Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

The ontogeny of lymphoid organs and IgM+ B-cells in ballan wrasse (Labrus bergylta) reveals a potential site for extrarenal B-cell lymphopoiesis: The pancreas.

Vaccination of farmed fish is the most effective prophylactic measure against contagious diseases but requires specific knowledge on when the adaptive immune system is fully developed. The present work describes kidney and spleen morphogenesis as well as B-cell development in the ballan wrasse (Labrus bergylta). The kidney was present at hatching (0 days pot hatching, dph) but was not lymphoid before larvae was 50-60 dph (stage 5), containing abundant Igµ+ cells. The spleen anlage was first observed in larvae at 20-30 dph and was later populated with B-cells. Unexpectedly, we found strong RAG1 signal together with abundant Igµ+ and IgM + cells in the exocrine pancreas of larvae from when the kidney was lymphoid and onwards, suggesting that B-cell lymphopoiesis occurs not only in the head kidney (HK) but also in pancreatic tissue. In this agastric fish, the pancreas is diffused along the intestine and the early presence of IgM+ B-cells in pancreatic tissue might have a role in maintain immune homeostasis in the peritoneal cavity, making a substantial contribution to early protection. IgM-secreting cells in HK indicate the presence of systemic IgM at stage 5, before the first IgM+ cells were identified in mucosal sites. This work together with our previous study on T-cell development in this species indicates that although T- and B-cells start to develop around the same time, B-cells migrate to mucosal tissues ahead of T-cells. This early migration likely involves the production of natural antibodies, contributing significantly to early protection. Moreover, a diet composed of barnacle nauplii did not result in an earlier onset of B-cell lymphopoiesis, as seen in the previous study analysing T-cell development. Nevertheless, components for adaptive immunity indicating putative immunocompetence is likely achieved in early juveniles (>100 dph). Additionally, maternal transfer of IgM to the offspring is also described. These findings provide important insights into the development of the immune system in ballan wrasse and lay the foundation for optimizing prophylactic strategies in the future. Furthermore, this work adds valuable information to broaden the knowledge on the immune system in lower vertebrates.

Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

MiR-9-1 controls osteoblastic regulation of lymphopoiesis.

The highly conserved MicroRNA-9 (miR-9) family consists of three members. We discovered that miR-9-1 deletion reduced mature miR-9 expression, causing 43% of the mice to display smaller size and postweaning lethality. MiR-9-1-deficient mice with growth defects experienced severe lymphopenia, but other blood cells were unaffected. The lymphopenia wasn't due to defects in hematopoietic progenitors, as mutant bone marrow (BM) cells underwent normal lymphopoiesis after transplantation into wild-type recipients. Additionally, miR-9-1-deficient mice exhibited impaired osteoblastic bone formation, as mutant mesenchymal stem cells (MSCs) failed to differentiate into osteoblastic cells (OBs). RNA sequencing revealed reduced expression of master transcription factors for osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Follistatin (Fst), an antagonist of bone morphogenetic proteins (BMPs), was found to be a direct target of miR-9-1. Its deficiency led to the up-regulation of Fst, inhibiting BMP signaling in MSCs, and reducing IL-7 and IGF-1. Thus, miR-9-1 controls osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

POT1a deficiency in mesenchymal niches perturbs B-lymphopoiesis.

Protection of telomeres 1a (POT1a) is a telomere binding protein. A decrease of POT1a is related to myeloid-skewed haematopoiesis with ageing, suggesting that protection of telomeres is essential to sustain multi-potency. Since mesenchymal stem cells (MSCs) are a constituent of the hematopoietic niche in bone marrow, their dysfunction is associated with haematopoietic failure. However, the importance of telomere protection in MSCs has yet to be elucidated. Here, we show that genetic deletion of POT1a in MSCs leads to intracellular accumulation of fatty acids and excessive ROS and DNA damage, resulting in impaired osteogenic-differentiation. Furthermore, MSC-specific POT1a deficient mice exhibited skeletal retardation due to reduction of IL-7 producing bone lining osteoblasts. Single-cell gene expression profiling of bone marrow from POT1a deficient mice revealed that B-lymphopoiesis was selectively impaired. These results demonstrate that bone marrow microenvironments composed of POT1a deficient MSCs fail to support B-lymphopoiesis, which may underpin age-related myeloid-bias in haematopoiesis.

