Deficiency of Rac1 and Rac2 GTPases Perturbs Erythroid Proliferation and Differentiation but Not Enucleation.

Abstract The small Rho GTPases Rac1 and Rac2 have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. However their role in erythropoiesis has not yet been fully elucidated. Using conditional gene-targeted mice we demonstrated that deficiency of Rac1 and Rac2 GTPases causes a significant phenotype in erythroid lineage. The mice develop anemia that is both hemolytic (abnormal structure of the erythrocyte cytoskeleton and decreased deformability; Kalfa et al. Blood 2006) and dyserythropoietic in nature. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished as previously described (Gu et al. Science, 2003) on a Rac2-null genetic background. Colony assays revealed that although BFU-E frequency was similar, Rac1−/ −;Rac2−/ − BFU-E colonies had a strikingly different morphology appearing as round, small, dense colonies with solid edges, likely a manifestation of migration defects associated with Rac GTPase deficiency. CFU-E formation from hematopoietic stem/progenitors (HSC/Ps) derived from bone marrow (BM) of Rac1−/ −;Rac2−/ − mice was decreased more than 50% in comparison to WT (p=0.01). On the other hand, Rac1−/ −;Rac2−/ − mice developed marked splenomegaly (2-fold enlargement) and low density spleen cells demonstrated a 2-fold increase in CFU-E frequency in comparison to WT (p=0.008). To further assess erythroblast differentiation, BM and spleen cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood, 2001). Flow cytometry analysis revealed that the BM content of proerythroblasts and basophilic erythroblasts was significantly decreased (>5-fold) in Rac1−/ −;Rac2−/ − vs. WT mice. In contrast, the same erythroblast populations were 4-fold increased in the spleens of Rac1−/ −;Rac2−/ − animals. However, the terminal differentiation to orthochromatic erythroblasts was comparable. No survival difference was found between WT and Rac1−/ −;Rac2−/ − erythroid precursors by flow cytometry with annexin-V, indicating that apoptosis was not contributing to the changes in erythropoiesis in Rac-deficient mice. Differentiation of Rac1−/ −;Rac2−/ − HSC/Ps to proerythroblasts and basophilic erythroblasts was delayed significantly at the early stages in ex vivo erythropoiesis culture (Giarratana et al. Nat Biotechnol, 2005) in the presence of SCF and erythropoietin. Later in the culture the cytokine-independent terminal differentiation to orthochromatic erythroblasts was similar between WT and Rac1−/ −;Rac2−/ − mice. The phosphorylation of AKT in WT and Rac1−/ −;Rac2−/ − erythroid precursors revealed by immunoblotting was similar, but the phosphorylation of extracellular signal-regulated kinase (ERK) (p42/p44) in Rac1−/ −;Rac2−/ − erythroid precursors was significantly decreased. The enucleation process was evaluated quantitatively, in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable DNA-staining dye. The frequency of enucleated red cells (SYTO16-negative, Ter119-positive population) was similar in the WT and Rac1−/ −;Rac2−/ − erythroid cultures. These data suggest that Rac1 and Rac2 deficiency does not affect enucleation but causes a significant decrease of early erythroid precursor populations in the bone marrow.

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Fas-Dependent Apoptosis in Early MDS Erythroid Precursors Involves Endoplasmic Reticulum.

Blood ◽

10.1182/blood.v110.11.3346.3346 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3346-3346

Author(s):

Emmanuel Gyan ◽

Emilie Frisan ◽

Odile Beyne-Rauzy ◽

Cecile Pierre-Eugene ◽

Jean-Christophe Deschemin ◽

...

