Publications by authors named "Sachiko Kajigaya"

74 Publications

Comprehensive network modeling from single cell RNA sequencing of human and mouse reveals well conserved transcription regulation of hematopoiesis.

BMC Genomics 2020 Dec 29;21(Suppl 11):849. Epub 2020 Dec 29.

Hematopoiesis and Bone Marrow Failure Laboratory, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, 20892, USA.

Background: Presently, there is no comprehensive analysis of the transcription regulation network in hematopoiesis. Comparison of networks arising from gene co-expression across species can facilitate an understanding of the conservation of functional gene modules in hematopoiesis.

Results: We used single-cell RNA sequencing to profile bone marrow from human and mouse, and inferred transcription regulatory networks in each species in order to characterize transcriptional programs governing hematopoietic stem cell differentiation. We designed an algorithm for network reconstruction to conduct comparative transcriptomic analysis of hematopoietic gene co-expression and transcription regulation in human and mouse bone marrow cells. Co-expression network connectivity of hematopoiesis-related genes was found to be well conserved between mouse and human. The co-expression network showed "small-world" and "scale-free" architecture. The gene regulatory network formed a hierarchical structure, and hematopoiesis transcription factors localized to the hierarchy's middle level.

Conclusions: Transcriptional regulatory networks are well conserved between human and mouse. The hierarchical organization of transcription factors may provide insights into hematopoietic cell lineage commitment, and to signal processing, cell survival and disease initiation.
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http://dx.doi.org/10.1186/s12864-020-07241-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771096PMC
December 2020

Eltrombopag monotherapy can improve hematopoiesis in patients with low to intermediate risk-1 myelodysplastic syndrome.

Haematologica 2020 12 1;105(12):2785-2794. Epub 2020 Dec 1.

National Heart, Lung, and Blood Institute.

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.
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http://dx.doi.org/10.3324/haematol.2020.249995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716353PMC
December 2020

Comprehensive analysis of single-cell RNA sequencing data from healthy human marrow hematopoietic cells.

BMC Res Notes 2020 Nov 10;13(1):514. Epub 2020 Nov 10.

Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Objective: Single cell methodology enables detection and quantification of transcriptional changes and unravelling dynamic aspects of the transcriptional heterogeneity not accessible using bulk sequencing approaches. We have applied single-cell RNA-sequencing (scRNA-seq) to fresh human bone marrow CD34 cells and profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from healthy donors to characterize lineage- and stage-specific transcription during hematopoiesis.

Results: Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations based on lineage specific genes. Reconstruction of differentiation trajectories in single cells revealed four committed lineages derived from HSCs, as well as dynamic expression changes underlying cell fate during early erythroid-megakaryocytic, lymphoid, and granulocyte-monocyte differentiation. A similar non-hierarchical pattern of hematopoiesis could be derived from analysis of published single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), consistent with a sequential relationship between chromatin dynamics and regulation of gene expression during lineage commitment (first, altered chromatin conformation, then mRNA transcription). Computationally, we have reconstructed molecular trajectories connecting HSCs directly to four hematopoietic lineages. Integration of long noncoding RNA (lncRNA) expression from the same cells demonstrated mRNA transcriptome, lncRNA, and the epigenome were highly homologous in their pattern of gene activation and suppression during hematopoietic cell differentiation.
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http://dx.doi.org/10.1186/s13104-020-05357-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653854PMC
November 2020

Sequencing of RNA in single cells reveals a distinct transcriptome signature of hematopoiesis in GATA2 deficiency.

Blood Adv 2020 06;4(12):2656-2670

Hematology Branch, National Heart, Lung, and Blood Institute.

Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes, including systemic infections, lymphedema, cytopenias, and myeloid neoplasms. Genotype-phenotype correlation is not well understood mechanistically in GATA2 deficiency. We performed whole transcriptome sequencing of single hematopoietic stem and progenitor cells from 8 patients, who had pathogenic GATA2 mutations and myelodysplasia. Mapping patients' cells onto normal hematopoiesis, we observed deficiency in lymphoid/myeloid progenitors, also evident from highly constrained gene correlations. HSPCs of patients exhibited distinct patterns of gene expression and coexpression compared with counterparts from healthy donors. Distinct lineages showed differently altered transcriptional profiles. Stem cells in patients had dysregulated gene expression related to apoptosis, cell cycle, and quiescence; increased expression of erythroid/megakaryocytic priming genes; and decreased lymphoid priming genes. The prominent deficiency in lympho-myeloid lineages in GATA2 deficiency appeared at least partly due to the expression of aberrant gene programs in stem cells prior to lineage commitment. We computationally imputed cells with chromosomal abnormalities and determined their gene expression; DNA repair genes were downregulated in trisomy 8 cells, potentially rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetic abnormalities showed defects in genes related to multilineage differentiation and cell cycle. Single-cell RNA sequencing is powerful in resolving transcriptomes of cell subpopulations despite a paucity of cells in marrow failure. Our study discloses previously uncharacterized transcriptome signatures of stem cells and progenitors in GATA2 deficiency, providing a broad perspective of potential mechanisms by which germline mutations modulate early hematopoiesis in a human disease. This trial was registered at www.clinicaltrials.gov as NCT01905826, NCT01861106, and NCT00001620.
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http://dx.doi.org/10.1182/bloodadvances.2019001352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322959PMC
June 2020

