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.

Molecular mechanisms underlying the coordination of isotype switching with plasma cell differentiation are poorly understood. We show that interferon regulatory factor-4 (IRF-4) regulates both processes by controlling the expression of the Aicda and Prdm1 genes, which encode AID and Blimp-1, respectively. Genome-wide analysis demonstrated that Irf4(-/-) B cells failed to induce the entire Blimp-1-dependent plasma cell program. Restoration of AID or Blimp-1 expression in Irf4(-/-) B cells promoted isotype switching or secretion, respectively. IRF-4 was expressed in a graded manner in differentiating B cells and targeted Prdm1. Higher concentration of IRF-4 induced Prdm1 and consequently the transition from a germinal center gene expression program to that of a plasma cell. We propose a gene-regulatory network in which graded expression of IRF-4 developmentally coordinates isotype switching with plasma cell differentiation.

Summary 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 co-bound 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.

Blimp1, a zinc-finger containing DNA-binding transcriptional repressor,functions as a master regulator of B cell terminal differentiation. Considerable evidence suggests that Blimp1 is required for the establishment of anteroposterior axis formation and the formation of head structures during early vertebrate development. In mouse embryos, Blimp1 is strongly expressed in axial mesendoderm, the tissue known to provide anterior patterning signals during gastrulation. Here, we describe for the first time the defects caused by loss of Blimp1 function in the mouse. Blimp1 deficient embryos die at mid-gestation, but surprisingly early axis formation, anterior patterning and neural crest formation proceed normally. Rather, loss of Blimp1 expression disrupts morphogenesis of the caudal branchial arches and leads to a failure to correctly elaborate the labyrinthine layer of the placenta. Blimp1mutant embryos also show widespread blood leakage and tissue apoptosis, and,strikingly, Blimp1 homozygous mutants entirely lack PGCs. At the time of PGC allocation around 7.25 days post coitum, Blimp1 heterozygous embryos exhibit decreased numbers of PCGs. Thus Blimp1 probably acts to turn off the default pathway that allows epiblast cells to adopt a somatic cell fate, and shifts the transcriptional program so that they become exclusively allocated into the germ cell lineage.

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 anti-viral 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 Il10 and Il33 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 molecular crosstalk between the interkeukin-7 receptor (IL-7R) and pre-BCR in B lymphopoiesis has been enigmatic. We demonstrate that in pre-B cells, the IL-7R, but not the pre-BCR, was coupled to the phosphatidylinositol-3-OH kinase (PI(3)K)–Akt module, signaling by which prevents Rag expression. Attenuation of IL-7 signaling resulted in up-regulation of Foxo1 and Pax5, which co-activated many pre-B cell genes, including Rag1,2 and Blnk. Induction of the latter gene enabled pre-BCR signaling via the Syk-BLNK module and promoted immunoglobulin light chain rearrangement. BLNK signaling also antagonized Akt activation, thereby augmenting Foxo1 and Pax5 accumulation. This self-reinforcing molecular circuit appears to sense limiting concentrations of IL-7 and functions to control the expansion and differentiation of pre-B cells.

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 β-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.

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.