Regulatory role of E3 ubiquitin ligases in normal B lymphopoiesis and B-cell malignancies.

E3 ubiquitin ligases play an essential role in protein ubiquitination, which is involved in the regulation of protein degradation, protein-protein interactions and signal transduction. Increasing evidences have shed light on the emerging roles of E3 ubiquitin ligases in B-cell development and related malignances. This comprehensive review summarizes the current understanding of E3 ubiquitin ligases in B-cell development and their contribution to B-cell malignances, which could help explore the molecular mechanism of normal B-cell development and provide potential therapeutic targets of the related diseases.

Human and mouse early B cell development: So similar but so different.

Early B cell development in the bone marrow ensures the replenishment of the peripheral B cell pool. Immature B cells continuously develop from hematopoietic stem cells, in a process guided by an intricate network of transcription factors as well as chemokine and cytokine signals. Humans and mice possess somewhat similar regulatory mechanisms of B lymphopoiesis. The continuous discovery of monogenetic defects that impact early B cell development in humans substantiates the similarities and differences with B cell development in mice. These differences become relevant when targeted therapeutic approaches are used in patients; therefore, predicting potential immunological adverse events is crucial. In this review, we have provided a phenotypical classification of human and murine early progenitors and B cell stages, based on surface and intracellular protein expression. Further, we have critically compared the role of key transcription factors (Ikaros, E2A, EBF1, PAX5, and Aiolos) and chemo- or cytokine signals (FLT3, c-kit, IL-7R, and CXCR4) during homeostatic and aberrant B lymphopoiesis in both humans and mice.

Distinct subsets of multi-lymphoid progenitors support ontogeny-related changes in human lymphopoiesis.

Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Hematopoietic stem cell-derived Tregs are essential for maintaining favorable B cell lymphopoiesis following posttransplant cyclophosphamide.

Posttransplant cyclophosphamide (PTCy) is associated with a low incidence of chronic graft-versus-host disease (cGVHD) following hematopoietic stem cell (HSC) transplantation. Previous studies have shown the important roles of B cell immunity in cGVHD development. Here, we investigated the long-term reconstitution of B lymphopoiesis after PTCy using murine models. We first demonstrated that the immune homeostatic abnormality leading to cGVHD is characterized by an initial increase in effector T cells in the bone marrow and subsequent B and Treg cytopenia. PTCy, but not cyclosporine A or rapamycin, inhibits the initial alloreactive T cell response, which restores intra-bone marrow B lymphogenesis with a concomitant vigorous increase in Tregs. This leads to profound changes in posttransplant B cell homeostasis, including decreased B cell activating factors, increased transitional and regulatory B cells, and decreased germinal center B cells. To identify the cells responsible for PTCy-induced B cell tolerance, we selectively depleted Treg populations that were graft or HSC derived using DEREG mice. Deletion of either Treg population without PTCy resulted in critical B cytopenia. PTCy rescued B lymphopoiesis from graft-derived Treg deletion. In contrast, the negative effect of HSC-derived Treg deletion could not be overcome by PTCy, indicating that HSC-derived Tregs are essential for maintaining favorable B lymphopoiesis following PTCy. These findings define the mechanisms by which PTCy restores homeostasis of the B cell lineage and reestablishes immune tolerance.

Cell circuits between leukemic cells and mesenchymal stem cells block lymphopoiesis by activating lymphotoxin beta receptor signaling.

Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1ß2 and activate lymphotoxin beta receptor (LTßR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1ß2 expression in leukemic cells. Genetic or pharmacological disruption of LTßR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage.

Heterogeneity of B cell lymphopoiesis in patients with premalignant and active myeloma.

