CD71 Surface Expression Research Articles

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta-cells

Abstract Objectives The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. Methods We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. Results Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. Conclusions This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta cells

ObjectivesThe main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. MethodsWe developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. ResultsPancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. ConclusionsThis study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

Glucocorticoids Induce the Maintenance and Expansion of an Immature CFU-E Erythroid Progenitor Population in Humans

Glucocorticoids are widely used to increase red cell production in a number of hematological disorders including Diamond Blackfan anemia (DBA). Despite their clinical effectiveness we do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. To improve the clinical management of these patients, we studied the mechanisms of dexamethasone (Dex) on human erythroid progenitors. We employed an erythroid culture system to differentiate primary human CD34+ cells isolated from patients with DBA and healthy controls as well as cord blood samples. We found that Dex increases the proliferation of CD34+ cells derived from peripheral blood but surprisingly not of CD34+ cells from cord blood. Furthermore, by mass spectrometry proteomic analysis, we found that Dex specifically altered the induction of several targets unique to peripheral blood including the transcription factor and cell cycle regulator NR4A1. When examining the cell populations expanded with Dex treatment by immunophenotyping, we detected a specific expansion of CFU-Es. Dex increased the average size of CFU-E colonies in methylcellulose colony forming assays in the presence of Epo alone, suggesting that CFU-Es are the most Dex responsive progenitor in humans. In support of this hypothesis, reticulocyte counts from patients with DBA treated with prednisone were found to increase approximately one week after treatment, consistent with the time course of differentiation of CFU-E to reticulocytes in bone marrow. In order to further resolve the progenitor target of steroid responsive progenitor we developed a new gating scheme that allows for increased resolution of BFU-E and CFU-E subpopulations by monitoring surface expression of CD71 and CD105. Using this system, we found that Dex treatment led to the maintenance of a novel immature transitional progenitor population in peripheral blood-derived cells that we term immature CFU-E. In methylcellulose colony forming assays, these peripheral blood-derived immature CFU-E treated with Dex produced larger colonies than the untreated controls and mature CFU-E. The expansion and proliferation of Dex treated immature CFU-Es derived from peripheral blood was associated with a decreased percentage of cells in the S phase of the cell cycle. No change was noted for either Dex treated BFU-E from peripheral blood or for BFU-E or CFU-E from cord blood. Importantly, Dex responsive peripheral blood-derived CFU-Es divided at a faster rate than untreated CFU-Es, revealing a dissociation in the link between differentiation and proliferation. Mechanistically, we found that Dex treated peripheral blood CFU-E exhibited increased expression of the negative cell cycle regulator p57Kip2. Moreover, this protein was also found to be dysregulated in CD34+ cells from transfusion-dependent patients with DBA, wherein p57Kip2 expression was not significantly altered following Dex treatment, indicating a role for p57Kip2 in the erythroid progenitor defects seen in these patients. To further evaluate the role of p57Kip2 in the Dex dependent alteration in CFU-E proliferation, peripheral blood-derived CD34+ cells were transduced with p57Kip2-specific shRNA lentiviral constructs. shRNA knockdown of p57Kip2 abrogated the effects of Dex on growth and induced a premature EPO-mediated differentiation. In summary, our findings provide new insights into functional heterogeneity of human erythroid progenitors and reveal mechanistic insights into the action of Dex in normal and disordered erythropoiesis. Disclosures Vlachos: Novartis: Other: Steering committee member.

Comprehensive Cell Surface Antigen Analysis Identifies Transferrin Receptor Protein-1 (CD71) as a Negative Selection Marker for Human Neuronal Cells.

Cell state‐, developmental stage‐, and lineage‐specific combinatorial expression of cluster of differentiation (CD) molecules enables the identification of cellular subsets via multicolor flow cytometry. We describe an exhaustive characterization of neural cell types by surface antigens, exploiting human pluripotent stem cell‐derived neural cell systems. Using multiwell screening approaches followed by detailed validation of expression patterns and dynamics, we exemplify a strategy for resolving cellular heterogeneity in stem cell paradigms. In addition to providing a catalog of surface antigens expressed in the neural lineage, we identified the transferrin receptor‐1 (CD71) to be differentially expressed in neural stem cells and differentiated neurons. In this context, we describe a role for N‐Myc proto‐oncogene (MYCN) in maintaining CD71 expression in proliferating neural cells. We report that in vitro human stem cell‐derived neurons lack CD71 surface expression and that the observed differential expression can be used to identify and enrich CD71− neuronal derivatives from heterogeneous cultures. stem cells 2019;37:1293–1306

Placental Galectins Are Key Players in Regulating the Maternal Adaptive Immune Response.

