The Function of E2A in B-Cell Development.

Helix-loop-helix (HLH) transcription factors (TFs) play a key role in various cellular differentiation and function through the regulation of enhancer activity. E2A, a member of the mammalian E-protein family (class I HLH protein), is well known to play an important role in hematopoiesis, especially in adaptive lymphocyte development. E2A instructs B- and T-cell lineage development through the regulation of enhancer activity for B- or T-cell signature gene expression, including Rag1 and Rag2 (Rag1/2) genes. In this chapter, we mainly focus on the function of E2A in B-cell development and on the roles of E2A in establishing the enhancer landscape through the recruitment of EP300/KAT3B, chromatin remodeling complex, mediator, cohesion, and TET proteins. Finally, we demonstrate how E2A orchestrates the assembly of the Rag1/2 gene super-enhancer (SE) formation by changing the chromatin conformation across the Rag gene locus.

Pre-B cell receptor acts as a selectivity switch for galectin-1 at the pre-B cell surface.

Galectins are glycan-binding proteins translating the sugar-encoded information of cellular glycoconjugates into physiological activities, including immunity, cell migration, and signaling. Galectins also interact with non-glycosylated partners in the extracellular milieu, among which the pre-B cell receptor (pre-BCR) during B cell development. How these interactions might interplay with the glycan-decoding function of galectins is unknown. Here, we perform NMR experiments on native membranes to monitor Gal-1 binding to physiological cell surface ligands. We show that pre-BCR interaction changes Gal-1 binding to glycosylated pre-B cell surface receptors. At the molecular and cellular levels, we identify α2,3-sialylated motifs as key targeted epitopes. This targeting occurs through a selectivity switch increasing Gal-1 contacts with α2,3-sialylated poly-N-acetyllactosamine upon pre-BCR interaction. Importantly, we observe that this switch is involved in the regulation of pre-BCR activation. Altogether, this study demonstrates that interactions to non-glycosylated proteins regulate the glycan-decoding functions of galectins at the cell surface.

Single-cell systems pharmacology identifies development-driven drug response and combination therapy in B cell acute lymphoblastic leukemia.

Leukemia can arise at various stages of the hematopoietic differentiation hierarchy, but the impact of developmental arrest on drug sensitivity is unclear. Applying network-based analyses to single-cell transcriptomes of human B cells, we define genome-wide signaling circuitry for each B cell differentiation stage. Using this reference, we comprehensively map the developmental states of B cell acute lymphoblastic leukemia (B-ALL), revealing its strong correlation with sensitivity to asparaginase, a commonly used chemotherapeutic agent. Single-cell multi-omics analyses of primary B-ALL blasts reveal marked intra-leukemia heterogeneity in asparaginase response: resistance is linked to pre-pro-B-like cells, with sensitivity associated with the pro-B-like population. By targeting BCL2, a driver within the pre-pro-B-like cell signaling network, we find that venetoclax significantly potentiates asparaginase efficacy in vitro and in vivo. These findings demonstrate a single-cell systems pharmacology framework to predict effective combination therapies based on intra-leukemia heterogeneity in developmental state, with potentially broad applications beyond B-ALL.

Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction.

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

EBF1, PAX5, and MYC: regulation on B cell development and association with hematologic neoplasms.

During lymphocyte development, a diverse repertoire of lymphocyte antigen receptors is produced to battle against pathogens, which is the basis of adaptive immunity. The diversity of the lymphocyte antigen receptors arises primarily from recombination-activated gene (RAG) protein-mediated V(D)J rearrangement in early lymphocytes. Furthermore, transcription factors (TFs), such as early B cell factor 1 (EBF1), paired box gene 5 (PAX5), and proto-oncogene myelocytomatosis oncogene (MYC), play critical roles in regulating recombination and maintaining normal B cell development. Therefore, the aberrant expression of these TFs may lead to hematologic neoplasms.

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.

Research Note: Isolation and immunomodulatory activity of bursal peptide, a novel peptide from avian immune system developments.