To better characterize the heterogeneity of multiple myeloma (MM), we profiled plasma cells (PCs) and their B cell lymphopoiesis in the BM samples from patients with monoclonal gammopathy of undetermined significance, smoldering MM, and active MM by mass cytometry (CyTOF) analysis. Characterization of intra- and interneoplastic heterogeneity of malignant plasmablasts and PCs revealed overexpression of the MM SET domain (MMSET), Notch-1, and CD47. Variations in upregulation of B cell signaling regulators (IFN regulatory factor 4 [IRF-4], CXCR4, B cell lymphoma 6 [Bcl-6], c-Myc, myeloid differentiation primary response protein 88 [MYD88], and spliced X box-binding protein 1 [sXBP-1]) and aberrant markers (CD319, CD269, CD200, CD117, CD56, and CD28) were associated with different clinical outcomes in clonal PC subsets. In addition, prognosis was related to heterogeneity in subclonal expression of stemness markers, including neuroepithelial stem cell protein (Nestin), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (KLF-4), and Nanog. Furthermore, we have defined significantly elevated levels of MMSET, MYD88, c-Myc, CD243, Notch-1, and CD47 from hematopoietic stem cells to PCs in myeloma B cell lymphopoiesis, noted even in premalignant conditions, with variably modulated expression of B cell development regulators, including IRF-4, Bcl-2, Bcl-6, and sXBP-1; aberrant PC markers (such as CD52, CD44, CD200, CD81, CD269, CD117, and CXCR4); and stemness-controlling regulators, including Nanog, KLF-4, octamer-binding transcription factor 3/4 (Oct3/4), Sox2, and retinoic acid receptor α2 (RARα2). This study provides the rationale for precise molecular profiling of patients with MM by CyTOF technology to define disease heterogeneity and prognosis.

Stimulation of B-Lymphopoiesis by Administration of a Trimecaine-Based Ionic Compound in Cyclophosphamide-Induced Hematopoietic-Depressive Model.

According to the WHO, the secondary form of hematopoietic-depressive status increases the risk of death in people with oncological, infectious, and hormonal diseases. The choice of drugs that stimulate the hematopoietic activity of B-lymphopoiesis is limited. The current leucopoiesis drugs have a number of side effects: thymic preparations stimulate the production of PGE2, which causes chronic inflammation and various autoimmune diseases through the differentiation of T helper 1 (Th1) cells, the proliferation of Th17 cells, and the production of IL-22 from Th22 cells through EP2 and EP4 receptors; cytokine preparations can cause uncontrolled immune reactions and impaired contractility of smooth and cardiac muscles; drugs based on nucleic acids can stimulate the division of all cells, including bacterial and cancerous ones. The use of oligonucleotides such as ribozymes and antisense oligodeoxynucleotides (AS-ODNs) shows promise as therapeutic moieties, but faces a number of challenges such as nuclease sensitivity, off-target effects, and efficient delivery. The search for substances that stimulate B-lymphopoiesis among ionic compounds was motivated by the discovery of the unique properties of lidocaine docusate, one of the first ionic liquid forms of the known drugs. The lidocaine docusate (protonated form of lidocaine (2-(diethylamino)-N-(2,6-dimethylphenyl) acetamide + docusate-anion (dioctylsulfosuccinate))) suppresses the division of pheochromocytoma cells and activates immunity in rats. The trimecaine-based ionic compound (TIC) demonstrates high B-lymphopoiesis-stimulating activity. The TIC compound stimulates an increase in the volume of transitional B cells, which play an important role for further differentiation and formation of a sufficient number of mature B1 cells and mature B2 cells, where mature B2 cells make up the bulk of the functional population of B lymphocytes. The TIC compound most strongly stimulated the restoration of the number of marginal zone B cells, follicular B cells, and activated germinal center B cells after the cytotoxic emptying of the follicular centers of the spleen induced cyclophosphamide. It significantly exceeds the activity of the comparison drug methyluracil. The TIC compound does not affect the level of pro-B, pre-B-I, or pre-B-II bone marrow cells, which prevents the risk of the formation of immature functionally defective cells.

Understanding the Roles of the Hedgehog Signaling Pathway during T-Cell Lymphopoiesis and in T-Cell Acute Lymphoblastic Leukemia (T-ALL).

