An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling

B cell receptor signaling controls the expression of IRF-4, a transcription factor required for B cell differentiation. This study shows that IRF-4 regulates divergent B cell fates via a ‘kinetic-control' mechanism that determines the duration of a transient developmental state

IRF4 Is a Suppressor of c-Myc Induced B Cell Leukemia

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator in B cell development and function. We have previously shown that IRF4, together with IRF8, orchestrates pre-B cell development by limiting pre-B cell expansion and by promoting pre-B cell differentiation. Here, we report that IRF4 suppresses c-Myc induced leukemia in EμMyc mice. Our results show that c-Myc induced leukemia was greatly accelerated in the IRF4 heterozygous mice (IRF4+/−Myc); the average age of mortality in the IRF4+/−Myc mice was only 7 to 8 weeks but was 20 weeks in the control mice. Our results show that IRF4+/−Myc leukemic cells were derived from large pre-B cells and were hyperproliferative and resistant to apoptosis. Further analysis revealed that the majority of IRF4+/−Myc leukemic cells inactivated the wild-type IRF4 allele and contained defects in Arf-p53 tumor suppressor pathway. p27kip is part of the molecular circuitry that controls pre-B cell expansion. Our results show that expression of p27kip was lost in the IRF4+/−Myc leukemic cells and reconstitution of IRF4 expression in those cells induced p27kip and inhibited their expansion. Thus, IRF4 functions as a classical tumor suppressor to inhibit c-Myc induced B cell leukemia in EμMyc mice

Memory B Cells in Transplantation

Much of the research on the humoral response to allografts has focused on circulating serum antibodies and the long-lived plasma cells that produce these antibodies. In contrast, the interrogation of the quiescent memory B cell compartment is technically more challenging and thus has not been incorporated into the clinical diagnostic or prognostic toolkit. In this review, we discuss new technologies that have allowed this heretofore enigmatic subset of B cells to be identified at quiescence and during a recall response. These technologies in experimental models are providing new insights into memory B cell heterogeneity with respect to their phenotype, cellular function, and the antibodies they produce. Similar technologies are also allowing for the identification of comparable memory alloreactive B cells in transplant recipients. Although much of the focus in transplant immunology has been on controlling the alloreactive B cell population, long-term transplant patient survival is also critically dependent on protection by pathogen-specific memory B cells. Techniques are available that allow the interrogation of memory B cell response to pathogen re-encounter. Thus, we are poised in our ability to investigate how immunosuppression affects allospecific and pathogen-specific memory B cells, and reason that these investigations can yield new insights that will be beneficial for graft and patient survival

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

B Cell-Intrinsic IRF4 Haploinsufficiency Impairs Affinity Maturation.

The germinal center (GC) reaction is a coordinated and dynamic ensemble of cells and processes that mediate the maturation and selection of high-affinity GC B cells (GCBs) from lower-affinity precursors and ultimately results in plasma cell and memory cell fates that exit the GC. It is of great interest to identify intrinsic and extrinsic factors that control the selection process. The transcription factor IRF4, induced upon BCR and CD40 signaling, is essential for the acquisition of plasma cell and GCB cell fates. We hypothesized that beyond this early requirement, IRF4 continuously operates at later phases of the B cell response. We show that IRF4 is expressed in GCBs at levels greater than seen in resting cells and plays a role in efficient selection of high-affinity GCBs. Halving Irf4 gene copy number in an Ag-specific murine B cell model, we found that Ag presentation, isotype switching, GC formation and zonation, somatic hypermutation rates, and proliferation were comparable with cells with a full Irf4 allelic complement. In contrast, Irf4 haploinsufficient GCBs exhibited impaired generation of high-affinity cells. Mechanistically, we demonstrate suboptimal Blimp-1 regulation among high-affinity Irf4 haploinsufficient GCBs. Furthermore, in cotransfer settings, we observed a marked disadvantage of Irf4 haploinsufficient cells for GC entry, evidential of ineffective recruitment of T cell help. We propose that, analogous to its role in early GC entry, IRF4 continues to function in the late phase of the Ab response to promote productive T follicular helper cell interactions and to activate optimal Blimp-1 expression during GC selection and affinity maturation

A Synchronous IRF4-Dependent Gene Regulatory Network in B and Helper T Cells Orchestrating the Antibody Response

Control of diverse pathogens requires an adaptive antibody response, dependent on cellular division of labor to allocate antigen-dependent B- and CD4+ T-cell fates that collaborate to control the quantity and quality of antibody. This is orchestrated by the dynamic action of key transcriptional regulators mediating gene expression programs in response to pathogen-specific environmental inputs. We describe a conserved, likely ancient, gene regulatory network that intriguingly operates contemporaneously in B and CD4+ T cells to control their cell fate dynamics and thus, the character of the antibody response. The remarkable output of this network derives from graded expression, designated by antigen receptor signal strength, of a pivotal transcription factor that regulates alternate cell fate choices