Deficit of circulating CD19 CD24 CD38 regulatory B cells in severe aplastic anaemia.

Br J Haematol 2020 08 20;190(4):610-617. Epub 2020 Apr 20.

Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA.

Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24 CD38 regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL-10). We investigated circulating B-cell subpopulations, including CD24 CD38 Bregs, as well as total B cells, CD4 T cells, CD8 T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24 CD38 Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL-10; total B-cell counts and the other B-cell subpopulations were similar to those of healthy individuals. CD24 CD38 Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B-cell counts were unexpectedly associated with IST response. Markedly reduced CD24 CD38 Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.
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http://dx.doi.org/10.1111/bjh.16651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496711PMC
August 2020

High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis.

J Immunol Methods 2020 02 11;477:112667. Epub 2019 Nov 11.

Translational Immunology Section, Office of Science Technology (OST), National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Fluorescent cell barcoding (FCB) is a multiplexing technique for high-throughput flow cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique because of inter-operator variability and differences in data analysis. FCB was implemented by combining two-dye barcoding (DyLight 350 plus Pacific Orange) with five-color surface marker antibody and intracellular staining for phosphoprotein signaling analysis. We proposed a robust method to measure intra- and inter-assay variability of FCB in T/B cells and monocytes by combining range and ratio of variability to standard statistical analyses. Data analysis was carried out by conventional and semi-automated workflows and built with R software. Results obtained from both analyses were compared to assess feasibility and reproducibility of FCB data analysis by machine-learning methods. Our results showed efficient FCB using DyLight 350 and Pacific Orange at concentrations of 0, 15 or 30, and 250 μg/mL, and a high reproducibility of FCB in combination with surface marker and intracellular antibodies. Inter-operator variability was minimized by adding an internal control bridged across matrices used as rejection criterion if significant differences were present between runs. Computational workflows showed comparable results to conventional gating strategies. FCB can be used to study phosphoprotein signaling in T/B cells and monocytes with high reproducibility across operators, and the addition of bridge internal controls can further minimize inter-operator variability. This FCB protocol, which has high throughput analysis and low intra- and inter-assay variability, can be a powerful tool for clinical trial studies. Moreover, FCB data can be reliably analyzed using computational software.
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http://dx.doi.org/10.1016/j.jim.2019.112667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981073PMC
February 2020

A novel homozygous RTEL1 variant in a consanguineous Lebanese family: phenotypic heterogeneity and disease anticipation.

Hum Genet 2019 Dec 1;138(11-12):1323-1330. Epub 2019 Nov 1.

LAU Gilbert and Rose-Marie Chagoury School of Medicine, LAUMC/RH, Zahar Street, Achrafieh, Beirut, 1110, Lebanon.

Phenotypic heterogeneity is often observed in patients with telomeropathies caused by pathogenic variants in telomere biology genes. However, the roles of recessive variants in these different phenotypes are not fully characterized. Our goal is to describe the biological roles of a novel homozygous RTEL1 variant identified in a consanguineous Lebanese family with unusual presentation of telomeropathies. A proband was screened for germline variants in telomere biology genes by whole exome sequencing. Leukocytes' telomere length was measured in the proband and eight relatives. We identified a novel homozygous p.E665K RTEL1 variant in the proband, his mother, and seven siblings that associated with telomere shortening and a broad spectrum of clinical manifestations, ranging from mild unspecific findings to severe phenotypes. Consanguinity in at least three family generations led to increased frequency of the homozygous p.E665K variant in the youngest generation and progressive telomere shortening. The increased frequency of the homozygous RTEL1 variant due to consanguinity in this Lebanese family allowed us to infer novel behaviors of recessive RTEL1 variants, as the expressivity and penetrance of this gene are very heterogenous between inter- and intra-generations. Progressive telomere shortening was associated with disease anticipation, first reported in recessive autosomal telomeropathies. Both genetic testing and telomere length measurement were critical for the clinical diagnosis of this family with telomere diseases marked by phenotypic heterogeneity.
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http://dx.doi.org/10.1007/s00439-019-02076-8DOI Listing
December 2019

Long noncoding RNAs of single hematopoietic stem and progenitor cells in healthy and dysplastic human bone marrow.

