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Erythroid cells are the most abundant cells in the human body. In addition to their established function in gas-transportation, erythroid cells at various stages of differentiation have recently been shown to have immunomodulatory roles. Red blood cells may serve as modulators of innate and adaptive immunity, while their immature counterparts, CD71 + erythroid cells (CECs) have important immunomodulatory functions in various contexts. CECs are abundant in human cord blood and placenta, where they contribute to fetomaternal tolerance. CECs also accumulate in patients with infections, tumors, and anemia, and effectively suppress T cells by producing high levels of arginase, reactive oxygen species, programmed death-ligand 1, transforming growth factor β, and/or interleukin-10. Here, we systematically summarize the immunomodulatory functions of erythroid cells and propose some potential therapeutic applications based on their characteristics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Niu and Zhang.)

The role of Erythroid cells in immune regulation and immunosuppression is one of the emerging topics in modern immunology that still requires further clarification as Erythroid cells from different tissues and different species express different immunoregulatory molecules. In this study, we performed a thorough investigation of human bone marrow Erythroid cells from adult healthy donors and adult acute lymphoblastic leukemia patients using the state-of-the-art single-cell targeted proteomics and transcriptomics via BD Rhapsody and cancer-related gene copy number variation analysis via NanoString Sprint Profiler. We found that human bone marrow Erythroid cells express the ARG1, LGALS1, LGALS3, LGALS9 , and C10orf54 (VISTA) immunosuppressive genes, CXCL5, CXCL8 , and VEGFA cytokine genes, as well as the genes involved in antimicrobial immunity and MHC Class II antigen presentation. We also found that ARG1 gene expression was restricted to the single erythroid cell cluster that we termed ARG1-positive Orthochromatic erythroblasts and that late Erythroid cells lose S100A9 and gain MZB1 gene expression in case of acute lymphoblastic leukemia. These findings show that steady-state erythropoiesis bone marrow Erythroid cells express myeloid signature genes even without any transdifferentiating stimulus like cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Perik-Zavodskii, Perik-Zavodskaia, Alrhmoun, Volynets, Shevchenko, Nazarov, Denisova and Sennikov.)

Background: The establishment of microbial communities in neonatal mammals plays a pivotal role in shaping their immune responses to infections and other immune-related conditions. This process is influenced by a combination of endogenous and exogenous factors. Previously, we reported that depletion of CD71 + erythroid cells (CECs) results in an inflammatory response to microbial communities in newborn mice., Results: Here, we systemically tested this hypothesis and observed that the small intestinal lamina propria of neonatal mice had the highest frequency of CECs during the early days of life. This high abundance of CECs was attributed to erythropoiesis niches within the small intestinal tissues. Notably, the removal of CECs from the intestinal tissues by the anti-CD71 antibody disrupted immune homeostasis. This disruption was evident by alteration in the expression of antimicrobial peptides (AMPs), toll-like receptors (TLRs), inflammatory cytokines/chemokines, and resulting in microbial dysbiosis. Intriguingly, these alterations in microbial communities persisted when tested 5 weeks post-treatment, with a more notable effect observed in female mice. This illustrates a sex-dependent association between CECs and neonatal microbiome modulation. Moreover, we extended our studies on pregnant mice, observing that modulating CECs substantially alters the frequency and diversity of their microbial communities. Finally, we found a significantly lower proportion of CECs in the cord blood of pre-term human newborns, suggesting a potential role in dysregulated immune responses to microbial communities in the gut., Conclusions: Our findings provide novel insights into pivotal role of CECs in immune homeostasis and swift adaptation of microbial communities in newborns. Despite the complexity of the cellular biology of the gut, our findings shed light on the previously unappreciated role of CECs in the dialogue between the microbiota and immune system. These findings have significant implications for human health. Video Abstract., (© 2024. The Author(s).)

CD71 + erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L -arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71 high CD235a dim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation., (© 2021. The Author(s).)

Single-cell RNA sequencing (scRNA-Seq) is an emerging strategy for characterizing immune cell populations. Compared to flow or mass cytometry, scRNA-Seq could potentially identify cell types and activation states that lack precise cell surface markers. However, scRNA-Seq is currently limited due to the need to manually classify each immune cell from its transcriptional profile. While recently developed algorithms accurately annotate coarse cell types (e.g. T cells versus macrophages), making fine distinctions (e.g. CD8+ effector memory T cells) remains a difficult challenge. To address this, we developed a machine learning classifier called ImmClassifier that leverages a hierarchical ontology of cell type. We demonstrate that its predictions are highly concordant with flow-based markers from CITE-seq and outperforms other tools (+15% recall, +14% precision) in distinguishing fine-grained cell types with comparable performance on coarse ones. Thus, ImmClassifier can be used to explore more deeply the heterogeneity of the immune system in scRNA-Seq experiments., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mashhouri, Koleva, Huynh, Okoye, Shahbaz and Elahi.)

Patients with myelodysplastic syndromes (MDSs) display severe anemia but the mechanisms underlying this phenotype are incompletely understood. Right open-reading-frame kinase 2 (RIOK2) encodes a protein kinase located at 5q15, a region frequently lost in patients with MDS del(5q). Here we show that hematopoietic cell-specific haploinsufficient deletion of Riok2 (Riok2 f/+ Vav1 cre ) led to reduced erythroid precursor frequency leading to anemia. Proteomic analysis of Riok2 f/+ Vav1 cre erythroid precursors suggested immune system activation, and transcriptomic analysis revealed an increase in p53-dependent interleukin (IL)-22 in Riok2 f/+ Vav1 cre CD4 + T cells (T H 22). Further, we discovered that the IL-22 receptor, IL-22RA1, was unexpectedly present on erythroid precursors. Blockade of IL-22 signaling alleviated anemia not only in Riok2 f/+ Vav1 cre mice but also in wild-type mice. Serum concentrations of IL-22 were increased in the subset of patients with del(5q) MDS as well as patients with anemia secondary to chronic kidney disease. This work reveals a possible therapeutic opportunity for reversing many stress-induced anemias by targeting IL-22 signaling.

