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Accumulating evidence has shown that cellular double-stranded RNAs (dsRNAs) induce antiviral innate immune responses in human normal and malignant cancer cells. However, it is not fully understood how endogenous ‘self’ dsRNA homeostasis is regulated in the cell. Here, we show that an RNA-binding protein, DEAD-box RNA helicase 3X (DDX3X), prevents the aberrant accumulation of cellular dsRNAs. Loss of DDX3X induces dsRNA sensor-mediated type I interferon signaling and innate immune response in breast cancer cells due to abnormal cytoplasmic accumulation of dsRNAs. Dual depletion of DDX3X and a dsRNA-editing protein, ADAR1 synergistically activates the cytosolic dsRNA pathway in the breast cancer cells. Moreover, inhibiting DDX3X enhances the antitumor activity by increasing tumor intrinsic-type I interferon response, antigen presentation, and tumor-infiltration of cytotoxic T cells as well as dendritic cells in breast tumors, which may lead to the development of breast cancer therapy by targeting DDX3X in combination with immune checkpoint blockade.

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 −/− ) to determine how the enzyme affects PC biology. Although Enpp1 −/− mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 −/− PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

IFN regulatory factor 8 (IRF8) is expressed in many types of blood cells and plays critical roles in cellular differentiation and function. However, the role of IRF8 in nonhematopoietic systems remains poorly understood. In this study, we provide evidence that IRF8 is a transcriptional modulator of the gastric mucosa necessary for limiting Helicobacter pylori colonization. H. pylori infection significantly upregulated expression of IRF8, which, in turn, promoted IFN-γ expression by gastric epithelial cells. Mice deficient in IRF8 exhibited increased H. pylori colonization and aborted induction of mucosal IFN-γ. Genome-wide analyses of IFN-γ–treated gastric epithelial cells by chromatin immunoprecipitation sequencing and RNA sequencing led to the identification of IRF8 target genes, with many belonging to the IFN-regulated gene family that was observed previously in immune cells. Our results identify the IRF8–IFN-γ circuit as a novel gastric innate immune mechanism in the host defense against infection with H. pylori.

The transcription factors PU.1 and IRF8 regulate an unknown number of gene programs for differentiation of many hematopoietic cells including B cells, dendritic cells and myeloid cells. Their roles in B cell development were previously studied using B cell-specific conditional deletion mouse models, such as PU.1flox/flox-CD19Cre, IRF8flox/flox-CD19Cre, IRF8−/−PU.1flox/flox-CD19Cre, or IRF8flox/flox-PU.1flox/flox-CD19Cre mice. While PU.1-deficient B cells are phenotypically normal, deletion of IRF8 in B cells caused a moderate expansion of marginal zone B cells. Double deletion of PU.1 and IRF8 caused moderate expansion of plasma cells (PCs) in vitro. In this report, we investigated IRF8 and PU.1 double deletion mice using Mb1-Cre - termed DKO mice. FACS analysis revealed normal levels of early stage B cells in the bone marrow and transitional B cells in the spleens of DKO mice. However, follicular B cells were markedly reduced and the MZ B cell compartment was modestly expanded in DKO mice. The peritoneal CD5+ B-1a cells, which are a major source of circulating IgM, were completely absent in DKO mice. Surprisingly, the serum levels of IgM were higher and the levels of IgG3 and IgA were slightly lower in DKO than control mice. While the levels of serum IgG1 were comparable between DKO and control mice, DKO mice had no serum IgG2b or IgG2c antibodies. More intriguingly, following immunization with NP-KLH/alum, although the DKO mice produced more IgM-secreting PCs early on, they failed to generate germinal centers and produced markedly reduced levels of NP-specific switched IgG antibodies. Taken together, these data revealed a critical role of IRF8 and PU.1 in differentiation of follicular and germinal center B cells.

The E3 ligase ARF-BP1 governs the balance of life and death decisions by directing the degradation of p53 and enhancing the transcriptional activity of MYC. We find B cells selectively deficient in ARF-BP1 have many defects in developing and mature B cells associated with increased expression of p53 and reduced expression of Myc. Overexpression of Myc results in suppression of p53 and complete reversal of defects induced by ARF-BP1 deficiency. These findings indicate that the dynamic balance between MYC and p53 required for normal B cell maturation and function is finely tuned and critically dependent on the activities of ARF-BP1.

Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53.

IRF8 is a transcription factor critically involved in the differentiation of dendritic cells (DCs) and B cells. IRF8 is expressed in early hematopoietic progenitors, but it is not clear how IRF8 promotes these progenitors to commit to one lineage while preventing the development of alternative lineages. To better understand the functions of IRF8, we generated an IRF8-EGFP reporter mouse by replacing the stop codon of the Irf8 locus with the EGFP sequence. Flow cytometric analyses revealed progressive increases in expression of IRF8-EGFP from hematopoietic stem cells to progenitors destined to the granulocyte-macrophage pathway and to the common lymphoid progenitor pathway but not to the megakaryocytic-erythroid progenitor pathway. Cells committed to the B lineage expressed high levels of IRF8-EGFP, which is in sharp contrast with thymic T cells that expressed negligible levels of IRF8-EGFP. Unexpectedly, IRF8-EGFP expression in pre-pro-B cells was extremely heterogeneous, ranging from background to 4 logs above background. Furthermore, we identified an IRF8-EGFPbri population that expresses B220 with the potential to generate B cells, plasmacytoid DCs and myeloid cells in vivo. Finally, DC progenitors including common DC progenitors and pre-cDCs were marked by extremely high expression of IRF8-EGFP. Taken together, these data revealed a sequential expression pattern of IRF8-EGFP in early hematopoietic progenitors coinciding with lineage priming and commitment.

G A A b st ra ct s an important role in the homeostasis of PP in response to the microbiota. However, we do not know the respective roles of Nod2 in the epithelial and immune compartments of PP. Methods: To investigate the part of Nod2 in epithelial and immune compartments within PP, chimeric mice were developed by reconstituting sublethally irradiated wild-type (WT) mice with bone marrow (BM) stem cells from Nod2 Knockout mice (KO) and vice versa. Three months following BM transplantation, paracellular permeability (FITC 4kD dextran flux) and E. coli translocation were assessed in Ussing chamber. The number of immune cells and cellular composition from PP of chimeric mice were determined by flow cytometry. Results: An excess of permeability and E. coli translocation was observed in PP of KO mice reconstituted by BM of KO mice (KO→KO) compared to WT mice reconstituted by BM WT mice (WT→WT). Permeability and E. coli translocation were also higher in PP from chimeric mice that did not express Nod2 in the immune compartment but expressed it in epithelial cells (KO→WT). These excesses of permeability and translocation in PP were comparable to Nod2 Knockout mice (KO→KO). Conversely, permeability and E. coli translocation from chimeric mice expressing Nod2 in immune cells but not in epithelial cells (WT→KO) were similar to controls. An excess of CD3+ T-cells but a decreased number of dendritic cells were observed in the PP from chimeric mice that did not express Nod2 in immune cells (KO→KO and KO→WT). Conclusion: Nod2 deficiency in the immune compartment is sufficient to alter the cellular composition of PP and the functionality of the follicle associated epithelium. These results highlight the importance of Nod2 in the dialogue between the immune and epithelial cells and may contribute to understand CD mechanisms.