RESUMEN
Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.
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Ciclosporina , Nitrilos , Pirazoles , Pirimidinas , Pirazoles/uso terapéutico , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Pirimidinas/uso terapéutico , Animales , Ratones , Ciclosporina/uso terapéutico , Ciclosporina/administración & dosificación , Quimioterapia Combinada , Trastornos de Fallo de la Médula Ósea/tratamiento farmacológico , Modelos Animales de EnfermedadRESUMEN
We previously reported that granulocytic myeloid-derived suppressor cells (G-MDSCs) suppressed T-cell activation and attenuated bone marrow failure (BMF) in a minor histocompatibility (minor-H) antigen mismatched murine aplastic anemia (AA) model. In the current study, we tested the hypothesis that exosomes, a subset of extracellular vesicles, are responsible at least partially for G-MDSCs' therapeutic efficacy. Indeed, exosomes isolated from GMDSCs (G-MDSC-exos) suppressed CD4+ and CD8+ T-cell proliferation in vitro and mildly attenuated immune BMF in the minor-H mismatched AA model. G-MDSC-exos treatment significantly increased red blood cells, hemoglobin, and total bone marrow (BM) cells, and moderately reduced BM CD8+ T cells. G-MDSC-exos' effects were associated with upregulations in an array of lymphocyte-suppression-related miRNAs such as hsa-miR-142-5p, miR-19a-3p, and miR-19b-3p in both BM CD4+ and CD8+ T cells. We concluded that G-MDSC-exos attenuate immune BMF via modulating the delivery of immunosuppressive miRNAs into activated T lymphocytes.
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Exosomas , MicroARNs , Células Supresoras de Origen Mieloide , Pancitopenia , Ratones , Animales , Linfocitos T CD8-positivos , Modelos Animales de Enfermedad , Granulocitos , Inmunosupresores/farmacología , MicroARNs/genética , Trastornos de Fallo de la Médula ÓseaRESUMEN
VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a pleiotropic, severe autoinflammatory disease caused by somatic mutations in the ubiquitin-like modifier activating enzyme 1 (UBA1) gene. To elucidate VEXAS pathophysiology, we performed transcriptome sequencing of single bone marrow mononuclear cells and hematopoietic stem and progenitor cells (HSPCs) from VEXAS patients. HSPCs are biased toward myeloid (granulocytic) differentiation, and against lymphoid differentiation in VEXAS. Activation of multiple inflammatory pathways (interferons and tumor necrosis factor alpha) occurs ontogenically early in primitive hematopoietic cells and particularly in the myeloid lineage in VEXAS, and inflammation is prominent in UBA1-mutated cells. Dysregulation in protein degradation likely leads to higher stress response in VEXAS HSPCs, which positively correlates with inflammation. TCR usage is restricted and there are increased cytotoxicity and IFN-γ signaling in T cells. In VEXAS syndrome, both aberrant inflammation and myeloid predominance appear intrinsic to hematopoietic stem cells mutated in UBA1.
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Células Madre Hematopoyéticas , Inflamación , Humanos , Proteolisis , Diferenciación Celular , Inflamación/genéticaRESUMEN
Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.
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Linfocitos T CD4-Positivos , Linfocitos T Reguladores , Ratones , Animales , Citometría de Flujo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/farmacologíaRESUMEN
Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that originate in the bone marrow (BM) and have immunoregulatory functions. MDSCs have been implicated in the pathogenesis of several autoimmune diseases but have not been investigated in immune aplastic anemia (AA). We examined the roles of granulocytic-MDSCs (G-MDSCs) in murine models of human AA and BM failure (BMF). As both prophylaxis and therapy, BM-derived G-MDSCs improved pancytopenia and BM cellularity and suppressed BM T-cell infiltration in major histocompatibility complex (MHC)-matched C.B10 BMF mice. These effects were not obtained in the MHC-mismatched CByB6F1 AA model, likely because of MHC disparity between G-MDSCs and donor T cells. Single-cell RNA sequencing demonstrated that G-MDSCs downregulated cell cycle-related genes in BM-infiltrated T cells, consistent with suppression of T-cell proliferation by G-MDSCs through reactive oxygen species pathways. Clearance of G-MDSCs in the MHC-mismatched CByB6F1 model using anti-Ly6G antibody facilitated T cell-mediated BM destruction, suggesting an intrinsic immunosuppressive property of G-MDSCs. However, the same anti-Ly6G antibody in the MHC-matched C.B10 AA model mildly mitigated BMF, associated with expansion of an intermediate Ly6G population. Our results demonstrate that G-MDSC eradication and therapeutic efficacy are immune context-dependent.
