abstractMyeproliferative neoplams (MPNs) constitute a group of hematopoietic malignancies that feature enhanced proliferation and/or survival of one or more myeloid lineage cells, including Essential Thrombocythemia (ET). MPN development is rare in children with an estimated annual incidence of ET of 0.09/106 children. The WHO defines ET as persistent platelet count >600 k/mcL in the absence of known cause of reactive/secondary thrombocytosis. The JAK2 V617F mutation is most commonly reported in both children and adults with ET although the reported frequency varies in pediatric populations from 0 to 36% of patients. Mutations in the thrombopoietin receptor (TPO receptor or MPL) intracellular domain, specifically W515K/L, have also been reported in both adult and pediatric ET patients. Here we report a novel mutation in the MPL extracellular domain, Y252H, causing mild thrombocytosis. The patient presented at 2 years of age with a platelet count of 765 k/mcL. During the 3-year follow-up period, she possessed platelet counts between 600–700k/mcL, without any obvious indication of reactive/secondary thrombocytosis. Because of the persistently increased platelet count, her bone marrow was evaluated and it demonstrated increased numbers of megakaryocytes with focal clustering. JAK2 mutation analysis was negative and cytogenetics did not show any clonal abnormalities. Sequencing of the MPL gene showed a missense variant at c.754 T>C resulting in a tyr252his amino acid substitution. To investigate if this Y252H mutation in MPL dysregulates TPO/MPL- mediated cell growth, we introduced it into cytokine-dependent BaF3 cells. Cells stably expressing the mutant MPL allele showed increased proliferation to TPO, in particular at low concentration, in comparison to cells expressing wildtype (WT) MPL. Upon cytokine withdrawal, BaF3 cells expressing the MPL Y252H mutant survived better than that of WT MPL. Primary bone marrow cells from this patient along with the healthy control were subjected to colony forming unit -megakaryocyte (CFU-meg) assays in response to a serial dose of TPO. The Y252H MPL bone marrow showed significantly increased megakaryocyte colonies at low dose of TPO when compared to control bone marrow (17.5 ± 2.5 colonies versus 4.75 ± 1.1 colonies at 15 ng/mL TPO, p<0.001). These results are consistent with the clinically mild thrombocytosis. In summary, our results suggest a novel MPL mutation, Y252H, results in pediatric ET. Further evaluation of the mechanisms of increased TPO sensitivity imparted by this mutation should contribute to our understanding of the molecular pathogenesis of ET. Disclosures:Lambert:Cangene: Honoraria.

Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3–23.3 (n=1), 9q33.1–34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31–36.33 (n=6), 17q21.2–q21.31 (n=5) and 17q25.1–25.3 (n=5) and deletions affecting 18p11.31–11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a ‘HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal.

Cytokine-dependent and cytokine-independent roles for Mcl-1: genetic evidence for multiple mechanisms by which Mcl-1 promotes survival in primary T lymphocytes

Myeloid cell leukemia sequence-1 (Mcl-1) is a critical anti-apoptotic factor in T lymphocytes. However, in spite of the many pro-apoptotic proteins with proposed binding to Mcl-1, the specific interactions by which Mcl-1 regulates primary T-cell survival under different conditions have not been fully explored. Further, how different trophic cytokines modulate the specific role(s) of Mcl-1 is unknown. Here, we use genetic mouse models to dissect the roles of Mcl-1 in primary T lymphocytes. Using the inducible Mcl-1-floxed estrogen receptor-Cre fusion protein (Mcl-1f/fERCre) deletion system in combination with genetic modification of other B-cell lymphoma 2 (Bcl-2) family members, we show that loss of pro-apoptotic Bcl-2 homologous antagonist/killer (Bak) rescues the survival of Mcl-1-deficient T cells in the presence of IL-7. Without IL-7, the survival of Mcl-1-deficient cells cannot be rescued by loss of Bak, but is partially rescued by overexpression of Bcl-2 or loss of Bcl-2-interacting mediator of cell death (Bim). Thus, Mcl-1 and Bcl-2 have a shared role, the inhibition of Bim, in promoting T-cell survival during cytokine withdrawal. Finally, we show that other common gamma-chain (γc) cytokines differentially modulate the roles of Mcl-1. IL-15 has effects similar to those of IL-7 in memory T cells and naïve CD8+ cells, but not naïve CD4+ cells. However, IL-4 maintains Mcl-1 and Bcl-2 but also upregulates Bim and Bcl-2-associated X protein (Bax), thus altering the cell's dependence on Mcl-1.