In inflammatory bowel disease (IBD), inflammation can occur beyond the intestine and spread systemically causing complications such as arthritis, cachexia, and anemia. Here, we determine the impact of CD45, a pan-leukocyte marker and tyrosine phosphatase, on IBD. Using a mouse model of T cell transfer colitis, CD25 - CD45RB high CD4 + T cells were transferred into Rag1-deficient mice (RAGKO) and CD45-deficient RAGKO mice (CD45RAGKO). Weight loss and systemic wasting syndrome were delayed in CD45RAGKO mice compared to RAGKO mice, despite equivalent inflammation in the colon. CD45RAGKO mice had reduced serum levels of TNF-α, and reduced TNF-α production by splenic myeloid cells. CD45RAGKO mice also had increased numbers of erythroid progenitors in the spleen, which had previously been shown to be immunosuppressive. Adoptive transfer of these erythroid progenitors into RAGKO mice reduced their weight loss and TNF-α expression by splenic red pulp macrophages. In vitro, erythroid cells suppressed TNF-α expression in red pulp macrophages in a phagocytosis-dependent manner. These findings show a novel role for erythroid progenitors in suppressing the pro-inflammatory function of splenic macrophages and cachexia associated with IBD., (© 2020 Wiley-VCH GmbH.)

CD45 + CD71 + erythroid cells generated through splenic extramedullary erythropoiesis have recently been found to suppress anti-infection and tumor immunity in neonates and adults with malignances. However, their role in tumor microenvironment has not been investigated. In the present study, we found that the number of CD45 + CD71 + erythroid cells was significantly elevated in hepatocellular carcinoma (HCC) tissues compared to that in paratumor region and circulation. Additionally, they were more abundant in HCC tissues compared to some immune suppressive cells as well as CD45 - CD71 + erythroid cells. CD45 + CD71 + erythroid cells suppressed T cells through generation of reactive oxygen species, IL-10, and TGF-β in a paracrine and cell-cell contact manner, and their suppressive effect was stronger than that of myeloid-derived suppressor cells. The abundance of CD45 + CD71 + erythroid cells in tumor tissue, as illustrated via immunofluorescence, predicted disease-free survival and overall survival, and its prognostic value was better than that of Cancer of the Liver Italian Program score. This study demonstrated that accumulation of intratumoral CD45 + CD71 + erythroid cells in HCC tissues could play a superior immunosuppressive role in tumor microenvironment and may serve as a valuable biomarker to predict recurrence of HCC., (Copyright © 2020. Published by Elsevier B.V.)

Aridity and heat are significant environmental stressors that affect sheep adaptation and adaptability, thus influencing immunity, growth, reproduction, production performance, and profitability. The aim of this study was to profile mRNA expression levels in the spleen of indigenous Kazakh sheep breed for comparative analysis with the exotic Suffolk breed. Spleen histomorphology was observed in indigenous Kazakh sheep and exotic Suffolk sheep raised in Xinjiang China. Transcriptome sequencing of spleen tissue from the two breeds were performed via Illumina high-throughput sequencing technology and validated by RT-qPCR. Blood cytokine and IgG levels differed between the two breeds and IgG and IL-1β were significantly higher in Kazakh sheep than in Suffolk sheep (p < 0.05), though spleen tissue morphology was the same. A total of 52.04 Gb clean reads were obtained and the clean reads were assembled into 67,271 unigenes using bioinformatics analysis. Profiling analysis of differential gene expression showed that 1158 differentially expressed genes were found when comparing Suffolk with Kazakh sheep, including 246 up-regulated genes and 912 down-regulated genes. Utilizing gene ontology annotation and pathway analysis, 21 immune- responsive genes were identified as spleen-specific genes associated with adaptive traits and were significantly enriched in hematopoietic cell lineage, natural killer cell-mediated cytotoxicity, complement and coagulation cascades, and in the intestinal immune network for IgA production. Four pathways and up-regulated genes associated with immune responses in indigenous sheep played indispensable and promoting roles in arid and hot environments. Overall, this study provides valuable transcriptome data on the immunological mechanisms related to adaptive traits in indigenous and exotic sheep and offers a foundation for research into adaptive evolution.

The biomechanical properties of the bone marrow microenvironment emerge from a combination of interactions between various extracellular matrix (ECM) structural proteins and soluble factors. Matrix stiffness directs stem cell fate, and both bone marrow stromal and hematopoietic cells respond to biophysical cues. Within the bone marrow, the megakaryoblasts and erythroblasts are thought to originate from a common progenitor, giving rise to fully mature magakaryocytes (the platelet precursors) and erythrocytes. Erythroid and megakaryocytic progenitors sense and respond to the ECM through cell surface adhesion receptors such as integrins and mechanosensitive ion channels. While hematopoietic stem progenitor cells remain quiescent on stiffer ECM substrates, the maturation of the erythroid and megakaryocytic lineages occurs on softer ECM substrates. This review surveys the major matrix structural proteins that contribute to the overall biomechanical tone of the bone marrow, as well as key integrins and mechanosensitive ion channels identified as ECM sensors in context of megakaryocytosis or erythropoiesis.

CD71 + erythroid cells (CECs) have a wide range of immunomodulatory properties. Here, we show that CECs are expanded in the peripheral blood of HIV patients, with a positive correlation between their frequency and the plasma viral load. CECs from HIV patients and human cord blood/placenta exacerbate HIV-1 infection/replication when cocultured with CD4 + T cells, and that preexposure of CD4 + T cells to CECs enhances their permissibility to HIV infection. However, mature red blood cells (RBCs) do not enhance HIV replication when cocultured with CD4 + T cells. We also found CECs express substantial levels of the NOX2 gene and via a mitochondrial reactive oxygen species (ROS)-dependent mechanism possibly upregulate NF-κB in CD4 + T cells once cocultured, which affects the cell cycle machinery to facilitate HIV-1 replication. The complement receptor-1 (CD35) and the Duffy antigen receptor for chemokines (DARC) as potential HIV target molecules are expressed significantly higher on CECs compared to mature red blood cells. Blocking CD35 or DARC substantially abolishes HIV-1 transmission by RBCs to uninfected CD4 + T cells but not by CECs. In contrast, we observed CECs bind to HIV-1 via CD235a and subsequently transfer the virus to uninfected CD4 + T cells, which can be partially blocked by the anti-CD235a antibody. More importantly, we found that CECs from HIV-infected individuals in the presence of antiretroviral therapy harbor infective viral particles, which mediate HIV-1 trans -infection of CD4 + T cells. Therefore, our findings provide a novel insight into the role of CECs in HIV pathogenesis as potential contributing cells in viral persistence and transmission. IMPORTANCE Immature red blood cells (erythroid precursors or CD71 + erythroid cells) have a wide range of immunomodulatory properties. In this study, we found that these erythroid precursors are abundant in the human cord blood/placental tissues, in the blood of HIV-infected and anemic individuals. We observed that these cells exacerbate HIV-1 replication/infection in target cells and even make HIV target cells more permissible to HIV infection. In addition, we found that HIV gets a free ride by binding on the surface of these cells and thus can travel to different parts of the body. In agreement, we noticed a positive correlation between the plasma viral load and the frequency of these cells in HIV patients. More importantly, we observed that infective HIV particles reside inside these erythroid precursors but not mature red blood cells. Therefore, these cells by harboring HIV can play an important role in HIV pathogenesis., (Copyright © 2019 Namdar et al.)