Galectins are potent immunomodulators that regulate maternal immune responses in pregnancy and prevent the rejection of the semi-allogeneic fetus that also occurs in miscarriages. We previously identified a gene cluster on Chromosome 19 that expresses a subfamily of galectins, including galectin-13 (Gal-13) and galectin-14 (Gal-14), which emerged in anthropoid primates. These galectins are expressed only by the placenta and induce the apoptosis of activated T lymphocytes, possibly contributing to a shifted maternal immune balance in pregnancy. The placental expression of Gal-13 and Gal-14 is decreased in preeclampsia, a life-threatening obstetrical syndrome partly attributed to maternal anti-fetal rejection. This study is aimed at revealing the effects of Gal-13 and Gal-14 on T cell functions and comparing the expression of these galectins in placentas from healthy pregnancies and miscarriages. First-trimester placentas were collected from miscarriages and elective termination of pregnancies, tissue microarrays were constructed, and then the expression of Gal-13 and Gal-14 was analyzed by immunohistochemistry and immunoscoring. Recombinant Gal-13 and Gal-14 were expressed and purified, and their effects were investigated on primary peripheral blood T cells. The binding of Gal-13 and Gal-14 to T cells and the effects of these galectins on apoptosis, activation marker (CD25, CD71, CD95, HLA-DR) expression and cytokine (IL-1β, IL-6, IL-8, IL-10, IFNγ) production of T cells were examined by flow cytometry. Gal-13 and Gal-14 are primarily expressed by the syncytiotrophoblast at the maternal-fetal interface in the first trimester, and their placental expression is decreased in miscarriages compared to first-trimester controls. Recombinant Gal-13 and Gal-14 bind to T cells in a population- and activation-dependent manner. Gal-13 and Gal-14 induce apoptosis of Th and Tc cell populations, regardless of their activation status. Out of the investigated activation markers, Gal-14 decreases the cell surface expression of CD71, Gal-13 increases the expression of CD25, and both galectins increase the expression of CD95 on T cells. Non-activated T cells produce larger amounts of IL-8 in the presence of Gal-13 or Gal-14. In conclusion, these results show that Gal-13 and Gal-14 already provide an immunoprivileged environment at the maternal-fetal interface during early pregnancy, and their reduced expression is related to miscarriages.

Delayed Globin Synthesis Leads to Excessive Heme and the Macrocytic Anemia of Diamond Blackfan Anemia and del(5q) Myelodysplastic Syndrome

Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med 8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1 -deletion) (J Clin Invest 125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1 -deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others. DisclosuresNo relevant conflicts of interest to declare.

Notch regulates Th17 differentiation and controls trafficking of IL-17 and metabolic regulators within Th17 cells in a context-dependent manner.

Th17 cells play critical roles in host defense and autoimmunity. Emerging data support a role for Notch signaling in Th17 cell differentiation but whether it is a positive or negative regulator remains unclear. We report here that T cell-specific deletion of Notch receptors enhances Th17 cell differentiation in the gut, with a corresponding increase in IL-17 secretion. An increase in Th17 cell frequency was similarly observed following immunization of T cell specific Notch mutant mice with OVA/CFA. However, in this setting, Th17 cytokine secretion was impaired, and increased intracellular retention of IL-17 was observed. Intracellular IL-17 co-localized with the CD71 iron transporter in the draining lymph node of both control and Notch-deficient Th17 cells. Immunization induced CD71 surface expression in control, but not in Notch-deficient Th17 cells, revealing defective CD71 intracellular transport in absence of Notch signaling. Moreover, Notch receptor deficient Th17 cells had impaired mTORC2 activity. These data reveal a context-dependent impact of Notch on vesicular transport during high metabolic demand suggesting a role for Notch signaling in the bridging of T cell metabolic demands and effector functions. Collectively, our findings indicate a prominent regulatory role for Notch signaling in the fine-tuning of Th17 cell differentiation and effector function.

Erythropoietin-regulated oxidative stress negatively affects enucleation during terminal erythropoiesis.

Differentiating erythroblasts are exposed to an oxidative environment. The dynamics of oxidative status during terminal erythropoiesis and how they affect cell differentiation in response to erythropoietin (Epo) are unclear. Here, we show that Epo induces reactive oxygen species (ROS) production in the early stages of terminal erythropoiesis. The levels of ROS correlate with CD71 surface expression and the uptake of iron and transferrin. ROS decreases in the late stages of terminal erythropoiesis, when the cells are preparing for enucleation. Consistently, treatment of erythroblasts with a low dose (5mM) of N-acetyl-cysteine (NAC), a ROS scavenger, promotes enucleation. However, a high dose (20mM) of NAC leads to significant cell death. Our study reveals an important function of Epo in regulating the dynamics ofoxidative status and enucleation.

