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.

Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination.

Class switch recombination generates distinct antibody isotypes critical to a robust adaptive immune system, and defects are associated with autoimmune disorders and lymphomagenesis. Transcription is required during class switch recombination to recruit the cytidine deaminase AID-an essential step for the formation of DNA double-strand breaks-and strongly induces the formation of R loops within the immunoglobulin heavy-chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here, we report that cells lacking two enzymes involved in R loop removal-senataxin and RNase H2-exhibit increased R loop formation and genome instability at the immunoglobulin heavy-chain locus without impacting its transcriptional activity, AID recruitment, or class switch recombination efficiency. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking senataxin or RNase H2B alone. We propose that senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.

The immune system is a complex network of cells and molecules, which helps to protect the body from invaders. The adaptive immune system can recognise millions of assailants, kill them, and 'learn' from this experience to mount an even quicker defence the next time the body is infected. To achieve this level of protection, specific immune cells, called B cells, divide when they come into contact with a molecule from a foreign particle, the antigen. The cloned B cells then produce millions of protective proteins, the antibodies, which patrol the blood stream and tag harmful particles for destruction. An antibody resembles a Y-shaped structure that contains a 'variable' region, which gives it the specificity to interact with an antigen, and a 'constant' region, which interacts with components of the immune system and determines the mechanisms used to destroy a pathogen. Based on the constant region, antibodies can be divided into five main classes. B cells are able to switch their production from one antibody class to another in an event known as class switch recombination, by making changes to the constant region. They do this by cutting out a portion of the genes for the constant region from their DNA and fusing the remaining DNA. The resulting antibodies still recognise the same target, but interact with different components of the immune system, ensuring that all the body's forces are mobilised. R-loops are temporary structures that form when a cell 'reads' the instructions in its DNA to make proteins. R-loops provide physical support by anchoring the transcription template to the DNA. They help control the activity of genes, but if they stay on the DNA for too long they could interfere with any form of. DNA repair ­ including the cutting and fusing mechanisms during class switch recombination. To find out more about this process, Zhao et al. used B-cells from mice lacking two specific proteins that usually help to remove R-loops. Without these proteins, the B cells generated more R-loops than normal. Nevertheless, the B-cells were able to undergo class switch recombination, even though their chromosomes showed large areas of DNA damage, and DNA sections that had been repaired contained several mistakes. Errors that occur during class switch recombination have been linked to immune disorders and B cell cancers. The study of Zhao et al. shows that even if R-loops do not affect some processes in B cells, they could still impact the overall health of their DNA. A next step would be to test if an inability to remove R-loops could indeed play a role in immune disorders and B-cell cancers.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function.

Interruption of the programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway is an established and effective therapeutic strategy in human oncology and holds promise for veterinary oncology. We report the generation and characterization of monoclonal antibodies specific for canine PD-1 and PD-L1. Antibodies were initially assessed for their capacity to block the binding of recombinant canine PD-1 to recombinant canine PD-L1 and then ranked based on efficiency of binding as judged by flow cytometry. Selected antibodies were capable of detecting PD-1 and PD-L1 on canine tissues by flow cytometry and Western blot. Anti-PD-L1 worked for immunocytochemistry and anti-PD-1 worked for immunohistochemistry on formalin-fixed paraffin embedded canine tissues, suggesting the usage of this antibody with archived tissues. Additionally, anti-PD-L1 (JC071) revealed significantly increased PD-L1 expression on canine monocytes after stimulation with peptidoglycan or lipopolysaccharide. Together, these antibodies display specificity for the natural canine ligand using a variety of potential diagnostic applications. Importantly, multiple PD-L1-specific antibodies amplified IFN-γ production in a canine peripheral blood mononuclear cells (PBMC) concanavlin A (Con A) stimulation assay, demonstrating functional activity.

Inhibition of IRF4 in dendritic cells by PRR-independent and -dependent signals inhibit Th2 and promote Th17 responses.

Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.

Igß ubiquitination activates PI3K signals required for endosomal sorting.

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

Transcription factor IRF4 drives dendritic cells to promote Th2 differentiation.

Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 antiviral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il-10 and Il-33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4-deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.

The use of self-adjuvanting nanofiber vaccines to elicit high-affinity B cell responses to peptide antigens without inflammation.

Balancing immunogenicity with inflammation is a central tenet of vaccine design, especially for subunit vaccines that utilize traditional pro-inflammatory adjuvants. Here we report that by using a nanoparticulate peptide-based vaccine, immunogenicity and local inflammation could be decoupled. Self-assembled ß-sheet-rich peptide nanofibers, previously shown to elicit potent antibody responses in mice, were found to be non-cytotoxic in vitro and, remarkably, elicited no measurable inflammation in vivo-with none of the swelling at the injection site, accumulation of inflammatory cells or cytokines, or production of allergic IgE that were elicited by an alum-adjuvanted vaccine. Nanofibers were internalized by dendritic cells and macrophages at the injection site, and only dendritic cells that acquired the material increased their expression of the activation markers CD80 and CD86. Immunization with epitope-bearing nanofibers elicited antigen-specific differentiation of T cells into T follicular helper cells and B cells into germinal center cells, as well as high-titer, high-affinity IgG that cross-reacted with the native protein antigen and was neutralizing in an in vitro influenza hemagglutination inhibition assay. These responses were superior to those induced by alum and comparable to those induced by complete Freund's adjuvant. Thus, nanoparticulate assemblies may provide a new route to non-inflammatory immunotherapies and vaccines.

Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4.