Haematologica 2019 05 13;104(5):894-906. Epub 2018 Dec 13.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health.

Long noncoding RNAs (lncRNAs) are regulators of cell differentiation and development. The lncRNA transcriptome in human hematopoietic stem and progenitor cells is not comprehensively defined. We investigated lncRNAs in 979 human bone marrow-derived CD34 cells by single cell RNA sequencing followed by transcriptome reconstruction. We identified 3,173 lncRNAs in total, among which 2,365 were previously unknown, and we characterized lncRNA stem, differentiation, and maturation signatures. lncRNA expression exhibited high cell-to-cell variation, which was only apparent in single cell analysis. lncRNA expression followed a lineage-specific and highly dynamic pattern during early hematopoiesis. lncRNAs in hematopoietic cells closely correlated with protein-coding genes of known functions in the regulation of hematopoiesis and cell fate decisions, and the potential regulatory roles of lncRNAs in hematopoiesis were imputed by projection from protein-coding genes with a "guilt-by-association" approach. We characterized lncRNAs preferentially expressed in hematopoietic stem cells and in various downstream differentiated lineage progenitors. We also profiled lncRNA expression in single cells from patients with myelodysplastic syndromes and in aneuploid cells in particular. Our study provides a global view of lncRNAs in human hematopoietic stem and progenitor cells. We observed a highly ordered pattern of lncRNA expression and participation in regulation of early hematopoiesis, and coordinate aberrant messenger RNA and lncRNA transcriptomes in dysplastic hematopoiesis. ().
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http://dx.doi.org/10.3324/haematol.2018.208926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518886PMC
May 2019

Pathogenic TERT promoter variants in telomere diseases.

Genet Med 2019 07 7;21(7):1594-1602. Epub 2018 Dec 7.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA.

Purpose: The acquisition of pathogenic variants in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes.

Methods: We screened patients with a broad spectrum of telomeropathies (n = 136), their relatives (n = 52), and controls (n = 195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay.

Results: Pathogenic -124 and -146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging.

Conclusion: We extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.
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http://dx.doi.org/10.1038/s41436-018-0385-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555700PMC
July 2019

Epidemiological, clinical and genetic characterization of aplastic anemia patients in Pakistan.

Ann Hematol 2019 Feb 13;98(2):301-312. Epub 2018 Nov 13.

Department of Biochemistry, Quaid-i-Azam University, Islamabad, 44000, Pakistan.

Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.
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http://dx.doi.org/10.1007/s00277-018-3542-zDOI Listing
February 2019

Interleukin-18 plays a dispensable role in murine and likely also human bone marrow failure.

Exp Hematol 2019 01 12;69:54-64.e2. Epub 2018 Oct 12.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Interleukin-18 (IL-18), also known as interferon-gamma (IFN-γ)-inducing factor, is involved in Th1 responses and regulation of immunity. Accumulating evidence implicates IL-18 in autoimmune diseases, but little is known of its role in acquired aplastic anemia (AA), the immune-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). IL-18 protein levels were significantly elevated in sera of severe AA (SAA) patients, including both responders and nonresponders assayed before treatment, and decreased after treatment. IL-18 receptor (IL-18R) was expressed on HSPCs. Co-culture of human BM CD34 cells from healthy donors with IL-18 upregulated genes in the helper T-cell and Notch signaling pathways and downregulated genes in the cell cycle regulation, telomerase, and IL-6 signaling pathways. Plasma IL-18 levels were also elevated in murine models of immune-mediated BM failure. However, deletion of IL-18 in donor lymph node cells or deletions of either IL-18 or IL-18R in recipients did not attenuate elevations of circulating IFN-γ, tumor necrosis factor-alpha, or IL-6, nor did they alleviate BM failure. In summary, our findings suggest that, although increased circulating IL-18 is a feature of SAA, it may reflect an aberrant immune response but be dispensable to the pathogenesis of AA.
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http://dx.doi.org/10.1016/j.exphem.2018.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309608PMC
January 2019

Aptamer-based proteomics of serum and plasma in acquired aplastic anemia.

Exp Hematol 2018 12 9;68:38-50. Epub 2018 Oct 9.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health (NIH), Bethesda, MD, USA.

