Post-transcriptional (re)programming of B lymphocyte development: From bench to bedside?

Hematopoiesis, a process which generates blood and immune cells, changes significantly during mammalian development. Definitive hematopoiesis is marked by the emergence of long-term hematopoietic stem cells (HSCs). Here, we will focus on the post-transcriptional differences between fetal liver (FL) and adult bone marrow (ABM) HSCs. It remains unclear how or why exactly FL HSCs transition to ABM HSCs, but we aim to leverage their differences to revive an old idea: in utero HSC transplantation. Unexpectedly, the expression of certain RNA-binding proteins (RBPs) play an important role in HSC specification, and can be employed to convert or reprogram adult HSCs back to a fetal-like state. Among other features, FL HSCs have a broad differentiation capacity that includes the ability to regenerate both conventional B and T cells, as well as innate-like or unconventional lymphocytes such as B-1a and marginal zone B (MzB) cells. This chapter will focus on RNA binding proteins, namely LIN28B and IGF2BP3, that are expressed during fetal life and how they promote B-1a cell development. Furthermore, this chapter considers a potential clinical application of synthetic co-expression of LIN28B and IGF2BP3 in 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.

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.

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.

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.

Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis.

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

Rag1 and Rag2 Gene Expressions Identify Lymphopoietic Tissues in Larvae of Rice-Field Eel (Monopterus albus).

In immature lymphocytes, recombination activating genes 1 and 2 are necessary for antigen receptor V (D) J recombination, representing immature lymphocyte biomarkers. Herein, we cloned and sequenced rice-field eel rag1 and rag2 genes. Their expressions in the thymus, liver, and kidney were significant from 0 days post hatching (dph) to 45 dph, peaking at 45 dph in these three tissues. In situ hybridization detected high rag1 and rag2 expressions in the liver, kidney, and thymus of rice-field eel from 2 to 45 dph, suggesting that multiple tissues of rice-field eel contain lymphocyte lineage cells and undergo lymphopoiesis. Tissue morphology was used to observe lymphopoiesis development in these three tissues. The thymus primordium began to develop at 2 dph, while the kidney and liver have generated. Our findings verified that the thymus is the primary lymphopoietic tissue and suggested that, in rice-field eel, lymphocyte differentiation also occurs in the liver and kidney.

BAP1 shapes the bone marrow niche for lymphopoiesis by fine-tuning epigenetic profiles in endosteal mesenchymal stromal cells.

Hematopoiesis occurs within a unique bone marrow (BM) microenvironment, which consists of various niche cells, cytokines, growth factors, and extracellular matrix components. These multiple components directly or indirectly regulate the maintenance and differentiation of hematopoietic stem cells (HSCs). Here we report that BAP1 in BM mesenchymal stromal cells (MSCs) is critical for the maintenance of HSCs and B lymphopoiesis. Mice lacking BAP1 in MSCs show aberrant differentiation of hematopoietic stem and progenitor cells, impaired B lymphoid differentiation, and expansion of myeloid lineages. Mechanistically, BAP1 loss in distinct endosteal MSCs, expressing PRX1 but not LEPR, leads to aberrant expression of genes affiliated with BM niche functions. BAP1 deficiency leads to a reduced expression of pro-hematopoietic factors such as Scf caused by increased H2AK119-ub1 and H3K27-me3 levels on the promoter region of these genes. On the other hand, the expression of myelopoiesis stimulating factors including Csf3 was increased by enriched H3K4-me3 and H3K27-ac levels on their promoter, causing myeloid skewing. Notably, loss of BAP1 substantially blocks B lymphopoiesis and skews the differentiation of hematopoietic precursors toward myeloid lineages in vitro, which is reversed by G-CSF neutralization. Thus, our study uncovers a key role for BAP1 expressed in endosteal MSCs in controlling normal hematopoiesis in mice by modulating expression of various niche factors governing lymphopoiesis and myelopoiesis via histone modifications.