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Anemia Aplásica , Células Supresoras de Origen Mieloide , Pancitopenia , Humanos , Animales , Ratones , Granulocitos , Células Mieloides , Trastornos de Fallo de la Médula Ósea/metabolismo , Anemia Aplásica/terapiaRESUMEN
Immune aplastic anemia (AA) is a severe blood disease characterized by T-lymphocyte- mediated stem cell destruction. Hematopoietic stem cell transplantation and immunosuppression are effective, but they entail costs and risks, and are not always successful. The Janus kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T-cell activation and inhibits cytokine production in models of graft-versus-host disease. We tested RUX in murine immune AA for potential therapeutic benefit. After infusion of lymph node (LN) cells mismatched at the major histocompatibility complex [C67BL/6 (B6)âCByB6F1], RUX, administered as a food additive (Rux-chow), attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, preserved hematopoietic progenitors, and prevented mortality, when used either prophylactically or therapeutically. RUX suppressed the infiltration, proliferation, and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. Similar effects were obtained when Rux-chow was fed to C.B10 mice in a minor histocompatibility antigen mismatched (B6âC.B10) AA model. RUX only modestly suppressed lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice. Our data support clinical trials of JAK/STAT inhibitors in human AA and other immune bone marrow failure syndromes.
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Anemia Aplásica , Enfermedades de la Médula Ósea , Pancitopenia , Ratones , Humanos , Animales , Pancitopenia/patología , Anemia Aplásica/patología , Trastornos de Fallo de la Médula Ósea/patología , Médula Ósea/patología , Enfermedades de la Médula Ósea/patología , Janus Quinasa 1RESUMEN
(1) Background: Single-cell RNA sequencing (scRNA-seq) data are useful for decoding cell-cell communication. CellCall is a tool that is used to infer inter- and intracellular communication pathways by integrating paired ligand-receptor (L-R) and transcription factor (TF) activities from steady-state data and thus cannot directly handle two-condition comparisons. For tumor and healthy status, it can only individually analyze cells from tumor or healthy tissue and examine L-R pairs only identified in either tumor or healthy controls, but not both together. Furthermore, CellCall is highly affected by gene expression specificity in tissues. (2) Methods: CellCallEXT is an extension of CellCall that deconvolutes intercellular communication and related internal regulatory signals based on scRNA-seq. Information on Reactome was retrieved and integrated with prior knowledge of L-R-TF signaling and gene regulation datasets of CellCall. (3) Results: CellCallEXT was successfully applied to examine tumors and immune cell microenvironments and to identify the altered L-R pairs and downstream gene regulatory networks among immune cells. Application of CellCallEXT to scRNA-seq data from patients with deficiency of adenosine deaminase 2 demonstrated its ability to impute dysfunctional intercellular communication and related transcriptional factor activities. (4) Conclusions: CellCallEXT provides a practical tool to examine intercellular communication in disease based on scRNA-seq data.
RESUMEN
In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.
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Anemia Aplásica , Pancitopenia , Adulto , Autoanticuerpos , Biomarcadores , Ciclooxigenasa 2 , Cadenas HLA-DRB1 , HumanosRESUMEN
T-cell large granular lymphocyte leukemia (T-LGLL) is a lymphoproliferative disease and bone marrow failure syndrome which responds to immunosuppressive therapies. We show single-cell TCR coupled with RNA sequencing of CD3+ T cells from 13 patients, sampled before and after alemtuzumab treatments. Effector memory T cells and loss of T cell receptor (TCR) repertoire diversity are prevalent in T-LGLL. Shared TCRA and TCRB clonotypes are absent. Deregulation of cell survival and apoptosis gene programs, and marked downregulation of apoptosis genes in CD8+ clones, are prominent features of T-LGLL cells. Apoptosis genes are upregulated after alemtuzumab treatment, especially in responders than non-responders; baseline expression levels of apoptosis genes are predictive of hematologic response. Alemtuzumab does not attenuate TCR clonality, and TCR diversity is further skewed after treatment. Inferences made from analysis of single cell data inform understanding of the pathophysiologic mechanisms of clonal expansion and persistence in T-LGLL.
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Leucemia Linfocítica Granular Grande , Alemtuzumab/uso terapéutico , Células Clonales , Humanos , Leucemia Linfocítica Granular Grande/genética , Receptores de Antígenos de Linfocitos T/genética , Análisis de Secuencia de ARNRESUMEN
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.