Single-stranded oligonucleotides containing deoxyuridine are aptamers (SOMAmers) that can bind proteins with high specificity and affinity and slow dissociation rates. SOMAscan, an aptamer-based proteomic technology, allows measurement of more than 1,300 proteins simultaneously for the identification of new disease biomarkers. The aim of the present study was to identify new serum and plasma protein markers for diagnosis of acquired aplastic anemia (AA) and response to immunosuppressive therapies (IST). SOMAscan was used to screen 1,141 serum proteins in 28 AA patients before and after therapy and 1,317 plasma proteins in seven SAA patients treated with standard IST and a thrombopoietin receptor agonist. From our analysis, 19 serum and 28 plasma proteins were identified as possible candidate diagnostic and prognostic markers. A custom immunobead-based multiplex assay with five selected serum proteins (BMP-10, CCL17, DKK1, HGF, and SELL) was used for validation in a verification set (n = 65) of samples obtained before and after IST and in a blinded validation cohort at baseline (n = 16). After technical validation, four biomarkers were employed to predict diagnosis (accuracy, 88%) and long-term response to IST (accuracy, 79%). In conclusion, SOMAscan is a powerful tool for the identification of new biomarkers. We propose further larger studies to validate new candidate serum and plasma diagnostic and prognostic markers of AA.
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http://dx.doi.org/10.1016/j.exphem.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748047PMC
December 2018

Circulating S100A8 and S100A9 protein levels in plasma of patients with acquired aplastic anemia and myelodysplastic syndromes.

Cytokine 2019 01 27;113:462-465. Epub 2018 Jun 27.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD, USA.

The alarmin family members S100A8 and S100A9 are acute phase inflammation proteins, but they also have been proposed as biomarkers in many malignant and non-malignant diseases. In this study, circulating S100A8 and S100A9 homodimers and S100A8/A9 heterodimers in plasma were systematically investigated by ELISA in aplastic anemia (AA) and myelodysplastic syndromes (MDS). Plasma was obtained from 58 severe AA (SAA) and 30 MDS patients, and from 47 age- and sex-matched healthy donors. In 40 out of the 58 AA subjects, S100A protein levels were measured before and 6 months after immunosuppressive therapy (IST). No differences were observed in AA patients at diagnosis compared to healthy controls for circulating S100A homodimers and heterodimers. After therapy, SAA-responders showed significantly increased circulating S100A8. Non-responding patients had significantly higher levels of circulating S100A8/A9 compared to responders and healthy controls, but without variations of S100A8 and S100A9 homodimers. In MDS patients, circulating S100A8 was significantly elevated compared to those of AA and/or healthy controls. By Pearson correlation analysis of protein levels and blood counts, multiple correlations were found. However, as S100A8 and S100A9 are abundantly present in white blood cells and platelets, correlations with blood counts likely mirror the higher number of cells in the blood of some patients. In conclusion, our findings indicate that circulating S100A8 is increased in MDS but not in AA, and that may be useful to distinguish these diseases in the differential diagnosis of bone marrow failure syndromes. Clinicaltrials.gov identifiers: NCT00260689, NCT00604201, NCT01328587, NCT01623167, NCT00001620, NCT00001397.
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http://dx.doi.org/10.1016/j.cyto.2018.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336387PMC
January 2019

Circulating exosomal microRNAs in acquired aplastic anemia and myelodysplastic syndromes.

Haematologica 2018 07 19;103(7):1150-1159. Epub 2018 Apr 19.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD, USA.

Exosomal microRNAs modulate cancer cell metabolism and the immune response. Specific exosomal microRNAs have been reported to be reliable biomarkers of several solid and hematologic malignancies. We examined the possible diagnostic and prognostic values of exosomal microRNAs in two human bone marrow failure diseases: aplastic anemia and myelodysplastic syndromes. After screening 372 microRNAs in a discovery set (n=42) of plasma exosome samples, we constructed a customized PCR plate, including 42 microRNAs, for validation in a larger cohort (n=99). We identified 25 differentially expressed exosomal microRNAs uniquely or frequently present in aplastic anemia and/or myelodysplastic syndromes. These microRNAs could be related to intracellular functions, such as metabolism, cell survival, and proliferation. Clinical parameters and progression-free survival were correlated to microRNA expression levels in aplastic anemia and myelodysplastic syndrome patients before and after six months of immunosuppressive therapy. One microRNA, mir-126-5p, was negatively correlated with a response to therapy in aplastic anemia: patients with higher relative expression of miR-126-5p at diagnosis had the shortest progression-free survival compared to those with lower or normal levels. Our findings suggest utility of exosomal microRNAs in the differential diagnosis of bone marrow failure syndromes. (Registered at ).
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http://dx.doi.org/10.3324/haematol.2017.182824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029526PMC
July 2018

CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation.