The Hedgehog (HH) signaling network is one of the main regulators of invertebrate and vertebrate embryonic development. Along with other networks, such as NOTCH and WNT, HH signaling specifies both the early patterning and the polarity events as well as the subsequent organ formation via the temporal and spatial regulation of cell proliferation and differentiation. However, aberrant activation of HH signaling has been identified in a broad range of malignant disorders, where it positively influences proliferation, survival, and therapeutic resistance of neoplastic cells. Inhibitors targeting the HH pathway have been tested in preclinical cancer models. The HH pathway is also overactive in other blood malignancies, including T-cell acute lymphoblastic leukemia (T-ALL). This review is intended to summarize our knowledge of the biological roles and pathophysiology of the HH pathway during normal T-cell lymphopoiesis and in T-ALL. In addition, we will discuss potential therapeutic strategies that might expand the clinical usefulness of drugs targeting the HH pathway in T-ALL.

The layered development of mouse B and T Cells.

Traditional models of lymphopoiesis present B and T cell development as a linear process that initiates in the fetus and continues after birth in the bone marrow and thymus, respectively. However, this view of lymphocyte development is not in accord with reports, dating back several decades, indicating that the types of lymphocytes generated before and after birth differ. In this regard, selected γδ T cells, and those that utilize the Vγ3 receptor in particular, and innate-like B-1 B cells preferentially arise during fetal blood cell development. This review synthesizes data from multiple laboratories, with an emphasis on our own work using mouse models, demonstrating that innate and conventional B and T cells emerge in hematopoietic stem cell independent and dependent waves of development that are differentially regulated. This layering of lymphocyte development has implications for understanding the composition of the adult immune system and may provide insights into the origin of various lymphocytic leukemias.

The splicing regulators TIA1 and TIAL1 are required for the expression of the DNA damage repair machinery during B cell lymphopoiesis.

B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins T cell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.

Adenosine deaminase acting on RNA-1 is essential for early B lymphopoiesis.

Adenosine deaminase acting on RNA-1 (ADAR1) is a ubiquitously expressed RNA deaminase catalyzing adenosine-to-inosine editing to prevent hyperactivated cytosolic double-stranded RNA (dsRNA) response mediated by MDA5. Here, we demonstrate that ADAR1 is essential for early B lymphopoiesis from late pro-B and large pre-B cell stages onward. ADAR1 exerts its requisite role via both MDA5-dependent and -independent pathways. Interestingly, the MDA5-dependent mechanisms regulate early pro-B to large pre-B cell transition by promoting early B cell survival. In contrast, the MDA5-independent mechanisms control large pre-B to small pre-B cell transition by regulating pre-B cell receptor (pre-BCR) expression. Moreover, we show that protein kinase R (PKR) and oligoadenylate synthetase/ribonuclease (OAS/RNase) L pathways are dispensable for ADAR1's role in early B lymphopoiesis. Importantly, we demonstrate that p150 isoform of ADAR1 exclusively accounts for ADAR1's function in early B lymphopoiesis, and its conventional dsRNA-binding, but not the Z-DNA/RNA-binding or the RNA-editing, activity is required for ADAR1's function in B cell development. Thus, our findings suggest that ADAR1 regulates early B lymphopoiesis through various mechanisms.

Lycopene induces bone marrow lymphopoiesis and differentiation of peritoneal IgA-producing cells.

Lycopene is a hydrocarbon-carotenoid commonly found in red fruits intake with major function correlated to antioxidative capacity in several pathological conditions, including cancer and cardiovascular diseases. Recently, lycopene has been associated with hematopoiesis, although the effects on B lymphocyte differentiation and antibody production are poorly understood. In this work, the principal aim was to investigate whether lycopene affects B lymphopoiesis and terminal differentiation into plasma cells. Distinct in vivo and in vitro strategies based on lycopene supplementation were used direct in Balb/c mice or in culture systems with cells derived of these mice. In the bone marrow, lycopene expanded B220+IgM- progenitor B cells and B220+IgM+ immature B lymphocytes. In the spleen, lycopene induced terminal CD138+ plasma cell generation. In the blood, we found prominent IgA and low IgM levels after lycopene administration. Interestingly, the pattern of peritoneal IgM+ and IgA+ B cells indicated a significant IgM-to-IgA class switching after lycopene injection. These data indicated that lycopene induces B cell differentiation into IgA-producing plasma cells. Thus, a new cellular function has been attributed to lycopene for B lymphocyte biology and possibly associated with humoral responses and mucosal immunity.