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Transducción de Señal , Sirolimus , Animales , Linfocitos B/metabolismo , Diferenciación Celular , Activación de Linfocitos , Ratones , Ratones Noqueados , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Exposure of young C57BL/6 (B6) mice to two courses of busulfan (BSF) injections or two rounds of sublethal total-body irradiation (TBI) induced significant damage to the function of hematopoietic stem and progenitor cells (HSPCs). Fifteen weeks after treatment, BSF- and TBI-treated mice had reduced white blood cells without significant change in red blood cells or platelets, indicating that BSF and TBI hematotoxicity was chronic, with leukocytes being the major targets. Hematopoietic damage induced by BSF or TBI persisted long term. Residual adverse effects were reflected by significantly decreased CD45R B cells and reduced recovery of total bone marrow cells, especially HSPCs carrying markers for KSL (Kit+Sca-1+Lin-) cells, multipotent progenitor (MPP) cells (KSLCD34+CD135+), myeloid progenitor (MP) cells (Kit+Sca-1-Lin-), and common lymphoid progenitor (CLP) cells 62 wk posttreatment. Transplantation of bone marrow (BM) cells from BSF and TBI donors at 49 weeks after treatment into lethally irradiated hosts resulted in decreased engraftment of CD45R B cells in blood and reduced reconstitution of BM HSPCs including KSL cells, short-term hematopoietic stem cells (KSLCD34+CD135-), MPP cells, and MP cell subsets. TBI donor had better reconstitution of CLP cells in recipients posttransplantation than did BSF donor, suggesting an impact of TBI and BSF on B cells at different development stages. In summary, BSF and TBI exposure produced long-lasting adverse effects on hematopoiesis with pronounced effects on mature B cells, immature ST-HSCs, and hematopoietic progenitor cells. Our results may have implications for therapy of human diseases.
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Busulfano/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Agonistas Mieloablativos/farmacología , Animales , Células de la Médula Ósea , Trasplante de Médula Ósea , Femenino , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Irradiación Corporal TotalRESUMEN
Deficiency of adenosine deaminase 2 (DADA2) is a monogenic vasculitis syndrome caused by autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1). Vasculitis, vasculopathy, and inflammation are dominant clinical features of this disease; the spectrum of manifestations includes immunodeficiency and lymphoproliferation as well as hematologic manifestations. ADA2 is primarily secreted by stimulated monocytes and macrophages. Aberrant monocyte differentiation to macrophages and neutrophils are important in the pathogenesis of DADA2, but little is known about T lymphocytes in this disease. We performed combined single-cell RNA sequencing and single-cell TCR sequencing in order to profile T cell repertoires in 10 patients with DADA2. Although there were no significant alterations of T cell subsets, we observed activation of both CD8+ and CD4+ T cells. There was no clonal expansion of T cells: most TCRs were expressed at basal levels in patients and healthy donors. TCR usage was private to individual patients and not disease specific, indicating as unlikely a common pathogenic background or predisposition to a common pathogen. We recognized activation of IFN pathways as a signature of T cells and STAT1 as a hub gene in the gene network of T cell activation and cytotoxicity. Overall, T cells in DADA2 patients showed distinct cell-cell interactions with monocytes, as compared with healthy donors, and many of these ligand-receptor interactions likely drove up-regulation of STAT1 in both T cells and other immune cells in patients. Our analysis reveals previously undercharacterized cell characteristics in DADA2.