Int J Oncol 2018 Apr 28;52(4):1209-1223. Epub 2018 Feb 28.

Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1202, USA.

Additional sex combs-like 1 (ASXL1) is a well‑known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis.
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http://dx.doi.org/10.3892/ijo.2018.4290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843401PMC
April 2018

PD-1 deficiency augments bone marrow failure in a minor-histocompatibility antigen mismatch lymphocyte infusion model.

Exp Hematol 2018 06 7;62:17-23. Epub 2018 Mar 7.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health Bethesda, MD, USA.

Although PD-1 blockade has revolutionized cancer immunotherapy, immune-related adverse events (irAEs) present life-threatening complications. Recent reports of aplastic anemia (AA) as irAEs implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche. Infusion of PD-1-deficient (PD-1 knockout [KO]) lymph node (LN) cells into minor-antigen mismatched mice resulted in early mortality, as well as more severe bone marrow (BM) hypoplasia, anemia, and BM microarchitecture disruption in PD-1 KO LN-infused mice relative to mice that received B6 LN cell infusion. Mice that received PD-1 KO LN cells had more CD8 T-cell infiltration of the BM and greater expansion of H60-specific CD8 T cells than did their B6 LN-infused counterparts. In the spleen, CD8 T cells were skewed to an effector memory phenotype, suggesting accelerated differentiation of PD-1 KO T cells. Our data suggest that PD-1 dysregulation has a role in murine BM failure and vigilance in irAE monitoring may be desirable to treat early AA and related cytopenias.
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http://dx.doi.org/10.1016/j.exphem.2018.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962409PMC
June 2018

Heterozygous variants in bone marrow failure and myeloid neoplasms.

Blood Adv 2018 01 4;2(1):36-48. Epub 2018 Jan 4.

Department of Haematological Medicine, King's College Hospital, London, United Kingdom.

Biallelic germline mutations in (regulator of telomere elongation helicase 1) result in pathologic telomere erosion and cause dyskeratosis congenita. However, the role of mutations in other bone marrow failure (BMF) syndromes and myeloid neoplasms, and the contribution of monoallelic mutations to disease development are not well defined. We screened 516 patients for germline mutations in telomere-associated genes by next-generation sequencing in 2 independent cohorts; one constituting unselected patients with idiopathic BMF, unexplained cytopenia, or myeloid neoplasms (n = 457) and a second cohort comprising selected patients on the basis of the suspicion of constitutional/familial BMF (n = 59). Twenty-three variants were identified in 27 unrelated patients from both cohorts: 7 variants were likely pathogenic, 13 were of uncertain significance, and 3 were likely benign. Likely pathogenic variants were identified in 9 unrelated patients (7 heterozygous and 2 biallelic). Most patients were suspected to have constitutional BMF, which included aplastic anemia (AA), unexplained cytopenia, hypoplastic myelodysplastic syndrome, and macrocytosis with hypocellular bone marrow. In the other 18 patients, variants were likely benign or of uncertain significance. Telomeres were short in 21 patients (78%), and 3' telomeric overhangs were significantly eroded in 4. In summary, heterozygous variants were associated with marrow failure, and telomere length measurement alone may not identify patients with telomere dysfunction carrying variants. Pathogenicity assessment of heterozygous variants relied on a combination of clinical, computational, and functional data required to avoid misinterpretation of common variants.
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http://dx.doi.org/10.1182/bloodadvances.2017008110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761623PMC
January 2018

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.

Blood Cells Mol Dis 2018 03 4;69:10-22. Epub 2018 Jan 4.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892-1202, USA.

DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.
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http://dx.doi.org/10.1016/j.bcmd.2017.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728079PMC
March 2018

Single-cell RNA-seq reveals a distinct transcriptome signature of aneuploid hematopoietic cells.