The bursa of Fabricius (BF) is pivotal for B lymphocyte ontogenesis. In the present investigation, a novel bursal peptide, designated BP7, was extracted from BF and was found to stimulate colony-forming unit pre-B (CFU pre-B) formation at various concentrations (1 µg/mL, P < 0.05; 5 µg/mL, P < 0.05; 25 µg/mL, P < 0.05). Moreover, BP7 modulated B cell differentiation pathways. The immunoregulatory potential of BP7 was further assessed in avian and murine models subjected to immunization with inactivated avian influenza virus (AIV, H9N2 subtype). BP7 significantly augmented AIV-specific antibody levels (Prime immunization: 5 mg/kg, P < 0.05; Boost immunization: 0.4, 1, and 5 mg/kg, P < 0.05) and cytokine secretion in the avian model (IL-4 and IFN-γ: 0.4, 1, and 5 mg/kg, P < 0.05). Similarly, in the murine model, AIV-specific antibody levels (Prime and Boost immunization: 0.4, 1, and 5 mg/kg, P < 0.05) and cytokine production (IL-4 and IFN-γ: 0.4, 1, and 5 mg/kg, P < 0.05) were notably enhanced. This study offers novel insights into the mechanisms underlying B cell maturation and holds implications for future immunopharmacological interventions.

Lipid metabolism in B cell biology.

In recent years, the field of immunometabolism has solidified its position as a prominent area of investigation within the realm of immunological research. An expanding body of scientific literature has unveiled the intricate interplay between energy homeostasis, signalling molecules, and metabolites in relation to fundamental aspects of our immune cells. It is now widely accepted that disruptions in metabolic equilibrium can give rise to a myriad of pathological conditions, ranging from autoimmune disorders to cancer. Emerging evidence, although sometimes fragmented and anecdotal, has highlighted the indispensable role of lipids in modulating the behaviour of immune cells, including B cells. In light of these findings, this review aims to provide a comprehensive overview of the current state of knowledge regarding lipid metabolism in the context of B cell biology.

Isolation and Analysis of B-cell Progenitors from Bone Marrow by Flow Cytometry.

B cells play a critical role in host defense, producing antibodies in response to microbial infection. An inability to produce an effective antibody response leaves affected individuals prone to serious infection; therefore, proper B-cell development is essential to human health. B-cell development begins in the bone marrow and progresses through various stages until maturation occurs in the spleen. This process involves several sequential, complex events, starting with pre- and pro-B cells, which rearrange the heavy and light chain genes responsible for producing clonally diverse immunoglobulin (Ig) molecules. These cells then differentiate into immature B cells, followed by mature B cells. The bone marrow is a complex ecological niche of supporting stromal cells, extracellular matrix components, macrophages, and hematopoietic precursor cells influencing B-cell development, maturation, and differentiation. Once fully mature, B cells circulate in peripheral lymphoid organs and can respond to antigenic stimuli. As specific cell surface markers are expressed during each stage of B-cell development, researchers use flow cytometry as a powerful tool to evaluate developmental progression. In this protocol, we provide a step-by-step method for bone marrow isolation, cell staining, and data analysis. This tool will help researchers gain a deeper understanding of the progression of B-cell development and provide a pertinent flow gating strategy.

Dysregulation of B lymphocyte development in the SKG mouse model of rheumatoid arthritis.

Rheumatoid arthritis is a chronic and systemic inflammatory disease that affects approximately 1% of the world's population and is characterised by joint inflammation, the destruction of articular cartilage and bone, and many potentially life-threatening extraarticular manifestations. B lymphocytes play a central role in the pathology of rheumatoid arthritis as the precursors of autoantibody secreting plasma cells, as highly potent antigen-presenting cells, and as a source of various inflammatory cytokines, however, the effects of rheumatoid arthritis on B lymphocyte development remain poorly understood. Here, we analyse B lymphocyte development in murine models of rheumatoid arthritis, quantifying all the subsets of B cell precursors in the bone marrow and splenic B cells using flow cytometry. We demonstrate a severe reduction in pre-B cells and immature B cells in the bone marrow of mice with active disease, despite no major effects on the mature naïve B cell numbers. The loss of B cell precursors in the bone marrow of the affected mice was associated with a highly significant reduction in the proportion of Ki67+ cells, indicating impaired cell proliferation, while the viability of the B cell precursors was not significantly affected. We also observed some mobilisation of the B cell precursor cells into the mouse spleen, demonstrated with flow cytometry and pre-B colony forming units assays. In summary, the current work demonstrates a severe dysregulation in B lymphocyte development in murine rheumatoid arthritis, with possible implications for B cell repertoire formation, tolerance induction, and disease mechanisms.

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.

NF-kB's contribution to B cell fate decisions.

NF-κB signaling is essential to an effective innate and adaptive immune response. Many immune-specific functional and developmental outcomes depend in large on NF-κB. The formidable task of sorting out the mechanisms behind the regulation and outcome of NF-κB signaling remains an important area of immunology research. Here we briefly discuss the role of NF-κB in regulating cell fate decisions at various times in the path of B cell development, activation, and the generation of long-term humoral immunity.