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Adenosina Desaminasa/deficiencia , Biomarcadores/metabolismo , Regulación de la Expresión Génica , Síndromes de Inmunodeficiencia/patología , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Enfermedades de la Piel/patología , Linfocitos T/patología , Enfermedades Vasculares/patología , Adenosina Desaminasa/genética , Adolescente , Adulto , Estudios de Casos y Controles , Células Cultivadas , Niño , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Factor de Transcripción STAT1/genética , Análisis de la Célula Individual , Enfermedades de la Piel/genética , Enfermedades de la Piel/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Enfermedades Vasculares/genética , Enfermedades Vasculares/inmunología , Adulto JovenRESUMEN
Pretreatment blood counts, particularly an absolute reticulocyte count ≥25×109/L, correlate with response to immunosuppressive therapy in severe aplastic anemia. In recent trials, eltrombopag combined with standard immunosuppressive therapy yielded superior responses than those to immunosuppressive therapy alone. Our single institution retrospective study aimed to elucidate whether historical predictors of response to immunosuppressive therapy alone were also associated with response to immunosuppressive therapy plus eltrombopag. We sought correlations of blood counts, thrombopoietin levels and the presence of paroxysmal nocturnal hemoglobinuria clones with both overall and complete responses in 416 patients with severe aplastic anemia, aged 2-82 years (median, 30 years), initially treated with immunosuppressive therapy plus eltrombopag between 2012 and 2019 (n=176) or with immunosuppressive therapy alone between 1999 and 2010 (n=240). Compared to non-responders, patients in the group of overall responders to immunosuppressive therapy plus eltrombopag had significantly higher pretreatment absolute reticulocyte counts, higher neutrophil counts and reduced thrombopoietin levels, as also observed for the group treated with immunosuppressive therapy alone. Addition of eltrombopag markedly improved the overall response in subjects with an absolute reticulocyte count between 10-30×109/L from 60% (54 of 90) to 91% (62 of 68). Absolute lymphocyte count correlated with complete response in the groups treated with immunosuppressive therapy with or without eltrombopag, especially in adolescents aged ≥10 years and adults, but the correlation was reversed in younger children. Platelet count and the presence of a paroxysmal nocturnal hemoglobinuria clone did not correlate with responses to immunosuppressive therapy. Blood counts remain the best predictors of response to nontransplant therapies in severe aplastic anemia. Addition of eltrombopag to immunosuppressive therapy shifted patients with a lower absolute reticulocyte count into a better prognostic category.
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Anemia Aplásica , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia Aplásica/diagnóstico , Anemia Aplásica/tratamiento farmacológico , Benzoatos/uso terapéutico , Niño , Preescolar , Humanos , Hidrazinas/uso terapéutico , Terapia de Inmunosupresión , Inmunosupresores/uso terapéutico , Persona de Mediana Edad , Pirazoles , Estudios Retrospectivos , Adulto JovenRESUMEN
Immune aplastic anemia (AA) features somatic loss of HLA class I allele expression on bone marrow cells, consistent with a mechanism of escape from T-cell-mediated destruction of hematopoietic stem and progenitor cells. The clinical significance of HLA abnormalities has not been well characterized. We examined the somatic loss of HLA class I alleles and correlated HLA loss and mutation-associated HLA genotypes with clinical presentation and outcomes after immunosuppressive therapy in 544 AA patients. HLA class I allele loss was detected in 92 (22%) of the 412 patients tested, in whom there were 393 somatic HLA gene mutations and 40 instances of loss of heterozygosity. Most frequently affected was HLA-B*14:02, followed by HLA-A*02:01, HLA-B*40:02, HLA-B*08:01, and HLA-B*07:02. HLA-B*14:02, HLA-B*40:02, and HLA-B*07:02 were also overrepresented in AA. High-risk clonal evolution was correlated with HLA loss, HLA-B*14:02 genotype, and older age, which yielded a valid prediction model. In 2 patients, we traced monosomy 7 clonal evolution from preexisting clones harboring somatic mutations in HLA-A*02:01 and HLA-B*40:02. Loss of HLA-B*40:02 correlated with higher blood counts. HLA-B*07:02 and HLA-B*40:01 genotypes and their loss correlated with late-onset of AA. Our results suggest the presence of specific immune mechanisms of molecular pathogenesis with clinical implications. HLA genotyping and screening for HLA loss may be of value in the management of immune AA. This study was registered at clinicaltrials.gov as NCT00001964, NCT00061360, NCT00195624, NCT00260689, NCT00944749, NCT01193283, and NCT01623167.
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Anemia Aplásica/genética , Genes MHC Clase I , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Mutación , Adolescente , Adulto , Alelos , Anemia Aplásica/inmunología , Evolución Clonal , Femenino , Eliminación de Gen , Expresión Génica , Antígenos HLA-A/inmunología , Antígenos HLA-B/inmunología , Humanos , Inmunidad , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
(1) Background: mouse models are fundamental to the study of hematopoiesis, but comparisons between mouse and human in single cells have been limited in depth. (2) Methods: we constructed a single-cell resolution transcriptomic atlas of hematopoietic stem and progenitor cells (HSPCs) of human and mouse, from a total of 32,805 single cells. We used Monocle to examine the trajectories of hematopoietic differentiation, and SCENIC to analyze gene networks underlying hematopoiesis. (3) Results: After alignment with Seurat 2, the cells of mouse and human could be separated by same cell type categories. Cells were grouped into 17 subpopulations; cluster-specific genes were species-conserved and shared functional themes. The clustering dendrogram indicated that cell types were highly conserved between human and mouse. A visualization of the Monocle results provided an intuitive representation of HSPC differentiation to three dominant branches (Erythroid/megakaryocytic, Myeloid, and Lymphoid), derived directly from the hematopoietic stem cell and the long-term hematopoietic stem cells in both human and mouse. Gene regulation was similarly conserved, reflected by comparable transcriptional factors and regulatory sequence motifs in subpopulations of cells. (4) Conclusions: our analysis has confirmed evolutionary conservation in the hematopoietic systems of mouse and human, extending to cell types, gene expression and regulatory elements.