Keyword(s):

Bone Marrow ◽

Endoplasmic Reticulum ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Caspase 8 ◽

Spontaneous Apoptosis ◽

Cytochrome C Release ◽

Erythroid Precursor ◽

Erythroid Precursors

Abstract The anemia that characterizes most early myelodysplastic syndromes (MDS) was proposed to involve a deregulation in cell death pathways leading to excessive apoptosis of bone marrow erythroid precursors. Pathways leading to this excess in MDS erythroid precursors have been partially depicted in ex vivo liquid cultures of patients CD34+ bone marrow cells induced to differentiate into red cells in the presence of various cytokines. For example, we have identified the Fas-dependent activation of caspase-8 as a key initiating event. In order to further understand the mechanisms of MDS erythroid precursor death, we explored the role of the endoplasmic reticulum (ER) in this process. We first observed that Fas-dependent activation of caspase-8 in these cells induced the cleavage of BAP-31, an ER protein that is associated to Bcl-2 at the ER surface and was demonstrated to be a caspase-8 substrate. We also detected a proteolysis of caspase-4, which was proposed to play a role in ER-mediated apoptosis. To further explore the role of the ER, we constructed a lentivirus expressing a Bcl-2 mutant targeted to the ER membrane. The specific expression of Bcl-2 at the ER level prevented BAP-31 and caspase-4 cleavage induced by Fas engagement at the surface of MDS erythroid precursors and inhibited Fas-dependent apoptosis. Interestingly, ER-targeted Bcl-2 also inhibited mitochondrial membrane permeabilization (MMP) and cytochrome c release in MDS erythroid precursors undergoing spontaneous or Fas-induced apoptosis. These data argued for a role of the ER in MDS erythroid precursor apoptosis, upstream of the mitochondria. MDS erythroid precursors also demonstrated elevated ER Ca2+ stores when compared to normal erythroid precursors cultured in the same conditions. Ca2+ chelation with BAPTA or treatment with pharmacologic Ca2+ inhibitors such as nicardipine prevented the spontaneous apoptosis of MDS erythroid precursors. Altogether, these data suggest that the ER is involved in the spontaneous apoptosis of MDS erythroid precursors, downstream of Fas and upstream of the mitochondria, through mechanisms that can be inhibited by Bcl-2 and that involve Ca2+ stores.

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Rac GTPases Regulate Erythropoiesis Both in the Early Steps of Differentiation and in Enucleation.

Blood ◽

10.1182/blood.v110.11.1714.1714 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1714-1714

Author(s):

Theodosia A. Kalfa ◽

Suvarnamala Pushkaran ◽

Jose A. Cancelas ◽

James F. Johnson ◽

Deidre Daria ◽

...

Keyword(s):