ARID5B regulates fatty acid metabolism and proliferation at the Pre-B cell stage during B cell development.

During B cell development in bone marrow, large precursor B cells (large Pre-B cells) proliferate rapidly, exit the cell cycle, and differentiate into non-proliferative (quiescent) small Pre-B cells. Dysregulation of this process may result in the failure to produce functional B cells and pose a risk of leukemic transformation. Here, we report that AT rich interacting domain 5B (ARID5B), a B cell acute lymphoblastic leukemia (B-ALL) risk gene, regulates B cell development at the Pre-B stage. In both mice and humans, we observed a significant upregulation of ARID5B expression that initiates at the Pre-B stage and is maintained throughout later stages of B cell development. In mice, deletion of Arid5b in vivo and ex vivo exhibited a significant reduction in the proportion of immature B cells but an increase in large and small Pre-B cells. Arid5b inhibition ex vivo also led to an increase in proliferation of both Pre-B cell populations. Metabolic studies in mouse and human bone marrow revealed that fatty acid uptake peaked in proliferative B cells then decreased during non-proliferative stages. We showed that Arid5b ablation enhanced fatty acid uptake and oxidation in Pre-B cells. Furthermore, decreased ARID5B expression was observed in tumor cells from B-ALL patients when compared to B cells from non-leukemic individuals. In B-ALL patients, ARID5B expression below the median was associated with decreased survival particularly in subtypes originating from Pre-B cells. Collectively, our data indicated that Arid5b regulates fatty acid metabolism and proliferation of Pre-B cells in mice, and reduced expression of ARID5B in humans is a risk factor for B cell leukemia.

DNAJA3 regulates B cell development and immune function.

BACKGROUND: DnaJ homolog subfamily A member 3 (DNAJA3), also known as the tumorous imaginal disc (Tid1), is shown to be crucial in T cell development. DNAJA3 functions as a tumor suppressor implicated in lymphocyte development and survival. However, the role of DNAJA3 in B cell development and immune function remains unknown. In this study, we utilized a mouse model of B cell-specific DNAJA3 knockout (CD19-Cre/+; DNAJA3flx/flx) to investigate the physiological function of DNAJA3 in B cell development and immune function. METHODS: We characterized B cell populations in various developmental stages and examined mitochondrial content and function between control and DNAJA3 KO using flow cytometry analysis. DNAJA3 and OXPHOS protein complexes in sorted B cells between mice groups were compared using immunoblot techniques. The activity of B cell blastogenesis in splenocytes was measured by performing CFSE and MTT assays. Furthermore, immunoglobulin production was detected using the ELISA method. RESULTS: DNAJA3 deficiency decreases from pro B cells to immature B cells. The overall B220+ population in the bone marrow and secondary immune organs also decreased. B cell subpopulations B1 (B1b) and B2 significantly decrease. The B cell blastogenesis activity and immunoglobulin production decreased in DNAJA3 KO mice. Mechanistically, DNAJA3 deficiency significantly increases dysfunctional mitochondria activity and decreases mitochondrial mass, membrane potential, and mitochondria respiratory complex proteins. These factors could have influenced B cell differentiation during development, differentiation to antibody-secreting cells, and immune activation. CONCLUSION: Overall, our study provides supportive evidence for the role of DNAJA3 in B cell development and function.

Enhanced protein synthesis is a defining requirement for neonatal B cell development.

The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b. Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.

Overview of human B-cell development and antibody deficiencies.

B cells are a key component of the humoral (antibody-mediated) immune response which is responsible for defense against a variety of pathogens. Here we provide an overview of the current understanding of B cell development and function and briefly describe inborn errors of immunity associated with B cell development defects which can manifest as immune deficiency, malignancy, autoimmunity, or allergy. The knowledge and application of B cell biology are essential for laboratory evaluation and clinical assessment of these B cell disorders.

CLL stereotyped B-cell receptor immunoglobulin sequences are recurrent in the B-cell repertoire of healthy individuals: Apparent lack of central and early peripheral tolerance censoring.