Blood 2017 12 13;130(25):2762-2773. Epub 2017 Oct 13.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

Cancer cells frequently exhibit chromosomal abnormalities. Specific cytogenetic aberrations often are predictors of outcome, especially in hematologic neoplasms, such as monosomy 7 in myeloid malignancies. The functional consequences of aneuploidy at the cellular level are difficult to assess because of a lack of convenient markers to distinguish abnormal from diploid cells. We performed single-cell RNA sequencing (scRNA-seq) to study hematopoietic stem and progenitor cells from the bone marrow of 4 healthy donors and 5 patients with bone marrow failure and chromosome gain or loss. In total, transcriptome sequences were obtained from 391 control cells and 588 cells from patients. We characterized normal hematopoiesis as binary differentiation from stem cells to erythroid and myeloid-lymphoid pathways. Aneuploid cells were distinguished from diploid cells in patient samples by computational analyses of read fractions and gene expression of individual chromosomes. We confirmed assignment of aneuploidy to individual cells quantitatively, by copy-number variation, and qualitatively, by loss of heterozygosity. When we projected patients' single cells onto the map of normal hematopoiesis, diverse patterns were observed, broadly reflecting clinical phenotypes. Patients' monosomy 7 cells showed downregulation of genes involved in immune response and DNA damage checkpoint and apoptosis pathways, which may contribute to the clonal expansion of monosomy 7 cells with accumulated gene mutations. scRNA-seq is a powerful technique through which to infer the functional consequences of chromosome gain and loss and explore gene targets for directed therapy.
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http://dx.doi.org/10.1182/blood-2017-08-803353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746166PMC
December 2017

Telomerase enzyme deficiency promotes metabolic dysfunction in murine hepatocytes upon dietary stress.

Liver Int 2018 01 19;38(1):144-154. Epub 2017 Aug 19.

Department of Internal Medicine, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil.

Background & Aims: Short telomeres and genetic telomerase defects are risk factors for some human liver diseases, ranging from non-alcoholic fatty liver disease and non-alcoholic steatohepatitis to cirrhosis. In murine models, telomere dysfunction has been shown to metabolically compromise hematopoietic cells, liver and heart via the activation of the p53-PGC axis.

Methods: Tert- and Terc-deficient mice were challenged with liquid high-fat diet. Liver metabolic contents were analysed by CE-TOFMS and liver fat content was confirmed by confocal and electronic microscopy.

Results: Tert-deficient but not Terc-deficient mice develop hepatocyte injury and frank steatosis when challenged with liquid high-fat diet. Upon high-fat diet, Tert hepatocytes fail to engage the citric acid cycle (TCA), with an imbalance of NADPH/NADP and NADH/NAD ratios and depletion of intermediates of TCA cycle, such as cis-aconitic acid. Telomerase deficiency caused an intrinsic metabolic defect unresponsive to environmental challenge. Chemical inhibition of telomerase by zidovudine recapitulated the abnormal Tert metabolic phenotype in Terc hepatocytes.

Conclusions: Our findings indicate that in telomeropathies short telomeres are not the only molecular trigger and telomerase enzyme deficiency provokes hepatocyte metabolic dysfunction, abrogates response to environmental challenge, and causes cellular injury and steatosis, providing a mechanism for liver damage in telomere diseases.
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http://dx.doi.org/10.1111/liv.13529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741503PMC
January 2018

Optimization and standardization of fluorescent cell barcoding for multiplexed flow cytometric phenotyping.

Cytometry A 2017 07 10;91(7):694-703. Epub 2017 Jul 10.

Center for Human Immunology, Autoimmunity, and Inflammation, NIH, Bethesda, Maryland, 20892-1202.

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4 and CD8 T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. Published 2017 by Wiley Periodicals, Inc., on behalf of International Society for Advancement of Cytometry. This article is a US government work and as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/cyto.a.23162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612408PMC
July 2017

T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways.

J Immunol 2017 07 19;199(2):477-488. Epub 2017 Jun 19.

Cell Biology Section, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder originating from hematopoietic stem cells and is a life-threating disease characterized by intravascular hemolysis, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene () on the X chromosome, which blocks synthesis of the glycolipid moiety and causes deficiency in GPI-anchored proteins. PNH is closely related to aplastic anemia, in which T cells mediate destruction of BM. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, we applied RNA-seq to examine the transcriptome of T cell subsets (CD4 naive, CD4 memory, CD8 naive, and CD8 memory) from PNH patients and healthy control subjects. Differentially expressed gene analysis in four different T cell subsets from PNH and healthy control subjects showed distinct transcriptional profiles, depending on the T cell subsets. By pathway analysis, we identified novel signaling pathways in T cell subsets from PNH, including increased gene expression involved in TNFR, IGF1, NOTCH, AP-1, and ATF2 pathways. Dysregulation of several candidate genes (, , , , , , , , and ) was validated by quantitative real-time RT-PCR and flow cytometry. We have demonstrated molecular signatures associated with positive and negative regulators in T cells, suggesting novel pathophysiologic mechanisms in PNH. These pathways may be targets for new strategies to modulate T cell immune responses in BM failure.
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http://dx.doi.org/10.4049/jimmunol.1601299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543699PMC
July 2017

Hematopoietic Aging Biomarkers in Mice.