The transcription factor IRF4 regulates immunoglobulin class switch recombination and plasma cell differentiation. Its differing concentrations appear to regulate mutually antagonistic programs of B and plasma cell gene expression. We show IRF4 to be also required for generation of germinal center (GC) B cells. Its transient expression in vivo induced the expression of key GC genes including Bcl6 and Aicda. In contrast, sustained and higher concentrations of IRF4 promoted the generation of plasma cells while antagonizing the GC fate. IRF4 cobound with the transcription factors PU.1 or BATF to Ets or AP-1 composite motifs, associated with genes involved in B cell activation and the GC response. At higher concentrations, IRF4 binding shifted to interferon sequence response motifs; these enriched for genes involved in plasma cell differentiation. Our results support a model of "kinetic control" in which signaling-induced dynamics of IRF4 in activated B cells control their cell-fate outcomes.

Plasma cell densities and glomerular filtration rates predict renal allograft outcomes following acute rejection.

The contribution of T cells and graft-reactive antibodies to acute allograft rejection is widely accepted, but the role of graft-infiltrating B and plasma cells is controversial. We examined 56 consecutive human renal transplant biopsies classified by Banff schema into T-cell-mediated (N = 21), antibody-mediated (N = 18), and mixed (N = 17) acute rejection, using standard immunohistochemistry for CD3, CD20, CD138, and CD45. In a predominantly African-American population (75%), neither Banff classification nor C4d deposition predicted the return to dialysis. Immunohistochemical analysis revealed CD3(+) T cells as the dominant cell type, followed by CD20(+) B cells and CD138(+) plasma cells in all acute rejection types. Using univariate Cox Proportional Hazard analysis, plasma cell density significantly predicted graft failure while B-cell density trended toward significance. Surprisingly T-cell density did not predict graft failure. The estimated glomerular filtration rate (eGFR) at diagnosis of acute rejection also predicted graft failure, while baseline eGFR ≥6 months prior to biopsy did not. Using multivariate analysis, a model including eGFR at biopsy and plasma cell density was most predictive of graft loss. These observations suggest that plasma cells may be a critical mediator and/or an independently sensitive marker of steroid-resistant acute rejection.

A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation.

The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.

IRF-8 extinguishes neutrophil production and promotes dendritic cell lineage commitment in both myeloid and lymphoid mouse progenitors.

While most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8(-/-) BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8(-/-) common myeloid progenitors and, unexpectedly, Irf8(-/-) ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context.

Discovering transcription factor regulatory targets using gene expression and binding data.

MOTIVATION: Identifying the target genes regulated by transcription factors (TFs) is the most basic step in understanding gene regulation. Recent advances in high-throughput sequencing technology, together with chromatin immunoprecipitation (ChIP), enable mapping TF binding sites genome wide, but it is not possible to infer function from binding alone. This is especially true in mammalian systems, where regulation often occurs through long-range enhancers in gene-rich neighborhoods, rather than proximal promoters, preventing straightforward assignment of a binding site to a target gene. RESULTS: We present EMBER (Expectation Maximization of Binding and Expression pRofiles), a method that integrates high-throughput binding data (e.g. ChIP-chip or ChIP-seq) with gene expression data (e.g. DNA microarray) via an unsupervised machine learning algorithm for inferring the gene targets of sets of TF binding sites. Genes selected are those that match overrepresented expression patterns, which can be used to provide information about multiple TF regulatory modes. We apply the method to genome-wide human breast cancer data and demonstrate that EMBER confirms a role for the TFs estrogen receptor alpha, retinoic acid receptors alpha and gamma in breast cancer development, whereas the conventional approach of assigning regulatory targets based on proximity does not. Additionally, we compare several predicted target genes from EMBER to interactions inferred previously, examine combinatorial effects of TFs on gene regulation and illustrate the ability of EMBER to discover multiple modes of regulation. AVAILABILITY: All code used for this work is available at http://dinner-group.uchicago.edu/downloads.html.

Multilineage transcriptional priming and determination of alternate hematopoietic cell fates.

Hematopoietic stem cells and their progenitors exhibit multilineage patterns of gene expression. Molecular mechanisms underlying the generation and refinement of these patterns during cell fate determination remain unexplored because of the absence of suitable experimental systems. Using PU.1(-/-) progenitors, we demonstrate that at subthreshold levels, this Ets transcription factor regulates a mixed pattern (macrophage/neutrophil) of gene expression within individual myeloid progenitors. Increased PU.1 levels refine the pattern and promote macrophage differentiation by modulating a novel regulatory circuit comprised of counter antagonistic repressors, Egr-1,2/Nab-2 and Gfi-1. Egr-1 and Egr-2 function redundantly to activate macrophage genes and to repress the neutrophil program. These results are used to assemble and mathematically model a gene regulatory network that exhibits both graded and bistable behaviors and accounts for the onset and resolution of mixed lineage patterns during cell fate determination.

Modular nature of Blimp-1 in the regulation of gene expression during B cell maturation.

The transcription factor Blimp-1 induces the maturation of B cells into Ab-secreting plasma cells. DNA microarrays were used to analyze the transcription profiles of both Blimp-1-transduced murine B cell lines and the inducible B cell line BCL(1). Hundreds of genes were differentially regulated, showing how Blimp-1 both restricts affinity maturation and promotes Ab secretion, homeostasis, migration, and differentiation. Strikingly, when different modes of plasma cell induction are used, very different genetic programs are used, suggesting that the transition from a B cell to plasma cell can occur in multiple ways, perhaps accounting for the different types of Ab-secreting cells observed in vivo. Furthermore, mutagenesis of Blimp-1 reveals multiple effector domains, which regulate distinct genes. This indicates that Blimp-1 subdivides the maturation program into select and tunable pathways.