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Hematopoyesis , Células Madre Hematopoyéticas , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Linaje de la Célula , Evolución Molecular , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , RatonesRESUMEN
Immune aplastic anemia (AA) results from T cell attack on hematopoietic cells, resulting in bone marrow hypocellularity and pancytopenia. Animal models have been successfully developed to study pathophysiological mechanisms in AA. While we have systemically defined the critical components of the adaptive immune response in the pathogenesis of immune marrow failure using this model, the role of innate immunity has not been fully investigated. Here, we demonstrate that lymph node (LN) cells from B6-based donor mice carrying IL-6, TLR2, or TLR4 gene deletions were fully functional in inducing severe pancytopenia and bone marrow failure (BMF) when infused into MHC-mismatched CByB6F1 recipients. Conversely, B6-based recipient mice with IL-6, TLR2, and TLR4 deletion backgrounds were all susceptible to immune-mediated BMF relative to wild-type B6 recipients following infusion of MHC-mismatched LN cells from FVB donors, but the disease appeared more severe in IL-6 deficient mice. We conclude that IL-6, TLR2, and TLR4, molecular elements important in maintenance of normal innate immunity, have limited roles in a murine model of immune-mediated BMF. Rather, adaptive immunity appears to be the major contributor to the animal disease.
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Trastornos de Fallo de la Médula Ósea/inmunología , Inmunidad Innata , Interleucina-6/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Animales , Trastornos de Fallo de la Médula Ósea/genética , Trastornos de Fallo de la Médula Ósea/patología , Modelos Animales de Enfermedad , Interleucina-6/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genéticaRESUMEN
The role of mammalian target of rapamycin and its suppressor sirolimus in the regulation of hematopoietic stem and progenitor cells (HSPCs) is controversial. We show here that sirolimus enhanced regeneration of HSPCs in mice exposed to sublethal total body irradiation (TBI) and other regenerative stressors. Sorted Lin- CD150+ bone marrow cells from sirolimus-treated TBI mice had increased expression of c-Kit and other hematopoietic genes. HSPCs from sirolimus-treated TBI mice were functionally competent when tested by competitive engraftment in vivo. Postradiation regeneration of HSPCs in mice treated with sirolimus was accompanied by decreased γ-H2AX levels detected by flow cytometry and increased expression of DNA repair genes by quantitative polymerase chain reaction. Reduction of cell death and DNA damage post-radiation by sirolimus was associated with enhanced clearance of cellular reactive oxygen species (ROS) in HSPCs. Increased HSPC recovery with sirolimus was also observed in mice injected with hematoxic agents, busulfan and 5-fluorouracil. In contrast, sirolimus showed no effect on HSPCs in normal mice at steady state, but stimulated HSPC expansion in mice carrying the Wv mutation at the c-Kit locus. In human to mouse xenotransplantation, sirolimus enhanced engraftment of irradiated human CD34+ cells. In summary, our results are consistent with sirolimus' acceleration of HSPC recovery in response to hematopoietic stress, associated with reduced DNA damage and ROS. Sirolimus might have clinical application for the treatment and prevention of hematopoietic injury.
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Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Inmunosupresores/farmacología , Sirolimus/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Fluorouracilo/toxicidad , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/efectos de los fármacos , Células Madre/fisiología , Células Madre/efectos de la radiación , Irradiación Corporal Total/efectos adversosRESUMEN
Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by cytopenia and a propensity to develop acute myeloid leukemia (AML). The management of lower-risk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag (EPAG), a thrombopoietin receptor agonist, can improve platelet counts in LR-MDS and tri-lineage hematopoiesis in aplastic anemia (AA). We conducted a phase 2 dose modification study to investigate the safety and efficacy of EPAG in LR-MDS. EPAG dose was escalated from 50 mg/day, to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a PNH clone, marrow hypocellularity, thrombocytopenia with or without other cytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with previous EPAG studies in AA; no patients discontinued drug due to adverse events. Three patients developed reversible grade-3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued EPAG after achieving a robust response (median time 16 months); four of them reinitiated EPAG due to declining counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to AML on study. In conclusion, EPAG was well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.