Flow Cytometry ◽

Actin Polymerization ◽

Ex Vivo ◽

Terminal Differentiation ◽

Annexin V ◽

Actin Dynamics ◽

Fluorescent Label ◽

Rac Gtpase ◽

Rac Gtpases

Abstract Rac GTPases (i.e. Rac1, Rac2 and Rac3), a subfamily of Rho GTPases, control actin organization and have overlapping as well as distinct roles in cell survival, proliferation, and differentiation in various hematopoietic cell lineages (Gu et al, Science 2003, Cancelas et al, Nature Med 2005). Using conditional gene-targeting in mice, we have previously demonstrated that Rac1 and Rac2 deficiency causes anemia with abnormal erythrocyte cytoskeleton and decreased deformability (Kalfa et al, Blood 2006). In the present studies, we found by colony assays that although bone marrow (BM) BFU-E activity was unaltered from that of the wild type (WT) mice, Rac1−/−;Rac2−/− erythroid bursts had a strikingly different morphology appearing as round, small, dense colonies, likely a manifestation of motility defects associated with Rac GTPase deficiency. Total CFU-Es recovered from Rac1−/−;Rac2−/− BM were as low as 25% of that in WT mice (p<0.05). To further assess erythroblast differentiation, BM cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood 2001). Flow cytometry analysis revealed that proerythroblasts and basophilic erythroblasts in the BM were significantly decreased in Rac1−/−;Rac2−/− (∼30–50% of WT content) while the terminal differentiation to orthochromatic erythroblasts was comparable. In vivo BrdU labeling and flow cytometry with 7-AAD and annexin-V in combination with staining for CD71 and Ter119 revealed no difference in proliferation or survival between WT and Rac1−/−;Rac2−/− erythroid cells after the proerythroblast stage. These data suggest that deficiency of Rac1 and Rac2 GTPases affect erythropoiesis mainly at the early stages of BFU-E and CFU-E formation but not during terminal differentiation to orthochromatic erythroblasts. Given the prominent role of Rac GTPases in regulating actin structure, we next evaluated the possible involvement of Rac GTPases in enucleation, the terminal step of erythropoiesis that likely requires significant actin remodeling. We performed quantitative analysis in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable nucleic acid-staining dye. The frequency of enucleated red cells (SYTO16-low, Ter119-positive population) was similar in the WT and the Rac1−/−;Rac2−/− erythroid cultures. However, application of a Rac GTPase inhibitor, NSC23766, to the WT or the Rac1−/−;Rac2−/− erythroid cultures during the enucleation phase resulted in an inhibition of enucleation up to 80% dose-dependently (figure 1). Rac1 and Rac2 deficiency led to a compensatory elevation of Rac3 activity that was effectively suppressed by NSC23766, as demonstrated by immunoblotting in the Rac1−/−;Rac2−/− erythroblasts and effector-domain pull-down studies. Moreover, NSC23766 inhibited Rac1, Rac2, and Rac3 activities as well as actin polymerization of the erythroblasts. Thus, Rac1, Rac2, and Rac3 have redundant but essential roles in supporting actin dynamics necessary for the nucleus extrusion during the enucleation process. Figure Figure

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RUNX1C-Mediated Reprogramming Of Human Bone Marrow Stromal Cells into Early Blood Progenitors

Blood ◽

10.1182/blood.v122.21.2463.2463 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2463-2463

Author(s):

Weihong Yin ◽

Christopher D Porada ◽

Stephen Walker ◽

Colin Bishop ◽

Graca Almeida-Porada

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Hematopoietic Cells ◽

Dermal Fibroblasts ◽

Hematopoietic Stem ◽

Surface Marker Expression ◽

Somatic Cell Reprogramming ◽

Hematopoietic Lineage

Abstract Somatic cell reprogramming to the hematopoietic lineage, either through a pluripotent state or directly, opens the possibility of production of a ready source of autologous hematopoietic stem cells (HSC) that can be used to treat/cure a wide variety of blood disorders. While it has previously been shown that dermal fibroblasts (HFF) can be directly reprogrammed to the hematopoietic lineage, the efficiency was relatively low and the resultant hematopoietic cells lacked multilineage differentiative potential. Stro1(+) isolated stromal progenitors (SIPs) can easily be isolated from the bone marrow (BM) and expanded ex-vivo to obtain clinically significant numbers of cells. In similarity to HSC, SIPs are derived from the mesoderm, and are intimately linked with HSC specification during ontogeny. As such, they are likely to be epigenetically closer to HSC than HFF, and therefore good candidates for reprogramming into hematopoietic cells. To verify the uniqueness of SIPs for reprogramming, we transduced SIPs and HFF with OCT4 and/or RUNX1C, a master transcription factor (TF) that triggers the developmental onset of definitive hematopoiesis, in the following combinations: 1) OCT4 alone; 2) RUNX1C alone; or 3) OCT4+RUNX1C. We then performed a timeline of gene/cell surface marker expression (using microarray, qRT-PCR, and flow cytometry) from day 3-16 post-transduction. Visual inspection of the cultures showed that, while reprogrammed colonies began to appear in SIPs cultures at day 9, no colonies were seen during this time period in HFF cultures. Flow cytometry and molecular analyses of colonies obtained from OCT4+RUNX1C combination demonstrated that expression of CD41, the earliest marker of commitment to the hematopoietic lineage, commenced within only 3-4 days and peaked at day 5-6, by which time ∼20% of SIPs expressed this marker. This peak in CD41 expression coincided with commencement of expression of CD34 and CD45, and maximal induction of several hematopoiesis-specific TFs and phenotypic markers such as PU.1, HOXB4, GATA2, MIXL, WNT3, KDR, CDX4, which occurred at 1-3 logs higher levels in SIPs than HFF. Further studies demonstrated that the chromatin remodeling function of OCT4 could be replaced with the histone methyltransferase inhibitor Bix-01294, with the combination of RUNX1C and Bix-01294 inducing levels of CD34 and CD41 expression by day 5 that were similar to those achieved with RUNX1C plus OCT4. The present studies thus take several important steps towards making the promise of producing autologous hematopoietic cells for transplantation via direct reprogramming a reality. Disclosures: No relevant conflicts of interest to declare.