Erythrocytic precursor cells show potent shear stress resistant adhesion and home to hematopoietic tissue in vivo

Transfusion of erythropoietic precursor cells has been suggested as an alternative to conventional red blood cells. However, little is known about the fate of transfused erythrocytic precursors after they enter the bloodstream. Erythrocytic precursors were classified by flow cytometry into different maturation stages. Precursors were enriched using cell surface expression of CD71 and Ter119 antigens and analyzed under shear stress in a parallel plate flow chamber and after fluorescence tagging with PKH and transfusion into anemic mice. We found that at all maturation stages, erythrocytic precursors expressed the adhesion receptor very late antigen (VLA)-4 with a frequency decreasing from 90% to approximately 60% during maturation. In contrast, expression of the beta(2)-integrins LFA-1 and Mac-1 and the rolling receptor P-selectin glycoprotein ligand-1 increased from 10% to 20% to approximately 50% during erythrocytic maturation. The chemokine receptor CXCR4 was expressed at low levels during differentiation stages. In vitro shear stress adhesion analysis showed that erythrocytic precursors can efficiently activate VLA-4 such that it binds its cognate ligand, vascular cell adhesion molecule (VCAM)-1. The coimmobilization of stromal cell-derived factor-1 alpha with VCAM-1 strengthened this adhesion. Transfusion of primitive (CD71+) or more mature (Ter119+) erythrocytic precursors into mice showed that both populations selectively and efficiently home to hematopoietic tissues. Our results demonstrate that erythrocytic precursor cells of different maturation stages are capable of homing to hematopoietic organs. This work has implications for the development of transfusion protocols that use ex vivo expanded, but not fully matured, erythrocytic precursors from cultured stem cell populations.

Notch-induced T cell development requires phosphoinositide-dependent kinase 1

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.

A Role for Mammalian Target of Rapamycin in Regulating T Cell Activation versus Anergy

Whether TCR engagement leads to activation or tolerance is determined by the concomitant delivery of multiple accessory signals, cytokines, and environmental cues. In this study, we demonstrate that the mammalian target of rapamycin (mTOR) integrates these signals and determines the outcome of TCR engagement with regard to activation or anergy. In vitro, Ag recognition in the setting of mTOR activation leads to full immune responses, whereas recognition in the setting of mTOR inhibition results in anergy. Full T cell activation is associated with an increase in the phosphorylation of the downstream mTOR target S6 kinase 1 at Thr(421)/Ser(424) and an increase in the mTOR-dependent cell surface expression of transferrin receptor (CD71). Alternatively, the induction of anergy results in markedly less S6 kinase 1 Thr(421)/Ser(424) phosphorylation and CD71 surface expression. Likewise, the reversal of anergy is associated not with proliferation, but rather the specific activation of mTOR. Importantly, T cells engineered to express a rapamycin-resistant mTOR construct are resistant to anergy induction caused by rapamycin. In vivo, mTOR inhibition promotes T cell anergy under conditions that would normally induce priming. Furthermore, by examining CD71 surface expression, we are able to distinguish and differentially isolate anergic and activated T cells in vivo. Overall, our data suggest that by integrating environmental cues, mTOR plays a central role in determining the outcome of Ag recognition.

Gene Expression Patterns Identify Novel Biologically Relevant Signaling and Transcriptional Pathways Involved in Terminal Erythroid Differentiation and Polycythemia Vera.