Deletion of Vhl in Dmp1-Expressing Cells Causes Microenvironmental Impairment of B Cell Lymphopoiesis.

The contributions of skeletal cells to the processes of B cell development in the bone marrow (BM) have not been completely described. The von-Hippel Lindau protein (VHL) plays a key role in cellular responses to hypoxia. Previous work showed that Dmp1-Cre;Vhl conditional knockout mice (VhlcKO), which deletes Vhl in subsets of mesenchymal stem cells, late osteoblasts and osteocytes, display dysregulated bone growth and reduction in B cells. Here, we investigated the mechanisms underlying the B cell defects using flow cytometry and high-resolution imaging. In the VhlcKO BM, B cell progenitors were increased in frequency and number, whereas Hardy Fractions B-F were decreased. VhlcKO Fractions B-C cells showed increased apoptosis and quiescence. Reciprocal BM chimeras confirmed a B cell-extrinsic source of the VhlcKO B cell defects. In support of this, VhlcKO BM supernatant contained reduced CXCL12 and elevated EPO levels. Intravital and ex vivo imaging revealed VhlcKO BM blood vessels with increased diameter, volume, and a diminished blood-BM barrier. Staining of VhlcKO B cells with an intracellular hypoxic marker indicated the natural existence of distinct B cell microenvironments that differ in local oxygen tensions and that the B cell developmental defects in VhlcKO BM are not initiated by hypoxia. Our studies identify novel mechanisms linking altered bone homeostasis with drastic BM microenvironmental changes that dysregulate B cell development.

Loss of Ribosomal Protein Paralog Rpl22-like1 Blocks Lymphoid Development without Affecting Protein Synthesis.

Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre-mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44-CD25+ double-negative stage. In vivo labeling with O-propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development.

IL7Rα, but not Flk2, is required for hematopoietic stem cell reconstitution of tissue-resident lymphoid cells.

Tissue-resident lymphoid cells (TLCs) span the spectrum of innate-to-adaptive immune function. Unlike traditional, circulating lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to further understand murine TLC development and the roles of Flk2 and IL7Rα, two cytokine receptors with known function in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled. Despite high labeling, loss of Flk2 minimally affected the generation of these cells. In contrast, loss of IL7Rα, or combined deletion of Flk2 and IL7Rα, dramatically reduced the number of B1a cells, MZBs, ILC2s and Tregs, both in situ and upon transplantation, indicating an intrinsic and essential role for IL7Rα. Surprisingly, reciprocal transplants of wild-type HSCs showed that an IL7Rα-/- environment selectively impaired reconstitution of TLCs when compared with TLC numbers in situ. Taken together, our data defined Flk2- and IL7Rα-positive TLC differentiation paths, and revealed functional roles of Flk2 and IL7Rα in TLC establishment.

Peroxisomes Are Critical for the Development and Maintenance of B1 and Marginal Zone B Cells but Dispensable for Follicular B Cells and T Cells.

Antioxidant systems maintain cellular redox (oxidation-reduction) homeostasis. In contrast with other key redox pathways, such as the thioredoxin system, glutathione, and NF-E2-related factor 2 (Nrf2), little is known about the function of the redox-sensitive organelle "peroxisome" in immune cells. In this study, we show that the absence of peroxisomes in conditional Pex5-deficient mice strikingly results in impaired homeostatic maintenance of innate-like B cells, namely, B1 and marginal zone B cells, which translates into a defective Ab response to Streptococcus pneumoniae Surprisingly, however, follicular B2 cell development, homeostatic maintenance, germinal center reactions, Ab production, class switching, and B cell memory formation were unaffected in Pex5-deficient animals. Similarly, T cell development and responses to viral infections also remained unaltered in the absence of Pex5 Thus, this study highlights the differential requirement of peroxisomes in distinct lymphocyte subtypes and may provide a rationale for specifically targeting peroxisomal metabolism in innate-like B cells in certain forms of B cell malignancies involving B1 cells.

Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline.

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

LncRNA00492 is required for marginal zone B-cell development.