Background: CD71 or Transferrin receptor is expressed on the surface of erythroid lineage cells. CD71 expression has been found to be significantly increased in rapidly proliferating cells., Methods: This cross-sectional study included 37 bone marrow samples of acute leukemia cases diagnosed between October 2016 to April 2018. The samples were analysed on BD FACS Canto II. We evaluated the expression of CD71 on leukemic blasts and compared median fluorescent intensities (MFI) of blasts in different types of acute leukemias., Results: The 37 cases comprised of 21 Acute Myeloid Leukemia (AML), 13 B-Acute Lymphoblastic Leukemia (B-ALL), 2 T- Acute Lymphoblastic Leukemia (T-ALL) and 1 mixed phenotypic acute leukemia (MPAL), T/Myeloid. CD 71 expression was noted in 70.3% (n= 26/37) of acute leukemia cases. CD71 expression was most commonly observed in AML (n= 15/21;71.4%), followed by B-ALL (n= 9/13;69.2%) and T-ALL (n= 1/2;50%). Single case of MPAL revealed blasts positive for CD71. MFI of leukemic blasts of single CD71 positive T-ALL was found to be highest, followed by AML, MPAL (T/Myeloid) and least in B ALL. Of the AML cases, the blasts of AML-M6, acute promyelocytic leukemia and AML-M1 showed higher CD71 expression in terms of MFI., Conclusions: Surface CD71 expression is not only found in erythroid lineage cells, but also in proliferating cells. CD71 MFI is highest in T lymphoblasts followed by leukemic erythroblasts, myeloblasts and least in B lymphoblasts., Competing Interests: There are no conflicts of interest

We used Casd1-deficient mice to confirm that this enzyme is responsible for 9-O-acetylation of sialic acids in vivo. We observed a complete loss of 9-O-acetylation of sialic acid on the surface of myeloid, erythroid and CD4+ T cells in Casd1-deficient mice. Although 9-O-acetylation of sialic acids on multiple hematopoietic lineages was lost, there were no obvious defects in hematopoiesis. Interestingly, erythrocytes from Casd1-deficient mice also lost reactivity to TER-119, a rat monoclonal antibody that is widely used to mark the murine erythroid lineage. The sialic acid glyco-epitope recognized by TER-119 on erythrocytes was sensitive to the sialic acid O-acetyl esterase activity of the hemagglutinin-esterase from bovine coronavirus but not to the corresponding enzyme from the influenza C virus. During erythrocyte development, TER-119+ Ery-A and Ery-B cells could be stained by catalytically inactive bovine coronavirus hemagglutinin-esterase but not by the inactive influenza C hemagglutinin-esterase, while TER-119+ Ery-C cells and mature erythrocytes were recognized by both virolectins. Although the structure of the sialoglycoconjugate recognized by TER-119 was not chemically demonstrated, its selective binding to virolectins suggests that it may be comprised of a 7,9-di-O-acetyl form of sialic acid. As erythrocytes mature, the surfaces of Ery-C cells and mature erythrocytes also acquire an additional distinct CASD1-dependent 9-O-acetyl sialic acid moiety that can be recognized by virolectins from both influenza C and bovine coronavirus., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- β, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-β compared to CD71+VISTA- erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells-compared to CD71+VISTA- and CD71+ erythroid cells from the VISTA KO mice-significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-β in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-β1, TGF-β2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond., Competing Interests: The authors have declared that no competing interests exist.

Aim: To understand the effects of delivery mode on the immune cells frequency and function in cord blood and placenta., Methods: We evaluated immunological differences in cord blood and placental tissues for a case of twins one of which delivered vaginally while the other delivered by caesarian section (C-section). Cord blood mononuclear cells were isolated and placenta tissues were processed for cell isolation. Immune phenotyping was performed by flow cytometry methods following staining for T cells, natural killer (NK) cells, monocytes, neutrophils and CD71 + erythroid cells in both cord blood and placenta tissues. In addition, fetal calprotectin of twins was measured 12 wk after birth., Results: We found lower percentages of immune cells ( e.g . T cells, monocytes and neutrophils) in the cord blood of C-section delivered compared to vaginally delivered newborn. In contrast, percentages of monocytes and neutrophils were > 2 folds higher in the placental tissues of C-section delivered newborn. More importantly, we observed lower percentages of CD71 + erythroid cells in both cord blood and placental tissues of C-section delivered case. Lower CD71 + erythroid cells were associated with a more pro-inflammatory milieu at the fetomaternal interface reflected by higher expression of inhibitory receptors on CD4 + T cells, higher frequency of monocytes and neutrophils. Furthermore, type of delivery impacted the gene expression profile in CD71 + erythroid cells. Finally, we found that C-section delivered child had > 20-fold higher FCP in his fecal sample at 12 wk of age., Conclusion: Mode of delivery impacted immune cells profile in cord blood/placenta. In particular frequency of immunosuppressive CD71 + erythroid cells was reduced in C-section delivered newborn., Competing Interests: Conflict-of-interest statement: All authors declare that there are no competing interests.

