HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses.

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

Contribution of IRF5 in B cells to the development of murine SLE-like disease through its transcriptional control of the IgG2a locus.

Interferon regulatory factor (IRF) 5 is a key transcription factor for the activation of innate immune responses downstream of Toll-like receptor signaling. Based on recent genetic analyses, IRF5 is a focus for its potential involvement in systemic lupus erythematosus (SLE), although how IRF5 contributes to SLE is uncertain. In this study, we demonstrate a requirement for IRF5 in the development of murine SLE via its role in B lymphocytes. We show that antinuclear autoantibodies and Ig glomerular deposits, hallmarks of SLE, are absent in Irf5(-/-) mice challenged to develop SLE by pristane injection. In particular, production of autoantibodies of the IgG2a subtype, the most prominent isotype in inducing autoimmunity, requires IRF5. Finally, we provide evidence for the critical role of this transcription factor in the secretion of pathogenic antibodies through its direct control of class switch recombination of the gamma2a locus. By demonstrating a B-cell-intrinsic role, this study places IRF5 in a context that may have implications for understanding the pathogenesis of human SLE.

B-1 B lymphocytes require Blimp-1 for immunoglobulin secretion.

B-1 B cells produce circulating natural antibodies that provide "innate-like" protection against bacterial and viral pathogens. They also provide adaptive responses to blood and air-borne pathogens. B lymphocyte-induced maturation protein 1 (Blimp-1) is a transcriptional repressor that is required for the formation of B-2-derived antibody-secreting plasma cells. In this study, we used mice lacking Blimp-1 in the B cell lineage to show that Blimp-1 is not necessary for the formation or self-renewal of B-1 B cells but that Blimp-1 is required for normal immunoglobulin (Ig) secretion by B-1 cells. B-1 cells lacking Blimp-1 do not repress Pax5 mRNA and do not induce X-box binding protein 1, and mu secreted mRNA normally, showing that B-1 and B-2 cells both use a common pathway for Ig secretion. Blimp-1-deficient B-1 B cells are also defective in providing early protection against influenza infection.

A selective contribution of the RIG-I-like receptor pathway to type I interferon responses activated by cytosolic DNA.

The activation of the innate immune responses by DNA exposed within the cytosol has gained much attention and, in this context, several cytosolic DNA sensors have been identified. However, previous studies revealed the operation of redundant and complex mechanisms and it still remains to be clarified how the DNA-mediated evocation of diverse innate immune responses can be achieved. Here we show that two RIG-I-like receptors (RLRs), RIG-I and MDA5, known as cytosolic RNA receptors, nonredundantly function as cytosolic DNA receptors that lead to the selective activation of type I IFN genes. We demonstrate that overexpression of otherwise IFN-inducible RIG-I or MDA5 in IFN signal-deficient cells results in a marked enhancement of type I IFN gene induction upon cytosolic DNA stimulation, while in their absence the induction is impaired. Interestingly, the DNA-mediated induction of other cytokine genes was barely affected by the absence of RLRs. Indeed, unlike the RNA-RLR pathway that activates the transcription factors IRF3 and NF-kappaB, the DNA-RLR pathway is primarily responsible for the IRF3 activation critical for type I IFN gene transcription, illustrating a deliberate divergence of the DNA signaling pathways. Expectedly, the RLR pathway also contributes to intricate innate immune responses against infection by a DNA virus. Our study may provide insights into the complexity of host defense mechanisms that thwart immune evasion by DNA-containing pathogens.

Blimp-1 is required for maintenance of long-lived plasma cells in the bone marrow.

Long-lived plasma cells, residing primarily in the bone marrow, continuously secrete antibody and provide an important component of humoral memory. However, when such cells secrete autoantibodies or become transformed, they can be pathogenic. We have shown recently that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) is required for the formation of plasma cells. To determine what role Blimp-1 might play in maintenance of plasma cells, we generated mice in which the gene encoding Blimp-1 could be deleted in an inducible manner. Deletion of Blimp-1 either in vitro or in vivo leads to loss of previously formed B220(LO)CD138(HI) plasma cells. Using BrdU incorporation, we confirmed that Blimp-1 is required for the maintenance of nondividing, long-lived plasma cells in the bone marrow. Blimp-1 is also required for long-term maintenance of antigen-specific immunoglobulin in serum. Thus Blimp-1 is required not only for the formation but also for the maintenance of long-lived plasma cells. This finding provides the possibility of new drug design strategies for autoimmunity and multiple myeloma focused on blocking Blimp-1 expression or activity.