Introduction: The leukemic cells of patients with chronic lymphocytic leukemia (CLL) are often unique, expressing remarkably similar IGHV-IGHD-IGHJ gene rearrangements, "stereotyped BCRs". The B-cell receptors (BCRs) on CLL cells are also distinctive in often deriving from autoreactive B lymphocytes, leading to the assumption of a defect in immune tolerance. Results: Using bulk and single-cell immunoglobulin heavy and light chain variable domain sequencing, we enumerated CLL stereotype-like IGHV-IGHD-IGHJ sequences (CLL-SLS) in B cells from cord blood (CB) and adult peripheral blood (PBMC) and bone marrow (BM of healthy donors. CLL-SLS were found at similar frequencies among CB, BM, and PBMC, suggesting that age does not influence CLL-SLS levels. Moreover, the frequencies of CLL-SLS did not differ among B lymphocytes in the BM at early stages of development, and only re-circulating marginal zone B cells contained significantly higher CLL-SLS frequencies than other mature B-cell subpopulations. Although we identified CLL-SLS corresponding to most of the CLL major stereotyped subsets, CLL-SLS frequencies did not correlate with those found in patients. Interestingly, in CB samples, half of the CLL-SLS identified were attributed to two IGHV-mutated subsets. We also found satellite CLL-SLS among the same normal samples, and they were also enriched in naïve B cells but unexpectedly, these were ~10-fold higher than standard CLL-SLS. In general, IGHV-mutated CLL-SLS subsets were enriched among antigen-experienced B-cell subpopulations, and IGHV-unmutated CLL-SLS were found mostly in antigen-inexperienced B cells. Nevertheless, CLL-SLS with an IGHV-mutation status matching that of CLL clones varied among the normal B-cell subpopulations, suggesting that specific CLL-SLS could originate from distinct subpopulations of normal B cells. Lastly, using single-cell DNA sequencing, we identified paired IGH and IGL rearrangements in normal B lymphocytes resembling those of stereotyped BCRs in CLL, although some differed from those in patients based on IG isotype or somatic mutation. Discussion: CLL-SLS are present in normal B-lymphocyte populations at all stages of development. Thus, despite their autoreactive profile they are not deleted by central tolerance mechanisms, possibly because the level of autoreactivity is not registered as dangerous by deletion mechanisms or because editing of L-chain variable genes occurred which our experimental approach could not identify.

BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells.

Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.

NONO regulates B-cell development and B-cell receptor signaling.

The paraspeckle protein NONO is a multifunctional nuclear protein participating in the regulation of transcriptional regulation, mRNA splicing and DNA repair. However, whether NONO plays a role in lymphopoiesis is not known. In this study, we generated mice with global deletion of NONO and bone marrow (BM) chimeric mice in which NONO is deleted in all of mature B cells. We found that the global deletion of NONO in mice did not affect T-cell development but impaired early B-cell development in BM at pro- to pre-B-cell transition stage and B-cell maturation in the spleen. Studies of BM chimeric mice demonstrated that the impaired B-cell development in NONO-deficient mice is B-cell-intrinsic. NONO-deficient B cells displayed normal BCR-induced cell proliferation but increased BCR-induced cell apoptosis. Moreover, we found that NONO deficiency impaired BCR-induced activation of ERK, AKT, and NF-κB pathways in B cells, and altered BCR-induced gene expression profile. Thus, NONO plays a critical role in B-cell development and BCR-induced B-cell activation.

BAFF induces CXCR5 expression during B cell differentiation in bone marrow.

B cell activating factor (BAFF) plays an important role in antibody production through differentiation and maturation of B cells mainly in secondary lymphoid organs. On the other hand, the role of BAFF in the bone marrow, the primary lymphoid organ of B cell development, has not been well elucidated. Here, effects of BAFF in bone marrow B cell development were examined by using BAFF-deficient mice. When mRNA expression levels of B cell differentiation markers including Cd19, Bcl2, Igµ, Il7r and Cxcr5 were compared between bone marrow of wild-type and BAFF-KO mice, a lower level of Cxcr5 expression was found in the KO mice. Additionally, protein expression of CXCR5 on IgM+ cells in the bone marrow was decreased by BAFF deficiency. In vitro studies also confirmed the effect of BAFF on CXCR5 by IgM+ cells; culturing bone marrow cells from BAFF-KO mice with BAFF in vitro increased the proportion of CXCR5+ cells in IgM+ cells compared with non-treated bone marrow cells. In addition, BAFF synergized with TNF-α and IL-6 to increase the expression of CXCR5+ on IgM+ cells. The BAFF-mediated up-regulation of CXCR5 expression was reproduced by using CD19+ cells purified from BAFF-KO bone marrow cells, suggesting that BAFF directly affects B-lineage cells in bone marrow to promote CXCR5 expression. Together, this study suggests that BAFF has an important role in B cell differentiation in bone marrow by directly inducing CXCR5 expression which affect their migration to secondary lymphoid organs.