J Aging Sci 2017 Apr 6;5(1). Epub 2017 Feb 6.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD USA.

We analyzed hematopoietic phenotypes in (PL) mice at young (2-9 months), middle (22-23 months) and old (33-46 months) ages aimed at characterizing age-associated changes in this unique rodent species. We found a significantly higher number of monocytes in old PL mice in peripheral blood, and higher proportions of CD44 cells in blood, spleen and bone marrow in old PL mice than in middle and young counterparts. We conclude that elevated blood monocyte counts and up-regulated hematopoietic cell CD44 expression are two useful aging biomarkers for PL mice.
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http://dx.doi.org/10.4172/2329-8847.1000169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469418PMC
April 2017

Whole transcriptome sequencing identifies increased CXCR2 expression in PNH granulocytes.

Br J Haematol 2017 04 1;177(1):136-141. Epub 2017 Feb 1.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA.

The aetiology of paroxysmal nocturnal haemoglobinuria (PNH) is a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIGA), resulting in global deficiency of glycosyl phosphatidylinositol-anchored proteins (GPI-APs). This study applied RNA-sequencing to examine functional effects of the PIGA mutation in human granulocytes. CXCR2 expression was increased in GPI-AP compared to GPI-AP granulocytes. Macrophage migration inhibitory factor, a CXCR2 agonist, was significantly higher in plasma of PNH patients. Nuclear factor-κB phosphorylation was upregulated in GPI-AP compared with GPI-AP granulocytes. Our data suggest novel mechanisms in PNH, not obviously predicted by decreased production of the GPI moiety.
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http://dx.doi.org/10.1111/bjh.14502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378616PMC
April 2017

Epigenetic landscape of the TERT promoter: a potential biomarker for high risk AML/MDS.

Br J Haematol 2016 Nov 19;175(3):427-439. Epub 2016 Jul 19.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Although recent observations implicate the importance of telomerase activity in acute myeloid leukaemia (AML), the roles of epigenetic regulations of the TERT gene in leukaemogenesis, drug resistance and clinical prognosis in AML are not fully understood. We developed a quantitative pyrosequencing-based methylation assay covering the TERT proximal promoter and a partial exon 1 (TERTpro/Ex1) region and tested both cell lines and primary leukaemia cells derived from AML and AML with preceding myelodysplastic syndrome (AML/MDS) patients (n = 43). Prognostic impact of methylation status of the upstream TERT promoter region was assessed by the Kaplan-Meier method. The activity of the telomerase inhibitor, imetelstat, was measured using leukaemia cell lines. The TERTpro/Ex1 region was highly methylated in all cell lines and primary leukaemia cells showed diverse methylation profiles. Most cases showed hypermethylated regions at the upstream TERTpro/Ex1 region, which were associated with inferior patient survival. TERTpro/Ex1 methylation status was correlated with the cytotoxicity to imetelstat and its combination with hypomethylating agent enhanced the cytotoxicity of imetelstat. AML cell lines and primary blasts harbour distinct TERTpro/Ex1 methylation profiles that could serve as a prognostic biomarker of AML. However, validation in a large cohort of patients is necessary to confirm our findings.
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http://dx.doi.org/10.1111/bjh.14244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245983PMC
November 2016

A plasma microRNA signature as a biomarker for acquired aplastic anemia.

Haematologica 2017 01 22;102(1):69-78. Epub 2016 Sep 22.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MA, USA.

Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.
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http://dx.doi.org/10.3324/haematol.2016.151076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210234PMC
January 2017

Alemtuzumab in T-cell large granular lymphocytic leukaemia: interim results from a single-arm, open-label, phase 2 study.

Lancet Haematol 2016 Jan 17;3(1):e22-9. Epub 2015 Dec 17.

Clinical Hematology, Antônio Ermírio de Moraes Cancer Center, Hospital São José and Beneficência Portuguesa, São Paulo, SP, Brazil. Electronic address:

Background: T-cell large granular lymphocytic leukaemia (T-LGL) is a lymphoproliferative disease that presents with immune-mediated cytopenias and is characterised by clonal expansion of cytotoxic CD3+ CD8+ lymphocytes. Use of methotrexate, ciclosporin, or cyclophosphamide as first therapy improves cytopenias in 50% of patients, but long-term use of these can lead to toxicity. We aimed to explore the activity and safety of alemtuzumab, an anti-CD52 monoclonal antibody, in patients with T-LGL.