Tissue-resident mast cells are associated with many inflammatory and physiological processes. Although mast cells arise from the yolk sac, the exact ontogeny of adult mast cells remains unclear. Here we have investigated the hematopoietic origin of mast cells using fate-mapping systems. We have shown that early erythro-myeloid progenitors (EMPs), late EMPs, and definitive hematopoietic stem cells (HSCs) each gave rise to mast cells in succession via an intermediate integrin β7 + progenitor. From late embryogenesis to adult, early EMP-derived mast cells were largely replaced by late EMP-derived cells in most connective tissues except adipose and pleural cavity. Thus, mast cells with distinct origin displayed tissue-location preferences: early EMP-derived cells were limited to adipose and pleural cavity and late EMP-derived cells dominated most connective tissues, while HSC-derived cells were a main group in mucosa. Therefore, embryonic origin shapes the heterogeneity of adult mast cells, with diverse functions in immunity and development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45 - Ter119 - CD31 - (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51 - TNCs. Ly6D + CD44 + CD51 - TNCs phenotypically and functionally resembled CD45 + pro-B lymphoid cells, whereas Ly6D - CD44 + CD51 - TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44 + CD51 - TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44 + CD51 - TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45 - hematopoietic progenitors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Survival of the allogeneic pregnancy depends on the maintenance of immune tolerance to paternal alloantigens at the fetomaternal interface. Multiple localized mechanisms contribute to the fetal evasion from the mother's immune rejection as the fetus is exposed to a wide range of stimulatory substances such as maternal alloantigens, microbes and amniotic fluids. In this article, we demonstrate that CD71 + erythroid cells are expanded at the fetomaternal interface and in the periphery during pregnancy in both humans and mice. These cells exhibit immunosuppressive properties, and their abundance is associated with a Th2 skewed immune response, as their depletion results in a proinflammatory immune response at the fetomaternal interface. In addition to their function in suppressing proinflammatory responses in vitro, maternal CD71 + erythroid cells inhibit an aggressive allogeneic response directed against the fetus such as reduction in TNF-α and IFN-γ production through arginase-2 activity and PD-1/programmed death ligand-1 (PDL-1) interactions. Their depletion leads to the failure of gestation due to the immunological rejection of the fetus. Similarly, fetal liver CD71 + erythroid cells exhibit immunosuppressive activity. Therefore, immunosuppression mediated by CD71 + erythroid cells on both sides (mother/fetus) is crucial for fetomaternal tolerance. Thus, our results reveal a previously unappreciated role for CD71 + erythroid cells in pregnancy and indicate that these cells mediate homeostatic immunosuppressive/immunoregulatory responses during pregnancy., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Neonatal CD71+ erythroid cells are thought to have immunosuppressive functions. Recently, we demonstrated that CD71+ erythroid cells from neonates born to women who underwent spontaneous preterm labor (PTL) are reduced to levels similar to those of term neonates; yet, their functional properties are unknown. Herein, we investigated the functionality of CD71+ erythroid cells from neonates born to women who underwent spontaneous preterm or term labor. CD71+ erythroid cells from neonates born to women who underwent PTL displayed a similar mRNA profile to that of those from term neonates. The direct contact between preterm or term neonatal CD71+ erythroid cells and maternal mononuclear immune cells, but not soluble products from these cells, induced the release of proinflammatory cytokines and a reduction in the release of TGF-β. Moreover, PTL-derived neonatal CD71+ erythroid cells (1) modestly altered CD8+ T cell activation; (2) inhibited conventional CD4+ and CD8+ T-cell expansion; (3) suppressed the expansion of CD8+ regulatory T cells; (4) regulated cytokine responses mounted by myeloid cells in the presence of a microbial product; and (5) indirectly modulated T-cell cytokine responses. In conclusion, neonatal CD71+ erythroid cells regulate neonatal T-cell and myeloid responses and their direct contact with maternal mononuclear cells induces a proinflammatory response. These findings provide insight into the biology of neonatal CD71+ erythroid cells during the physiologic and pathologic processes of labor., (©2018 Society for Leukocyte Biology.)