B-cell development undergoes a series of steps from the bone marrow to the secondary lymphoid organs. A defect in B-cell development can lead to immunodeficiency or malignant disorders, such as leukaemia or lymphoma. Long non-coding RNAs have been reported to act as important regulators of many pathological processes. However, very little is known regarding the role of lncRNAs during B-cell development and the regulation of their expression. In this study, we explored the expression and role of lncRNA Gme00492 in B-cell development. We observed that lnc00492 was highly expressed in B-cell development and primarily expressed in the nucleus. Lnc00492-deficient mice had fewer marginal zone B cells in the spleen, likely due to a developmental block. Importantly, lnc00492 interacts with CTBP1 and targets it for ubiquitination and degradation during B-cell development, whereas the transcriptional corepressor factor CTBP1 plays a critical role in Notch2 signalling. Thus, we identified a novel regulatory axis between lnc00492 and CTBP1 in B cells, suggesting that lnc00492 is essential for marginal zone B-cell development.

Modeling human yolk sac hematopoiesis with pluripotent stem cells.

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors.

Regeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

ß2 Integrin CD11d/CD18: From Expression to an Emerging Role in Staged Leukocyte Migration.

CD11d/CD18 is the most recently discovered and least understood ß2 integrin. Known CD11d adhesive mechanisms contribute to both extravasation and mesenchymal migration - two key aspects for localizing peripheral leukocytes to sites of inflammation. Differential expression of CD11d induces differences in monocyte/macrophage mesenchymal migration including impacts on macrophage sub-set migration. The participation of CD11d/CD18 in leukocyte localization during atherosclerosis and following neurotrauma has sparked interest in the development of CD11d-targeted therapeutic agents. Whereas the adhesive properties of CD11d have undergone investigation, the signalling pathways induced by ligand binding remain largely undefined. Underlining each adhesive and signalling function, CD11d is under unique transcriptional control and expressed on a sub-set of predominately tissue-differentiated innate leukocytes. The following review is the first to capture the nearly three decades of CD11d research and discusses the emerging role of CD11d in leukocyte migration and retention during the progression of a staged immune response.

Microbiota Signals Suppress B Lymphopoiesis With Aging in Mice.

Aging is associated with significant changes in hematopoiesis that include a shift from lymphopoiesis to myelopoiesis and an expansion of phenotypic hematopoietic stem cells (HSCs) with impaired self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SPF mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. The expansion of phenotypic and functional HSCs that occurs with aging is similar in SPF and GF mice. However, HSCs from young GF mice have increased lymphoid lineage output, and the aging-associated expansion of myeloid-biased HSCs is significantly attenuated in GF mice. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress B lymphopoiesis at multiple stages of development and contribute to the expansion of myeloid-biased HSCs that occurs with aging.

RAG1 splicing mutation causes enhanced B cell differentiation and autoantibody production.

Hypomorphic RAG1 or RAG2 mutations cause primary immunodeficiencies and can lead to autoimmunity, but the underlying mechanisms are elusive. We report here a patient carrying a c.116+2T>G homozygous splice site mutation in the first intron of RAG1, which led to aberrant splicing and greatly reduced RAG1 protein expression. B cell development was blocked at both the pro-B to pre-B transition and the pre-B to immature B cell differentiation step. The patient B cells had reduced B cell receptor repertoire diversity and decreased complementarity determining region 3 lengths. Despite B cell lymphopenia, the patient had abundant plasma cells in the BM and produced large quantities of IgM and IgG Abs, including autoantibodies. The proportion of naive B cells was reduced while the frequency of IgD-CD27- double-negative (DN) B cells, which quickly differentiated into Ab-secreting plasma cells upon stimulation, was greatly increased. Immune phenotype analysis of 52 patients with primary immunodeficiency revealed a strong association of the increased proportion of DN B and memory B cells with decreased number and proportion of naive B cells. These results suggest that the lymphopenic environment triggered naive B cell differentiation into DN B and memory B cells, leading to increased Ab production.