Methods: We did this single-arm, phase 2 trial in consecutively enrolled adults with T-LGL referred to the National Institutes of Health in Bethesda, MD, USA. Alemtuzumab was given intravenously at 10 mg per day for 10 days. The primary endpoint was haematological response at 3 months after infusion. A complete response was defined as normalisation of all affected lineages, and a partial response was defined in neutropenic patients as 100% increase in the absolute neutrophil count to more than 5 × 10(8) cells per L, and in those with anaemia, as any increase in haemoglobin of 20 g/L or higher observed in at least two serial measurements 1 week apart and sustained for 1 month or longer without exogenous growth factors support or transfusions. Analysis was by intention to treat. We report results from the first stage of this Simon two-stage design trial; enrolment into the second stage is continuing. This study is registered with ClinicalTrials.gov, number NCT00345345.

Findings: From Oct 1, 2006, to March 1, 2015, we enrolled 25 patients with T-LGL. 14 patients (56%; 95% CI 35-76) had a haematological response at 3 months. Four patients with associated myelodysplastic syndrome and two who had received haemopoietic stem cell transplantation had either no response or were not evaluable, meaning 14 (74% [49-91]) of the 19 patients with classic T-LGL responded. All patients had an infusion reaction (24 [96%] patients grade 1-2, one [4%] patient grade 3), which improved with symptomatic therapy. All patients developed lymphopenia, with 22 (88%) patients having grade 3 or 4 lymphopenia. The other most common grade 3 and 4 adverse events were leukopenia (eight [32%]) and neutropenic infections (five [20%]). Seven patients died; all were non-responders.

Interpretation: This is the largest and only prospective study of alemtuzumab in patients with T-LGL. The activity reported with a single course of a lymphocytotoxic drug in patients with mainly relapsed and refractory disease suggests that haematological response can be achieved without continued use of oral immunosuppression.

Funding: National Heart, Lung, and Blood Institute.
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http://dx.doi.org/10.1016/S2352-3026(15)00227-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721315PMC
January 2016

Memory Stem T Cells in Autoimmune Disease: High Frequency of Circulating CD8+ Memory Stem Cells in Acquired Aplastic Anemia.

J Immunol 2016 Feb 13;196(4):1568-78. Epub 2016 Jan 13.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892;

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4(+) and CD8(+) T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8(+) TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8(+) TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8(+) TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8(+) and CD4(+) T cell subsets in AA patients, including CD8(+) and CD4(+) TSCMs. CD8(+) TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4(+) and CD8(+) TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8(+) TSCM subset is a novel biomarker and a potential therapeutic target for AA.
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http://dx.doi.org/10.4049/jimmunol.1501739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744506PMC
February 2016

PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function.

Haematologica 2016 Jan 20;101(1):57-67. Epub 2015 Nov 20.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically "fatless" mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation.
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http://dx.doi.org/10.3324/haematol.2014.121632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697892PMC
January 2016

Identification of novel microRNA signatures linked to acquired aplastic anemia.

Haematologica 2015 Dec 9;100(12):1534-45. Epub 2015 Sep 9.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA.

Emerging evidence indicates that microRNA control and modulate immunity. MicroRNA have not been investigated in acquired aplastic anemia, a T-cell-mediated immune disease. Analysis of 84 microRNA expression levels in CD4(+) and CD8(+) T cells of patients with aplastic anemia revealed concurrent down-regulation of miR-126-3p, miR-145-5p, miR-223-3p, and miR-199a-5p (>3-fold change, P<0.05) in both T-cell populations, which were unique in aplastic anemia compared to other hematologic disorders. MiR-126-3p and miR-223-3p were down-regulated in CD4(+) T effector memory cells, and miR-126-3p, miR-145-5p, and miR-223-3p were down-regulated in CD8(+) T effector memory and terminal effector cells. Successful immunosuppressive therapy was associated with restoration to normal expression levels of miR-126-3p, miR-145-5p, and miR-223-3p (>2-fold change, P<0.05). In CD4(+) and CD8(+) T cells in aplastic anemia patients, MYC and PIK3R2 were up-regulated and proved to be targets of miR-145-5p and miR-126-3p, respectively. MiR-126-3p and miR-145-5p knockdown promoted proliferation and increased interferon-γ and granzyme B production in both CD4(+) and CD8(+) T cells. Our work describes previously unknown regulatory roles of microRNA in T-cell activation in aplastic anemia, which may open a new perspective for development of effective therapy. Clinicaltrials.gov identifier: NCT 01623167.
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http://dx.doi.org/10.3324/haematol.2015.126128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666329PMC
December 2015