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self-renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34 + cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway-PD0325901 (PD)-significantly reduces the expansion of CD34 + and CD34 +  CD38 - cells, while there is no change in the expression of stemness-related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34 + cells. Notably, when ERK pathway is blocked in UCB-MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst-forming unit-erythroid colony (BFU-E) as well as enhancement of erythroid glycophorin-A marker. These results are in total conformity with up-regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down-regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self-renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB-haematopoietic progenitor cells., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Understanding the pathophysiology and associated host parasite interactions of the malaria infection is the prerequisite for developing effective prevention and treatment strategies. The exact mechanism underlying malaria associated ineffective and dyserythropoiesis is not yet fully understood. Being an important protein, haemoglobin serves as the main amino acid reservoir available to the intra-erythrocytic plasmodium. It is important to check the expression profiling of globin genes which may help us to understand host parasite interactions and its potential contribution to both infection and disease. Here, an in-vitro culture system was used to study the effect of different doses of Plasmodium falciparum on haematopoietic stem cell expansion, differentiation and expression of globin genes. Upon exposure to the different doses of P. falciparum parasites of strains 3D7, Dd2 and RKL9 (intact and lysed form) at different stages of erythroid development, cells demonstrated suppression in growth and differentiation. At almost all stages of erythroid development upon parasite exposure, the γ globin gene was found to be downregulated and the α/β as well as α/non- α globin mRNA ratios in late stage erythroid cells were found to be reduced (p < .01) compared to the untreated controls. The imbalance in globin chain expression might be considered as one of the factors involved in malaria associated inappropriate erythropoietic responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Newborns are highly susceptible to infection. The underlying mechanism of neonatal infection susceptibility has generally been associated with neonatal immune cell immaturity. In this study, we challenged this notion and built upon our recent discovery that neonates are physiologically enriched with erythroid TER119 + CD71 + cells (Elahi et al. 2013. Nature 504: 158-162). We have used Bordetella pertussis , a common neonatal respiratory tract infection, as a proof of concept to investigate the role of these cells in newborns. We found that CD71 + cells have distinctive immune-suppressive properties and suppress innate immune responses against B. pertussis infection. CD71 + cell ablation unleashed innate immune response and restored resistance to B. pertussis infection. In contrast, adoptive transfer of neonatal CD71 + cells into adult recipients impaired their innate immune response to B. pertussis infection. Enhanced innate immune response to B. pertussis was characterized by increased production of protective cytokines IFN-γ, TNF-α, and IL-12, as well as recruitment of NK cells, CD11b + , and CD11c + cells in the lung. Neonatal and human cord blood CD71 + cells express arginase II, and this enzymatic activity inhibits phagocytosis of B. pertussis in vitro. Thus, our study challenges the notion that neonatal infection susceptibility is due to immune cell-intrinsic defects and instead highlights active immune suppression mediated by abundant CD71 + cells in the newborn. Our findings provide additional support for the novel theme in neonatal immunology that immunosuppression is essential to dampen robust immune responses in the neonate. We anticipate that our results will spark renewed investigation in modulating the function of these cells and developing novel strategies for enhancing host defense to infections in newborns., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Infant's immune system cannot control infection or respond to vaccination as efficiently as older individuals, a phenomenon that has been attributed to immunological immaturity. Recently, we challenged this notion and proposed the presence of actively immunosuppressive and physiologically enriched CD71 + erythroid cells in neonates. Here we utilized Bordetella pertussis, a common neonatal respiratory tract pathogen, as a proof of concept to investigate the role of these cells in adaptive immunity. We observed that CD71 + cells have distinctive immunosuppressive properties and prevent recruitment of immune cells to the mucosal site of infection. CD71 + cells ablation unleashed induction of B. pertussis-specific protective cytokines (IL-17 and IFN-γ) in the lungs and spleen upon re-infection or vaccination. We also found that CD71 + cells suppress systemic and mucosal B. pertussis-specific antibody responses. Enhanced antigen-specific adaptive immunity following CD71 + cells depletion increased resistance of mice to B. pertussis infection. Furthermore, we found that human cord blood CD71 + cells also suppress T and B cell functions in vitro. Collectively, these data provide important insight into the role of CD71 + erythroid cells in adaptive immunity. We anticipate our results will spark renewed investigation in modulating the function of these cells to enhance host defense to infections in newborns.

Problem: Preterm neonates are highly susceptible to infection. Neonatal host defense against infection seems to be maintained by the temporal presence of immunosuppressive CD71+ erythroid cells. The aim of this study was to investigate whether umbilical cord CD71+ erythroid cells are reduced in neonates born to women who undergo spontaneous preterm labor/birth., Method of Study: Umbilical cord blood samples (n=155) were collected from neonates born to women who delivered preterm with (n=39) and without (n=12) spontaneous labor or at term with (n=82) and without (n=22) spontaneous labor. Time-matched maternal peripheral blood samples were also included (n=111). Mononuclear cells were isolated from these samples, and CD71+ erythroid cells were identified and quantified as CD3-CD235a+CD71+ cells by flow cytometry., Results: (i) The proportion of CD71+ erythroid cells was 50-fold higher in cord blood than in maternal blood; (ii) a reduced number and frequency of umbilical cord CD71+ erythroid cells were found in neonates born to women who underwent spontaneous preterm labor compared to those born to women who delivered preterm without labor; (iii) umbilical cord CD71+ erythroid cells were fewer in neonates born to term pregnancies, regardless of the process of labor, than in those born to women who delivered preterm without labor; and (iv) no differences were seen in umbilical cord CD71+ erythroid cells between neonates born to women who underwent spontaneous preterm labor and those born to women who delivered at term with labor., Conclusion: Umbilical cord CD71+ erythroid cells are reduced in neonates born to women who had undergone spontaneous preterm labor., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background and Objectives: Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals., Materials and Methods: We carried out in vitro erythroid differentiation of CD34(+) cells from peripheral blood of a Bm individual harbouring a 3.0-kb deletion including an erythroid cell-specific regulatory element, named the +5.8-kb site, in intron 1 of the human ABO blood group gene., Results: During the in vitro differentiation of CD34(+) cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5.8-kb site in cultured erythroid cells expressing ABO., Conclusion: It is likely that the +5.8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1., (© 2014 International Society of Blood Transfusion.)

Refractory cytopenia of childhood is the most common type of childhood myelodysplastic syndrome. Because the majority of children with refractory cytopenia have a normal karyotype and a hypocellular bone marrow, differentiating refractory cytopenia from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia can be challenging. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable diagnostic tool in differentiating myelodysplastic syndrome from non-clonal cytopenias in adults. Here, we performed the first comprehensive flow cytometric analysis of immature myeloid, lymphoid cells and erythroid cells, and granulocytes, monocytes, and lymphoid cells in bone marrow obtained from a large prospective cohort of 81 children with refractory cytopenia. Children with refractory cyotopenia had a strongly reduced myeloid compartment, but not as severe as children with aplastic anemia. Furthermore, the number of flow cytometric abnormalities was significantly higher in children with refractory cytopenia than in healthy controls and in children with aplastic anemia, but lower than in advanced myelodysplastic syndrome. We conclude that flow cytometric immunophenotyping could be a relevant addition to histopathology in the diagnosis of refractory cytopenia of childhood. (The multi-center studies EWOG-MDS RC06 and EWOG-MDS 2006 are registered at clinicaltrials.gov identifiers 00499070 and 00662090, respectively)., (Copyright© Ferrata Storti Foundation.)

Myelodysplastic syndromes is a heterogeneous group of bone marrow diseases ranging from low risk to high risk subtypes that may rapidly evolve to acute myeloid leukemia. Flow cytometry (FCM) is added as a recommended tool for diagnostic purposes in MDS. In recent studies FCM has also shown applicable to predict prognosis and treatment response. This review summarizes current data about the diagnostic, prognostic and therapeutic value of FCM in MDS. The high sensitivity of FCM in the detection of dysplasia in myelo-/monocytic and erythroid cell lineages makes it a valuable tool to distinguish possible clonal causes of cytopenia(s) from non-clonal causes, and to detect multi-lineage dysplasia in addition to cytomorphology. The utility of FCM in prediction of treatment response is promising. Therefore, FCM is an essential tool in standard diagnostic strategies in case of suspected MDS, and ready for general application., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71(+) erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71(+) cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71(+) cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71(+) cell-mediated susceptibility to infection is counterbalanced by CD71(+) cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71(+) cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these results will spark renewed investigation into the need for immunosuppression in neonates, as well as improved strategies for augmenting host defence in this vulnerable population.