Blimp-1/PRDM1 mediates transcriptional suppression of the NLR gene NLRP12/Monarch-1.

NLR (nucleotide-binding domain, leucine-rich repeat) proteins are intracellular regulators of host defense and immunity. One NLR gene, NLRP12 (NLR family, pyrin domain containing 12)/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in human myeloid cells. This is supported by genetic analysis linking the loss of a functional NLRP12 protein to hereditary periodic fever. NLRP12 transcription is diminished by specific TLR stimulation and myeloid cell maturation, consistent with its role as a negative regulator of inflammation. The NLRP12 promoter contains a novel Blimp-1 (B lymphocyte-induced maturation protein-1)/PRDM1 (PR domain-containing 1, with ZNF domain) binding site, and Blimp-1 reduces NLRP12 promoter activity, expression, and histone 3 acetylation. Blimp-1 associates with the endogenous NLRP12 promoter in a TLR-inducible manner and mediates the down-regulation of NLRP12 expression by TLR agonists. As expected, the expression of NLRP12 and Blimp-1 is inversely correlated. Analysis of Blimp-1(-/-) murine myeloid cells provides physiologic evidence that Blimp-1 reduces NLRP12 gene expression during cell differentiation. This demonstrates a novel role for Blimp-1 in the regulation of an NLR gene.

Regulation of immunity and oncogenesis by the IRF transcription factor family.

Nine interferon regulatory factors (IRFs) compose a family of transcription factors in mammals. Although this family was originally identified in the context of the type I interferon system, subsequent studies have revealed much broader functions performed by IRF members in host defense. In this review, we provide an update on the current knowledge of their roles in immune responses, immune cell development, and regulation of oncogenesis.

Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens.

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting T cell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific T cell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory T cell expansion in mice.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

Regulation of the cytosolic DNA-sensing system in innate immunity: a current view.

The sensing of nucleic acids by pattern recognition receptors is a central feature of innate immunity and is mediated by two types of receptors, Toll-like receptors and cytosolic receptors. Indeed, DNA, be it pathogen-derived or self-derived, can potently trigger the innate immune system; and much attention has been focused on the regulation of the DNA-sensing system(s) in the context of protective and pathological immune responses. An accumulating body of evidence has demonstrated that in addition to the membrane-bound type DNA-sensing receptor TLR9, there are cytosolic DNA receptors that can also evoke these responses. In particular, DAI (DLM-1/ZBP1), Trex1, and other regulators of the cytosolic DNA-sensing system have recently been identified and characterized. Here, we summarize our current understanding of how cytosolic DNA receptors contribute to the regulation of innate immune responses and discuss the complexity of the cytosolic DNA-sensing system as well as its future prospects.

The IRF family transcription factors in immunity and oncogenesis.

The interferon regulatory factor (IRF) family, consisting of nine members in mammals, was identified in the late 1980s in the context of research into the type I interferon system. Subsequent studies over the past two decades have revealed the versatile and critical functions performed by this transcription factor family. Indeed, many IRF members play central roles in the cellular differentiation of hematopoietic cells and in the regulation of gene expression in response to pathogen-derived danger signals. In particular, the advances made in understanding the immunobiology of Toll-like and other pattern-recognition receptors have recently generated new momentum for the study of IRFs. Moreover, the role of several IRF family members in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers.

Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1.

The cornified layer is a compacted lattice of lipid-embedded corneocytes that provides an organism's barrier to the external environment. Cornification is the final differentiative step for epidermal keratinocytes and involves dramatic cell condensation before death. Using conditional gene deletion in mice, we identified the transcriptional repressor Blimp-1 (B lymphocyte-induced maturation protein-1) as an important regulator of keratinocyte transition from the granular to the cornified layer. More than 250 genes are misregulated in conditional knockout epidermis, including those encoding transcription factors, signal transduction components, proteinases, and enzymes involved in lipid metabolism. Steady-state mRNA and ChIP analyses of a subset of these genes provide evidence that nfat5, fos, prdm1, and dusp16 are novel direct targets of Blimp-1. Identifying nfat5 as a target of Blimp-1 repression indicates that cornification involves suppression of normal osmotic regulation in granular cells. Consistently, conditional knockout mice have delayed barrier formation as embryos, enlarged granular layer cells and corneocytes, and a morphologically abnormal cornified layer. These studies provide insight into cornification, identifying transcriptional regulatory circuitry and indicating the importance of blocking osmotic homeostasis.