Hematopoietic proliferation, lineage commitment, and terminal differentiation are characterized by the emergence of a cell type-specific gene expression and transcriptional programs that determine the specific phenotype and function of cells in the erythroid lineage. Our objectives in this study were to identify unique gene expression patterns that characterize the transcriptional program of normal primary human erythroid precursors during terminal differentiation, and define the gene expression patterns seen in erythroblasts (EBL) of patients with polycythemia vera (PV). Homogenous populations of primary proEBL were generated from purified liquid cultures of CD34+ cells collected from healthy volunteers and PV patients. All patients with PV were diagnosed based on established criteria and had the JAK2-V617F mutation. Morphologic examination and surface expression of CD71 confirmed the purity of proEBL cell populations. ProEBL from normal individuals were induced to terminally differentiate generating orthochromatic EBL. RNA was extracted from normal proEBL, PV proEBL, and normal orthochromatic EBL. Affymetrix U133 Plus 2.0 arrays representing approximately 39,000 human genes were used for gene expression analysis. Four replicates from four independent primary cell cultures were analyzed for each comparison group (e.g. undifferentiated proEBL versus terminally differentiated orthochromatic EBL). Unsupervised hierarchical clustering showed distinct gene expression profiles in the proEBL and terminally differentiated EBL lineages. 1109 genes (2.0 fold change, P<0.01) were found to be differentially expressed. Numerous erythroid genes were found to be upregulated during terminal differentiation [e.g. globin genes, erythropoietin receptor, heme synthesis enzymes (ferrochelatase, ALAS2) erythrocyte membrane proteins (band 3, ankyrin, protein 4.1) and transcription factors (NFE2, Kruppel-like factors, myb, GATA2)]. As a proof of validation, the differential expression of 7 genes was verified by Northern blotting. To better understand the biologic role of the gene sets identified, using Ingenuity pathway analysis, individual genes were integrated into specific regulatory and signaling pathway networks. A total of 19 networks with significant scores (>23) were identified. Biological functions of the identified networks included RNA post-transcriptional regulation, cell cycle control, translational regulation, DNA replication and repair and cellular assembly/organization. In a proof of principle study, gene expression patterns in PV proEBL (n=6) were compared to normal proEBL (n=5). Unsupervised hierarchical clustering showed a distinct gene expression profile for PV. A binary regression predictive model was also developed to find gene expression patterns predictive for PV. Using this model a 150 gene predictor was found that could predict PV patients from control at 100% accuracy. Ingenuity pathways analysis of a subset of gene subsets demonstrated several biologically relevant networks that were distinct in patients with PV, including myc, CDC2, and JAK2. Deregulation of normal transcriptional mechanisms in hematopoietic cells is associated with the pathogenesis of PV. Further, our data shows that genomic studies provide new insights into transcriptional programs that govern erythroid differentiation, and identify biologically relevant deregulated pathways as potential targets for therapy in PV.

Hydroquinone and catechol interfere with T cell cycle entry and progression through the G 1 phase

Cigarette smoking causes profound suppression of pulmonary T cell responses, which is associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. Hydroquinone (HQ) and catechol, at concentrations comparable to those found in cigarette smoke, are potent inhibitors of T cell activation and proliferation. We have previously shown that HQ and catechol inhibit ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis. In this report we demonstrate that HQ and catechol also inhibit blastogenesis by interfering with T cell cycle entry and progression through the G 1 phase. In an attempt to localize the point in the cell cycle where arrest occurred, a set of key markers of activation and cell cycle progression were examined, including induction of c-Myc, up regulation of RNA synthesis, surface expression of CD71, and induction of E2F-dependent gene expression. Addition of HQ or catechol prior to stimulation inhibited each of these events without decreasing cell viability. However, production of IL-2 and surface expression of CD69 and CD25 were not affected, indicating that HQ and catechol inhibit only certain cell cycle events. These studies provide further indication of the regulatory pathways by which cigarette smoke inhibits T cell responses in the lungs of smokers.

Interleukin-18 enhances HIV-1 production in a human chronically-infected T cell line (H9-V).

Interleukin-18 (IL-18) is a recently identified immunoregulatory cytokine expressed by activated macrophages, that induces production of interferon-gamma (IFN-gamma) and Th-1 development. Recently some investigators reported controversial in vitro data on IL-18 stimulation of HIV-1 replication in several cell lines. In the present study the effect of IL-18 on HIV replication in a human chronically HIV-1-infected lymphocytic T cell line (H9-V) was investigated. HIV-1 replication was determined by an immunoassay method in order to evaluate the content of p24 antigen in the cell culture supernatants. Stimulation of H9-V cells with IL-18 resulted in increased production of p24, especially at concentrations of 0.01 microg ml(-1) and 0.10 microg ml(-1). Moreover a significant and persistent IL-18 stimulation of HIV-1 replication was observed at a concentration of 0.01 microg ml(-1) during a 7-day period. Pre-treatment of IL-18 with a specific neutralizing monoclonal antibody significantly reduced HIV-1 replication. These experiments show that IL-18 promotes the increase of HIV-1 replication in human chronically-infected lymphocytic T cells and confirm the role of IL-18 as a proimflammatory cytokine in stimulating and maintaining HIV-1 replication during the course of the disease. In a successive set of experiments, since one of the main activities of IL-18 is the induction of IFN-gamma, we evaluated the effect of this biological modifier on H9-V cells. In particular, IFN-gamma shows a significant effect on cell replication and on reduction of CD4 and CD71 surface expression.