Background: Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs., Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues., Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells., Conclusions: The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status., (© 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Because immune responses simultaneously defend and injure the host, the immune system must be finely regulated to ensure the host's survival. Here, we have shown that when injected with high Toll-like receptor ligand doses or infected with lymphocytic choriomeningitis virus (LCMV) clone 13, which has a high viral turnover, inflammatory monocyte-derived dendritic cells (Mo-DCs) engulfed apoptotic erythroid cells. In this process, called hemophagocytosis, phosphatidylserine (PS) served as an "eat-me" signal. Type I interferons were necessary for both PS exposure on erythroid cells and the expression of PS receptors in the Mo-DCs. Importantly, hemophagocytosis was required for interleukin-10 (IL-10) production from Mo-DCs. Blocking hemophagocytosis or Mo-DC-derived IL-10 significantly increased cytotoxic T cell lymphocyte activity, tissue damage, and mortality in virus-infected hosts, suggesting that hemophagocytosis moderates immune responses to ensure the host's survival in vivo. This sheds light on the physiological relevance of hemophagocytosis in severe inflammatory and infectious diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Within the myelodysplastic/myeloproliferative neoplasm (MDS/MPN) category of the WHO (2008), only chronic myelomonocytic leukemia was so far evaluated by multiparameter flow cytometry (MFC)., Methods: To investigate the potential of MFC for MDS/MPNs, unclassifiable (MDS/MPNu), and refractory anemia associated with ring sideroblasts and marked thrombocytosis (RARS-T), we studied 91 patients with these entities (60 males/31 females; 35.3-87.4 years) for MDS-related aberrant immunophenotypes (≥ 2 different cell lineages with ≥ 3 aberrantly expressed antigens). Data were correlated with cytomorphology and cytogenetics., Results: MFC identified MDS-related immunophenotypes in 54/91 (59.3%) of patients. Patients with or without MDS-related immunophenotype did not differ significantly by demographic characteristics, blood values, or median overall survival. MDS-related immunophenotype cases showed a higher number of aberrantly expressed antigens (mean ± SD, 4.9 ± 2.4 vs. 2.0 ± 1.4; P < 0.001). Aberrant karyotypes showed a similar frequency in patients with and without MDS-related immunophenotype (11/54; 20.4% vs. 7/37; 18.9%; P = n.s.)., Conclusions: MDS-related immunophenotype are present in more than half of patients with MDS/MPNu and RARS-T. MFC therefore may be helpful to separate cases into more "MDS-like" or "MPN-like" subgroups., (Copyright © 2012 International Clinical Cytometry Society.)

The reason why delayed RBC engraftment and pure red cell aplasia (PRCA) develop only in some but not all recipients of major ABO-incompatible hematopoietic stem cell transplantation (HSCT) remains elusive and the underlying mechanisms are not fully understood. Understanding how incompatible erythroid blood group antibodies (Abs) interact with ABH antigens (Ags) of grafts, and investigating how to induce artificially accommodation of grafts are of obvious importance in transplantation immunology. The effects of anti-H on proliferation, apoptosis, and α-(1,2)-fucosyltransferase gene (FUT1) expression in erythroid differentiated K562 cells were analyzed by the MTT assay, Annexin V/PI staining, and quantitative RT-PCR method. The growth of erythroid differentiated K562 cells was significantly suppressed when anti-H dilution was ≤ 1:8 (P<0.001, as compared with 1:16). Under the complement-free culture conditions, the apoptotic ratio of erythroid differentiated K562 cells was significantly increased when anti-H dilution was ≤ 1:16 (P<0.05, as compared with 1:32). The apoptosis was not only closely associated with anti-H dilution (F=138.991, P<0.001), but also correlated with treated time (F=583.249, P<0.001), which indicated typical dose- and time-dependent effects. Under the complement-free culture conditions, the FUT1 mRNA expression level was also suppressed when anti-H dilution was ≤ 1:16 (P<0.05, as compared with 1:32), which also manifested in typical dose-dependent (F=130.356, P<0.001) and time-dependent (F=1432.00, P<0.001) effects. The results confirm that anti-H can trigger apoptosis and down-regulate FUT1 expression in erythroid differentiated K562 cells without complement mediation. The findings suggest that anti-H could accommodate grafts through triggering apoptosis and down-regulating Fut1 expression to reduce ABH antigens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To observe the relationship between erythropoietin receptor (EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cells (BMMNC)-Coomb's test(+) hemocytopenia (immunorelated pancytopenia (IRP)) and explore the probable autoantigens of auto-Ab in IRP., Methods: A total of 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without) and 18 healthy controls were enrolled. The EPOR expressions on their nuclear erythrocytes were tested with flow cytometry (FCM) to observe the relationship between EPOR and auto-Ab. EPOR mRNA was detected by reverse transcription (RT)-PCR. Stat5 and P-Stat5 proteins in nucleated erythrocytes were measured by Western blot. EPOR expressions on nucleated erythrocytes membrane were re-tested after stripping autoantibodies with glycine buffer., Results: (1) EPOR of auto-Ab(+) group (1.6% ± 0.9%)was significantly lower than that of auto-Ab(-) group (4.6% ± 4.1%, P < 0.01)and the latter was significantly higher than that of normal controls (2.3% ± 1.8%, P < 0.05). EPOR of IRP patients was inversely correlated with their auto-Ab (r = -0.543, P = 0.000). (2) EPOR mRNA of auto-Ab(+) group (0.68 ± 0.14)was significantly higher than that of auto-Ab(-) group (0.55 ± 0.12, P < 0.01) and normal controls (0.58 ± 0.12, P < 0.05). (3) Protein Stat5 of auto-Ab(+) group (1.45 ± 0.94) was significantly higher than that of normal controls (0.54 ± 0.36, P < 0.05). While P-Stat5 of auto-Ab(+) group (0.42 ± 0.18)was significantly lower than that of normal controls (0.85 ± 0.38, P < 0.05). (4) EPOR expression increased significantly after auto-Ab stripping., Conclusions: The auto-Ab of some IRP patients blocks or competitively inhibits EPOR on the membrane of erythropoietic cells. And EPOR may be one of autoantigens in IRP.