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Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Blood ◽

10.1182/blood.v130.suppl_1.81.81 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 81-81

Author(s):

Silvana Di Giandomenico ◽

Pouneh Kermani ◽

Nicole Molle ◽

Mia Yabut ◽

Fabienne Brenet ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Progenitor Cells ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Effector Cells ◽

Erythroid Precursors ◽

Stem And Progenitor Cells

Abstract Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation). Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs. We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo. Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Exploration of a 3D Scaffold Culture System for Ex Vivo Blood Production

Blood ◽

10.1182/blood.v124.21.4371.4371 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4371-4371

Author(s):

Charlotte E Severn ◽

Ashley M Toye

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Peripheral Blood ◽

Ex Vivo ◽

Small Sample ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Adult Peripheral Blood

Abstract The human body produces 2.5 million red blood cells (RBC) every second in the bone marrow, where differentiation of immature erythroblasts to reticulocytes occurs largely within erythroblastic islands. Erythropoiesis can be routinely replicated in 2D liquid culture using hematopoietic stem cells (HSC) isolated from adult peripheral blood. However current 2D culture methodologies eventually exhaust the input of HSC. The use of 3D scaffolds to better mimic erythropoiesis in the bone marrow would increase the RBC yield and longevity of ex vivo cultures, whilst reducing use of exogenous cytokines and minimizing handling requirements. As a starting point we are utilizing a porous collagen coated synthetic polyurethane (PU) scaffold (0.5cm by 0.5cm) for static 3D cultures provided by the Mantalaris group at Imperial College London (Mortera-Blanco et al., 2011). The PU scaffolds were seeded with lineage depleted or CD34+ population isolated from adult peripheral blood and maintained in serum-free erythroid expansion media with SCF, IL-3 and Dexamethasone, alone, or with erythropoietin (EPO) or thrombopoietin (TPO). Advantages of using the lineage depleted population is that it provides a larger diversity of stem cells for establishment of the niche, potentially facilitating the use of rare patient blood samples which may only be available in small sample volumes with low numbers of CD34+ cells. Scaffolds are productive using both cellular inputs, with significant cellular egress for up to 5 weeks regardless of whether exogenous EPO or TPO were included. As anticipated, the highest increase in cell production from the scaffolds was observed using CD34+ in the presence of EPO, which also provided a significant reduction in cell death. Histology and immunofluorescence were used to explore the cell populations within the scaffold. No mature macrophages were detected but GPA+ cells within the scaffold was observed until the end of culture, suggesting that cellular expansion is occurring without establishment of the classical macrophage niche. We also characterized the cells that continually egress from the CD34+ scaffold cultures using flow cytometry. Typically the cellular output exhibited approximately 20-60% CD34 positivity dropping to <15% post day 24 of culture and 10-30% GPA positivity, the remainder of the population was largely CD61 positive, with a contingent of CD14 positive monocytes. Mature cell surface markers for erythroid and megakaryocytic lineages were detected in up to half of the population, when the harvested 3D egress cells were further cultured in erythroid or megakaryocyte 2D culture systems. This suggests a high proportion of the cells that egress from the scaffold are megakaryocyte erythroid progenitors (MEPs) that are consistently expanding within the scaffold environment for the entire culture period. Since CD34+ expansion here has been achieved in the absence of macrophages, we wanted to explore their effects in 2D with the intention of introducing macrophages or macrophage derived functionality into our next generation scaffolds. Importantly, macrophage inclusion significantly increased proliferation of expanding erythroblasts compared to erythroblasts alone. Expansion of CD34+ cells in co-culture gave a statistically significant average fold increase of 528 compared to 301 for the control at day 7 (p = 0.0126 and 0.0162 for days 5 and 7 respectively (n=5)). Flow cytometry at the endpoint of the experiment showed a larger CD34+ population and a reduced GPA+ population when cells are in co-culture, suggesting the CD34+ cells are maintained in a more primitive state for longer. Therefore, co-culture has the additional benefit of improving early erythroblast expansion, alongside the recently reported enhanced expansion of erythroblasts during terminal differentiation (Ramos et al., 2013). In summary, we have demonstrated that a basic static PU scaffold can be utilized to increase hematopoietic stem cell culture longevity and facilitate generation of megakaryocyte or erythroid progenitors with expansion potential. This occurs in the absence of any detectable macrophage niche generation. We have also shown that co-culture with macrophages enhances erythroblast expansion in 2D. Further work is needed to determine whether inclusion of macrophages or macrophage derived proteins in our scaffolds will effectively boost progenitor production. Disclosures No relevant conflicts of interest to declare.

