Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.
- MeSH
- Cell Self Renewal MeSH
- Drug Resistance, Neoplasm * genetics MeSH
- DNA Methyltransferase 3A * genetics MeSH
- DNA (Cytosine-5-)-Methyltransferases * genetics metabolism MeSH
- Hematopoietic Stem Cells * metabolism pathology drug effects MeSH
- Interferon-alpha * pharmacology MeSH
- Janus Kinase 2 * genetics metabolism MeSH
- Humans MeSH
- Myeloproliferative Disorders * genetics pathology drug therapy metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Polyethylene Glycols pharmacology MeSH
- Recombinant Proteins MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA Methyltransferase 3A * MeSH
- DNA (Cytosine-5-)-Methyltransferases * MeSH
- DNMT3A protein, human MeSH Browser
- Dnmt3a protein, mouse MeSH Browser
- Interferon-alpha * MeSH
- JAK2 protein, human MeSH Browser
- Jak2 protein, mouse MeSH Browser
- Janus Kinase 2 * MeSH
- peginterferon alfa-2a MeSH Browser
- Polyethylene Glycols MeSH
- Recombinant Proteins MeSH
Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.
- Keywords
- Bioinformatic, Genomic, Plant, Variant, Virus,
- MeSH
- Genome, Viral genetics MeSH
- Polymorphism, Single Nucleotide * genetics MeSH
- Humans MeSH
- Computational Biology MeSH
- High-Throughput Nucleotide Sequencing * MeSH
- Knowledge MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.
- MeSH
- Iron Deficiencies * MeSH
- Thrombocythemia, Essential * genetics MeSH
- Phenotype MeSH
- Hemoglobins genetics MeSH
- Janus Kinase 2 genetics MeSH
- Mutation MeSH
- Myeloproliferative Disorders * drug therapy genetics diagnosis MeSH
- Mice MeSH
- Polycythemia Vera * genetics MeSH
- Iron MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Hemoglobins MeSH
- Janus Kinase 2 MeSH
- Iron MeSH
We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.
- MeSH
- Point Mutation drug effects MeSH
- Gene Knock-In Techniques MeSH
- Hematopoietic Stem Cells cytology metabolism MeSH
- Interferon-alpha therapeutic use MeSH
- Janus Kinase 2 genetics MeSH
- Humans MeSH
- Megakaryocytes cytology metabolism MeSH
- Myeloproliferative Disorders drug therapy genetics MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Platelet Membrane Glycoprotein IIb genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Interferon-alpha MeSH
- JAK2 protein, human MeSH Browser
- Jak2 protein, mouse MeSH Browser
- Janus Kinase 2 MeSH
- Platelet Membrane Glycoprotein IIb MeSH
Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing T-cell receptor repertoires and using retrogenic monoclonal T-cell populations, we demonstrate that the virtual memory T-cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self-reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T cells. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity.
- Keywords
- T‐cell receptor repertoire, gene expression profiling of T‐cell subsets, retrogenic T cell, self‐reactivity, virtual memory T cells,
- MeSH
- Cell Differentiation * MeSH
- CD8-Positive T-Lymphocytes immunology MeSH
- Homeostasis MeSH
- Immunologic Memory * MeSH
- Mice MeSH
- Receptors, Antigen, T-Cell analysis MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Receptors, Antigen, T-Cell MeSH