Multiple transcription factors regulate B-cell commitment, which is coordinated with myeloid-erythroid lineage differentiation. NF-κB has long been speculated to regulate early B-cell development; however, this issue remains controversial. IκB kinase-α (IKKα) is required for splenic B-cell maturation but not for BM B-cell development. In the present study, we unexpectedly found defective BM B-cell development and increased myeloid-erythroid lineages in kinase-dead IKKα (KA/KA) knock-in mice. Markedly increased cytosolic p100, an NF-κB2-inhibitory form, and reduced nuclear NF-κB p65, RelB, p50, and p52, and IKKα were observed in KA/KA splenic and BM B cells. Several B- and myeloid-erythroid-cell regulators, including Pax5, were deregulated in KA/KA BM B cells. Using fetal liver and BM congenic transplantations and deleting IKKα from early hematopoietic cells in mice, this defect was identified as being B cell-intrinsic and an early event during hematopoiesis. Reintroducing IKKα, Pax5, or combined NF-κB molecules promoted B-cell development but repressed myeloid-erythroid cell differentiation in KA/KA BM B cells. The results of the present study demonstrate that IKKα regulates B-lineage commitment via combined canonical and noncanonical NF-κB transcriptional activities to target Pax5 expression during hematopoiesis.

Environmental factors likely regulate neonatal immunity and self-tolerance. However, evidence that the neonatal immune system is suppressed or deviated is varied depending on the antigen and the timing of antigen exposure relative to birth. These disparate findings may be related to the availability of the appropriate antigen presenting cells but also point to the possibility of homeostatic changes in non-lymphoid cells in the relevant lymphoid tissues. Here we show that, while leukocytes are the most abundant cell population present in spleen during the first 4-5 days after birth, a massive accumulation of nucleated immature erythroid population in the spleen takes places on day 6 after birth. Although the relative frequency of these immature erythorid cells slowly decreases during the development of neonates, they remain one of the most predominant populations up to three weeks of age. Importantly, we show that the immature erythroid cells from neonate spleen have the capacity to modulate the differentiation of CD4 T cells into effector cells and provide a bias towards a Th2 type instead of Th1 type. These nucleated erythroid cells can produce cytokines that participate in the Th2/Th1 balance, an important one being IL-6. Thus, the selective accumulation of immature erythroid cells in the spleen during a specific period of neonatal development may explain the apparent differences observed in the type(s) of immune responses generated in infants and neonates. These findings are potentially relevant to the better management of immune deficiency in and to the design of vaccination strategies for the young.

Deregulated expression of microRNAs is associated with neoplasia. Here, we show that mature miR-16 levels are abnormally increased in CD34(+) cells of patients with polycythemia vera as a consequence of preferential expression of miR-16-2 on chromosome 3 rather than of miR-16-1 on chromosome 13. Forced expression of miRNA-16 in normal CD34(+) cells stimulated erythroid cell proliferation and maturation. Conversely, exposure of polycythemia vera CD34(+) cells to small interfering RNA against pre-miR-16-2 reduced erythroid colonies and largely prevented formation of erythropoietin-independent colonies; myeloid progenitors remained unaffected. Experiments with knock down of JAK2 indicated that overexpression of miR-16 was independent of JAK/STAT pathway activation. Mice injected with an miR-16 antagomir showed a blunted erythroid response to exogenous erythropoietin, which indicates a role of miR-16 in normal erythropoiesis. These data suggest that deregulation of miR-16-2 contributes to abnormal expansion of erythroid lineage in polycythemia vera. However, the mechanisms for miR-16-2 overexpression remain to be elucidated, because no genetic abnormalities at the miR-16-2 locus were discovered.

The effects of immobilized granulocyte colony-stimulating factor (mediated by cells of the hemopoiesis-inducing microenvironment) on hemopoietic precursors of various classes were studied on the model of cytostatic-induced myelosuppression (administration of cyclophosphamide). The action of this preparation was compared with that of the standard preparation of granulocyte colony-stimulating factor. Thy 1,2(+)cells potentiated the effects of immobilized and standard granulocyte colony-stimulating factors on granulocyte-erythroid-macrophage-megakaryocyte precursors. Stromal cells were shown to potentiate the influence of these agents on granulocyte precursors. Induction of proliferation of precursor cells by the immobilized factor mediated by cells of the hemopoiesis-inducing microenvironment persisted for a longer period compared to that induced by the standard product.

Context: Acute erythroid leukemia (AEL) is an uncommon type of acute myeloid leukemia (AML), representing less than 5% of all cases. Acute erythroid leukemia is characterized by a predominant erythroid proliferation, and in the current World Health Organization (WHO) classification scheme there are 2 subtypes: erythroleukemia (erythroid/myeloid leukemia) and pure erythroid leukemia. Morphologic findings are most important for establishing the diagnosis. The erythroleukemia subtype, which is most common, is defined as the presence of 50% or more erythroid precursors and 20% or more blasts in the nonerythroid component. The pure erythroid leukemia subtype is composed of 80% or more immature erythroblasts. Although these morphologic criteria appear straightforward, AEL overlaps with other types of AML and myelodysplastic syndrome that are erythroid rich., Objective: To provide an update of AEL, including clinical presentation, morphologic features, immunophenotype, and cytogenetic and molecular data. As the erythroleukemia subtype is most common, the literature and this review are biased towards this subtype of AEL., Data Sources: Clinicopathologic, cytogenetic, and molecular information were extracted from our review of pertinent literature and a subset of AEL cases in the files of The University of Texas M. D. Anderson Cancer Center (Houston) and University of South Alabama (Mobile)., Conclusions: The current WHO criteria for establishing the diagnosis of AEL reduce the frequency of this entity, as cases once classified as the erythroleukemia subtype are now reclassified as other types of AML, particularly AML with myelodysplasia-related changes and therapy-related AML. This reclassification also may have prognostic significance for patients with the erythroleukemia subtype of AEL. In contrast, the current WHO criteria appear to have little impact on the frequency and poor prognosis of patients with the pure erythroid leukemia subtype of AEL. Molecular studies, preferably using high-throughput methods, are needed for a better understanding of the pathogenesis of AEL, and for developing diagnostic and prognostic markers.