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The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells

Cell Biology International ◽

10.1002/cbin.10483 ◽

2015 ◽

Vol 39 (10) ◽

pp. 1099-1110 ◽

Cited By ~ 4

Author(s):

Iordanis Pelagiadis ◽

Eftichia Stiakaki ◽

Christianna Choulaki ◽

Maria Kalmanti ◽

Helen Dimitriou

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem

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Multiparameter Flow Cytometry Analysis of the Human Spleen Applied to Studies of Plasma-Derived EVs From Plasmodium vivax Patients

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.596104 ◽

2021 ◽

Vol 11 ◽

Author(s):

Melisa Gualdrón-López ◽

Míriam Díaz-Varela ◽

Haruka Toda ◽

Iris Aparici-Herraiz ◽

Laura Pedró-Cos ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Responses ◽

Plasmodium Vivax ◽

Size Exclusion ◽

Multiparameter Flow Cytometry ◽

Spleen Cells ◽

Human Spleen ◽

Hematopoietic Stem ◽

Ethical Implications ◽

Cellular Analysis

The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45+ cells included B (30%), CD4+ T (16%), CD8+ T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria.

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CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽

10.1182/blood-2019-132138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1054-1054 ◽

Cited By ~ 1

Author(s):

Hongxing Liu

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Lymph Nodes ◽

Conflicts Of Interest ◽

Sh2 Domain ◽

Janus Kinase ◽

Gingival Hyperplasia ◽

Hematopoietic Stem ◽

T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

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T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.4580.4580 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4580-4580

Author(s):

Monica M Rivera Franco ◽

Eucario Leon Rodriguez ◽

Diana Gomez Martin ◽

Javier Merayo Chalico ◽

Jorge Alcocer Varela

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Th17 Cells ◽

Mononuclear Cells ◽

Chronic Gvhd ◽

The Other ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

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Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-142963 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Gordon G. L. Wong ◽

Gabriela Krivdova ◽

Olga I. Gan ◽

Jessica L. McLeod ◽

John E. Dick ◽

...

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Myeloid Differentiation ◽

Hematopoietic Stem ◽

Erythroid Precursor ◽

Lineage Differentiation ◽

Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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