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Early-life immune exposures can profoundly impact lifelong health. However, functional mechanisms underlying fetal immune development remain incomplete. Erythrocytes are not typically considered active immune mediators, primarily because erythroid precursors discard their organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Erythroid progenitors and precursors circulate in human fetuses and neonates. Although there is limited evidence that erythroid precursors are immunomodulatory, our understanding of the underlying mechanisms remains inadequate. To define the immunobiological role of fetal and perinatal erythroid progenitors and precursors, we analyzed single cell RNA-sequencing data and found that transcriptomics support erythroid progenitors as putative immune mediators. Unexpectedly, we discovered that human erythroid progenitors constitutively express Major Histocompatibility Complex (MHC) class II antigen processing and presentation machinery, which are hallmarks of specialized antigen presenting immune cells. Furthermore, we demonstrate that erythroid progenitors internalize and cleave foreign proteins into peptide antigens. Unlike conventional antigen presenting cells, erythroid progenitors express atypical costimulatory molecules and immunoregulatory cytokines that direct the development of regulatory T cells, which are critical for establishing maternal-fetal tolerance. Expression of MHC II in definitive erythroid progenitors begins during the second trimester, coinciding with the appearance of mature T cells in the fetus, and is absent in primitive progenitors. Lastly, we demonstrate physical and molecular interaction potential of erythroid progenitors and T cells in the fetal liver. Our findings shed light on a unique orchestrator of fetal immunity and provide insight into the mechanisms by which erythroid cells contribute to host defense.
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The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient numbers of in vitro-derived red blood cells (RBC) for clinical purposes. We and others have determined that BMI1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts (SREs). However, the mechanisms of BMI1 action remain poorly understood. BMI1 overexpression led to 10 billion-fold increase BMI1-induced (i)SRE self-renewal. Despite prolonged culture and BMI1 overexpression, human iSREs can terminally mature and agglutinate with typing reagent monoclonal antibodies against conventional RBC antigens. BMI1 and RING1B occupancy, along with repressive histone marks, were identified at known BMI1 target genes, including the INK-ARF locus, consistent with an altered cell cycle following BMI1 inhibition. We also identified upregulated BMI1 target genes with low repressive histone modifications, including key regulator of cholesterol homeostasis. Functional studies suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. These findings support the hypothesis that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand the pool of immature erythroid precursors for eventual clinical uses.
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ABSTRACT: During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.
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Células-Tronco Pluripotentes Induzidas , Humanos , Eritropoese/genética , Eritrócitos , Diferenciação Celular/genética , Eritroblastos/metabolismoRESUMO
Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild-type GATA1 (wtGATA1) or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and derepression of its downstream targets. Notably, DYRK1A loss had no effect in T21 iPSCs or megakaryocytes with wtGATA1. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in T21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.
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Síndrome de Down , Leucemia Megacarioblástica Aguda , Humanos , Síndrome de Down/genética , Síndrome de Down/complicações , Fator de Transcrição GATA1/genética , Leucemia Megacarioblástica Aguda/complicações , Leucemia Megacarioblástica Aguda/genética , Trombopoese/genéticaRESUMO
Red blood cell (RBC) transfusion is one of the most common medical treatments, with more than 10 million units transfused per year in the United States alone. Alloimmunization to foreign Rh proteins (RhD and RhCE) on donor RBCs remains a challenge for transfusion effectiveness and safety. Alloantibody production disproportionately affects patients with sickle cell disease who frequently receive blood transfusions and exhibit high genetic diversity in the Rh blood group system. With hundreds of RH variants now known, precise identification of Rh antibody targets is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen phenotypes. Using a combination of human-induced pluripotent stem cell (iPSC) reprogramming and gene editing, we designed a renewable source of cells with unique Rh profiles to facilitate the identification of complex Rh antibodies. We engineered a very rare Rh null iPSC line lacking both RHD and RHCE. By targeting the AAVS1 safe harbor locus in this Rh null background, any combination of RHD or RHCE complementary DNAs could be reintroduced to generate RBCs that express specific Rh antigens such as RhD alone (designated D--), Goa+, or DAK+. The RBCs derived from these iPSCs (iRBCs) are compatible with standard laboratory assays used worldwide and can determine the precise specificity of Rh antibodies in patient plasma. Rh-engineered iRBCs can provide a readily accessible diagnostic tool and guide future efforts to produce an alternative source of rare RBCs for alloimmunized patients.
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Antígenos de Grupos Sanguíneos , Células-Tronco Pluripotentes , Medicina Transfusional , Alelos , Antígenos de Grupos Sanguíneos/genética , Humanos , Sistema do Grupo Sanguíneo Rh-Hr/genéticaRESUMO
Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an â¼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.
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Transfusão de Sangue , Duplicação Gênica , Variação Genética , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Anemia Falciforme/genética , Anemia Falciforme/terapia , Quebra Cromossômica , Biologia Computacional/métodos , Frequência do Gene , Heterogeneidade Genética , Ligação Genética , Genômica/métodos , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodosRESUMO
The COVID-19 pandemic has created major disruptions in health care delivery, including a severe blood shortage. The inventory of Rh and K antigen-negative red cell units recommended for patients with hemoglobinopathies became alarmingly low and continues to be strained. Because patients with sickle cell disease requiring chronic red cell exchange (RCE) incur a large demand for red cell units, we hypothesized that implementation of 2 measures could reduce blood use. First, obtaining the pretransfusion hemoglobin S (HbS) results by procedure start time would facilitate calculation of exact red cell volume needed to achieve the desired post-RCE HbS. Second, as a short-term conservation method, we identified patients for whom increasing the targeted end procedure hematocrit up to 5 percentage points higher than the pretransfusion level (no higher than 36%) was not medically contraindicated. The goal was to enhance suppression of endogenous erythropoiesis and thereby reduce the red cell unit number needed to maintain the same target HbS%. These 2 measures resulted in an 18% reduction of red cell units transfused to 50 patients undergoing chronic RCE during the first 6 months of the COVID-19 pandemic. Despite reduction of blood use, pretransfusion HbS% target goals were maintained and net iron accumulation was low. Both strategies can help alleviate a shortage of Rh and K antigen-negative red cells, and, more generally, transfusing red cell units based on precise red cell volume required can optimize patient care and judicious use of blood resources.
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Anemia Falciforme , COVID-19 , Anemia Falciforme/terapia , Transfusão de Eritrócitos , Humanos , Pandemias , SARS-CoV-2RESUMO
Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein ß-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the ß-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate ß-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases ß-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.