DNase II is an endonuclease which plays a fundamental role in the degradation of DNA from both apoptotic cells, and nuclei extruded from red blood cells during erythropoiesis: important tasks, considering that everyday 10(8)-10(9) cells undergo apoptosis, and 10(11) red blood cells are produced in the adult human. The DNase II-null mouse demonstrates embryonic lethality due to type I interferon-mediated erythroid precursor cell death triggered by undegraded nucleic acids. However, the mechanisms leading to such cytotoxicity are poorly understood. A study in the current issue of the European Journal of Immunology investigates the role of the death ligand TRAIL in this process. Although TRAIL is shown to be dispensable for the interferon-induced apoptosis of erythroid cells in DNAse II(-/-) embryos, the authors have developed a useful strategy for further exploring this question in future studies. Interestingly, earlier studies by the same group showed that crossing the DNase II-null mouse with a mouse deficient for the type I interferon receptor can rescue the lethal anaemia observed in the DNase II-null embryos, but only at the cost of developing autoimmunity.

Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1(-/-) fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages.

Background: Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood., Design and Methods: The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor., Results: Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro., Conclusions: Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.

The purpose of this study was to establish the simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination. The bone marrow cells were stained with an anthraquinone fluorescent DNA stain (DRAQ5) and fluorescence-labeled antibodies, and were analyzed quantitatively using a flow cytometer in the presence of internal standard beads. DRAQ5 distinguished populations of nucleated cells. The absolute counts of nucleated cells were determined using an internal standard, and were equivalent to that measured by the electrical resistance method. The population of nucleated cells was classified into myeloids and erythroids by labeling with CD11b/c and CD71 antibodies, respectively. In a separate examination, T- and B-lymphocytes were also classified by labeling with CD3 and CD45RA antibodies, respectively. The classification of each cell lineage was identical with that of the alternative flow-cytometric method in which cells were differentiated according to cellular size and the fluorescence of a peroxidase indicator, 2',7'-dichlorofluorescin. The ratios of cell lineage, together with myeloid/erythroid ratio (ME), were the same as those obtained by a manual microscopic method. The present flow cytometric method enables the simultaneous measurement of the total nucleated cell counts and cellular differentials of rat bone marrow cells, allowing for rapid and highly quantitative bone marrow examination in rats.

Sox6, a member of the Sox transcription factor family, is essential for the silencing of epsilon y globin gene expression in definitive erythropoiesis of mice. Homozygous Sox6-null mice are neonatally lethal, precluding analysis at later stages. We created adult mice that are deficient in Sox6 specifically in hematopoietic tissues by transplanting embryonic liver stem cells from Sox6-deficient mice into lethally irradiated congenic wild-type adult mice. The mice receiving mutant stem cells (mutant engrafted) showed high expression levels of epsilon y in bone marrow, spleen, and circulating blood compared with mice receiving wild-type and heterozygous stem cells (control engrafted). The level of expression of epsilon y in circulating blood was directly correlated with the percentage of successful mutant donor cell engraftment. Additionally, the mutant engrafted adult mice showed an increase in erythroid precursor cells in bone marrow, spleen, and blood. Thus, Sox6 continues to function as a major regulator of epsilon y in adult definitive erythropoiesis and is required for normal erythrocyte maturation. Therefore, Sox6 may provide a novel therapeutic target by reactivating epsilon y in patients with hemoglobinopathies such as sickle cell anemia and beta-thalassemia.

Anaemia is an important global health problem. Therefore, it is crucial to understand its pathophysiology in various genetic or infectious diseases where dyserythropoiesis is a key pathological feature. To this effect, reproducible and reliable models of erythropoiesis in vitro are much needed as investigative tools. We have developed a serum-free liquid culture model of erythropoiesis using human umbilical cord blood CD34(+) cells cultured in the cytokine combination, interleukin-3 (IL-3), IL-6, stem cell factor (SCF) and erythropoietin (Epo), over 14 days. We found that these culture conditions favored erythroid differentiation over the expansion of the more primitive erythroid precursors. With an initiating culture density of 5x10(4) cells per ml, the nucleated cell fold expansion increased from 7.9+/-3.9 (range 4.5 to 11.1) after 4 days to 2990.2+/-1936.1 (range 626.6 to 6912.0) after 14 days in culture. Day-14 burst-forming unit-erythroid (BFU-E) frequencies peaked at day 4 (24.0+/-8.9%), with a marked decrease in BFU-E burst size as the cultures progressed. Time-course immunophenotypical profiles were characteristically erythroid with a decrease in CD34 expression (from 96.8+/-3.0% at day 0 to 0.8+/-0.8% at day 14), and a concomitant increase in the expression of erythroid-specific markers, CD36, glycophorin A (GpA) and CD71 (from 14.8+/-5.0%, 1.7+/-1.0% and 37.9+/-18.0% to 93.0+/-7.0%, 82.1+/-14.0% and 95.7+/-3.0%, respectively). Morphological studies revealed the presence of normoblasts with the complete absence of reticulocytes and mature erythrocytes after 14 days in culture. Once the culture conditions were optimized, we scaled down our culture model from 24-well plate (large-scale) to 96-well plate cultures (small-scale). We found that the small-scale cultures compared favorably with their large-scale counterpart in terms of erythroid progenitor cell proliferation and differentiation, particularly at low CD34(+) initiating cell doses. By using tumor necrosis factor-alpha (TNF-alpha), a known inhibitor of erythropoiesis, we validated our model system and showed a dose-dependent inhibition of erythroid differentiation with TNF-alpha in our cultures. Therefore, our results demonstrate a small-scale serum-free liquid culture model of erythropoiesis that is comparable with and complements our well-defined large-scale model. Our system would prove useful for screening the effects of exogenous factors on erythropoiesis in vitro.

EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation (IP) qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation (ChIP) assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells (MEL) in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase II, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE (HS26). This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.