New bioengineering insights into human neural precursor cell expansion in culture

Understanding initial cell growth, interactions associated with the process of expansion of human neural precursor cells (hNPCs), and cellular events pre- and postdifferentiation are important for developing bioprocessing protocols to reproducibly generate multipotent cells that can be used in basic research or the treatment of neurodegenerative disorders. Herein, we report the in vitro responses of telencephalon hNPCs grown in a serum-free growth medium using time-lapse live imaging as well as cell-surface marker, aggregate size, and immunocytochemical analyses. Time-lapse analysis of hNPC initial expansion indicated that cell-surface attachment in stationary culture and the frequency of cell-cell interaction in suspension conditions are important for subsequent aggregate formation and hNPC growth. In the absence of cell-surface attachment in low-attachment stationary culture, large aggregates of cells were formed and expansion was adversely affected. The majority of the telencephalon hNPCs expressed CD29, CD90, and CD44 (cell surface markers involved in cell-ECM and cell-cell interactions to regulate biological functions such as proliferation), suggesting that cell-surface attachment and cell-cell interactions play a significant role in the subsequent formation of cell aggregates and the expansion of hNPCs. Before differentiation, about 90% of the cells stained positive for nestin and expressed two neural precursor cells surface markers (CD133 and CD24). Upon withdrawal of growth cytokines, hNPCs first underwent cell division and then differentiated preferentially towards a neuronal rather than a glial phenotype. This study provides key information regarding human NPC behavior under different culture conditions and favorable culture conditions that are important in establishing reproducible hNPC expansion protocols.

Somatic KRAS mutations associated with a human nonmalignant syndrome of autoimmunity and abnormal leukocyte homeostasis

AbstractSomatic gain-of-function mutations in members of the RAS subfamily of small guanosine triphosphatases are found in > 30% of all human cancers. We recently described a syndrome of chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity associated with a mutation in NRAS affecting hematopoietic cells, and initially we classified the disease as a variant of the autoimmune lymphoproliferative syndrome. Here, we demonstrate that somatic mutations in the related KRAS gene can also be associated with a nonmalignant syndrome of autoimmunity and breakdown of leukocyte homeostasis. The activating KRAS mutation impaired cytokine withdrawal–induced T-cell apoptosis through the suppression of the proapoptotic protein BCL-2 interacting mediator of cell death and facilitated proliferation through p27kip1 down-regulation. These defects could be corrected in vitro by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 or phosphatidyl inositol-3 kinase inhibition. We suggest the use of the term RAS-associated autoimmune leukoproliferative disease to differentiate this disorder from autoimmune lymphoproliferative syndrome.

MzServer: Web-based Programmatic Access for Mass Spectrometry Data Analysis

Continued progress toward systematic generation of large-scale and comprehensive proteomics data in the context of biomedical research will create project-level data sets of unprecedented size and ultimately overwhelm current practices for results validation that are based on distribution of native or surrogate mass spectrometry files. Moreover, the majority of proteomics studies leverage discovery-mode MS/MS analyses, rendering associated data-reduction efforts incomplete at best, and essentially ensuring future demand for re-analysis of data as new biological and technical information become available. Based on these observations, we propose to move beyond the sharing of interpreted spectra, or even the distribution of data at the individual file or project level, to a system much like that used in high-energy physics and astronomy, whereby raw data are made programmatically accessible at the site of acquisition. Toward this end we have developed a web-based server (mzServer), which exposes our common API (mzAPI) through very intuitive (RESTful) uniform resource locators (URL) and provides remote data access and analysis capabilities to the research community. Our prototype mzServer provides a model for lab-based and community-wide data access and analysis.

Role for BH3-Only Protein NOXA In Growth-Factor Deprivation and Early Erythropoiesis

AbstractAbstract 4235Red blood cell production is a strictly regulated process and homeostatic maintenance of the erythropoietic system requires equilibrium between the rate of erythroid cell production and red blood cell destruction. Hematopoietic cytokines play a crucial role in regulating expansion, differentiation and survival of erythrocyte progenitors. Shortage of growth factors triggers the mitochondrial apoptosis pathway, which is critically dependent on Bcl-2 family members. However, the contribution of this mechanism in the regulation of erythropoiesis remains ill-defined. This prompted us to screen for candidate genes involved in this process in erythroid progenitors. We found that the expression of Noxa, a pro-apoptotic Bcl-2 family member, is upregulated during erythroid differentiation and following cytokine-withdrawal in erythroid progenitor cells. Knockdown or deletion of Noxa in IL-3 dependent human and murine erythroid progenitor cell lines increased Mcl-1 levels, which correlated with markedly decreased apoptosis following cytokine withdrawal. Importantly, Noxa ablation in mice increased extra-medullary erythropoiesis, resulting in enhanced numbers of early splenic erythroblasts and circulating reticulocytes. Noxa-deficient hematopoietic progenitors were more resistant to apoptosis induced by growth factor deprivation and displayed increased colony-forming potential. In addition, combined loss of Noxa and Bim resulted in enhanced resistance of erythroid progenitors to cytokine withdrawal compared to WT or single Bim knockouts, suggesting a non-redundant role for Noxa and Bim in regulating survival of erythroid progenitors in response to cytokine deprivation. Finally, in a model of acute haemolytic anaemia, deletion of Noxa enhanced subsequent hematocrit recovery. Together, these findings identify a non-redundant role for BH3-only protein Noxa in the regulation of erythroblast survival during early erythropoiesis. Therefore, Noxa may be a novel component to control red blood cell numbers and modulation of this pathway could be envisaged in therapeutic options for treatment of anaemia. Disclosures:No relevant conflicts of interest to declare.