The detection of intracellular microbial DNA is critical to an appropriate innate immune response, however current knowledge on how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor mediating interferon-β (IFNβ)-induction. IFI16 directly associated with IFNβ-inducing viral DNA motifs. STING, a critical mediator of IFNβ responses to DNA, was recruited to IFI16 after DNA stimulation. Reduction of expression of IFI16, or its murine ortholog p204, by RNA interference inhibited DNA- and herpes simplex virus (HSV)-1-induced gene induction and IRF3 and NFκB activation. IFI16/p204 is the first PYHIN protein shown to be involved in IFNβ induction, and thus together with AIM2, a PYHIN protein that senses DNA for caspase 1 activation, is part of a new family of innate DNA sensors which we term AIM2-like receptors (ALRs).

The mammalian target of rapamcyin complex 1 (mTORC1) is a key regulator of cellular metabolism and also has fundamental roles in controlling immune responses. Emerging evidence suggests that these two functions of mTORC1 are integrally linked. However, little is known regarding mTORC1 function in controlling the metabolism and function of natural killer (NK) cells, lymphocytes that play key roles in anti-viral and anti-tumour immunity. This study investigated the hypothesis that mTORC1-controlled metabolism underpins normal NK cell pro-inflammatory function. We demonstrate that mTORC1 is robustly stimulated in NK cells activated in vivo and in vitro. This mTORC1 activity is required for the production of the key NK cell effector molecules IFNγ, important in delivering antimicrobial and immunoregulatory functions, and granzyme B, a critical component of NK cell cytotoxic granules. The data reveal that NK cells undergo dramatic metabolic reprogramming upon activation, up-regulating rates of glucose uptake and glycolysis, and that mTORC1 activity is essential for attaining this elevated glycolytic state. Directly limiting the rate of glycolysis is sufficient to inhibit IFNγ production and granzyme B expression. This study provides the highly novel insight that mTORC1-mediated metabolic reprogramming of NK cells is a prerequisite for the acquisition of normal effector functions.

Human NK cells can be classified into phenotypically and functionally distinct subsets based on levels of CD56 receptor. CD56dim cells are generally considered more cytotoxic, whereas the CD56bright cells are potent producers of IFN-γ. In this study, we define the metabolic changes that occur in peripheral blood NK cells in response to cytokine. Metabolic analysis showed that NK cells upregulate glycolysis and oxidative phosphorylation in response to either IL-2 or IL-12/15 cytokine combinations. Despite the fact that both these cytokine combinations robustly upregulated mammalian Target of Rapamycin Complex 1 in human NK cells, only the IL-2–induced metabolic changes were sensitive to mammalian Target of Rapamycin Complex 1 inhibition by rapamycin. Interestingly, we found that CD56bright cells were more metabolically active compared with CD56dim cells. They preferentially upregulated nutrient receptors and also differed substantially in terms of their glucose metabolism. CD56bright cells expressed high levels of the glucose uptake receptor, Glut1 (in the absence of any cytokine), and had higher rates of glucose uptake compared with CD56dim cells. Elevated levels of oxidative phosphorylation were required to support both cytotoxicity and IFN-γ production in all NK cells. Finally, although elevated glycolysis was not required directly for NK cell degranulation, limiting the rate of glycolysis significantly impaired IFN-γ production by the CD56bright subset of cells. Overall, we have defined CD56bright NK cells to be more metabolically active than CD56dim cells, which supports their production of large amounts of IFN-γ during an immune response.

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFB signaling axis, recent data have shown that it is dispensable for NFB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFB activation pathway. The poxviral TLR antagonist A52 inhibited NFB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFB. Toll-like receptors (TLRs)3 recognize specific pathogen-associated molecular patterns found on infectious agents (1). TLRs are part of the larger IL-1R/TLR superfamily, defined by the presence of a cytoplasmic Toll-IL-1R-resistance (TIR) signaling domain, which also includes the IL-1, IL-18, and IL-33 receptors. Upon engagement of distinct TLRs by specific pathogen-associated molecular patterns, such as bacterial lipoprotein (for TLR2), viral dsRNA (for TLR3), or LPS (for TLR4), intracellular signaling cascades mediate activation of transcription factors such as NFB and interferon regulatory factors (IRFs) leading to gene induction and the production of pro-inflammatory cytokines, chemokines and interferons.Upon activation, IL-1R and all of the TLRs, excluding only TLR3, recruit the TIR domain-containing adaptor molecule myeloid differentiation factor 88 (MyD88) through a TIR-TIR homotypic protein interaction, leading to NFB activation (2). For TLR2 and TLR4, the TIR adaptor Mal (3) recruits MyD88 to the receptor complex (4). TLR3 utilizes another TIR adaptor known as TIR domain-containing adaptor inducing interferon ␤ (TRIF) (5, 6). TLR4 can also signal through TRIF, via a bridging adaptor called TRIF-related adaptor molecule (TRAM), resulting in a delayed MyD88-independent NFB response (7). Formation of the TLR⅐adaptor complex leads to the recruitment of interleukin-1 receptor associated kinases (IRAKs) (8). Four IRAKs have been identified namely IRAK-1, IRAK-2, IRAK-M, and IRAK-4. Both IRAK-1 and IRAK-4 are active Ser/ Thr kinases, and phosphorylation of IRAK-1 by IRAK-4 is crucial for IRAK-1 activation during TLR signaling (9, 10). Mice lacking IRAK-4 are completely resistant to LPS t...

No abstract

Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV+ B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.

Epstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II؉ B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature ␣␤Ii heterotrimers and mature ␣␤-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitt's lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity. Epstein-Barr virus (EBV)is a large DNA virus belonging to the family Herpesviridae, subfamily Gammaherpesviriniae, genus Lymphocryptovirus. Like other herpesviruses, EBV persists for life, establishing a latent infection in B lymphocytes with occasional viral reactivation (29). Approximately 90% of the adult population carries EBV DNA. EBV is the causative agent of infectious mononucleosis and is associated with malignancies that originate from lymphoid cells (e.g., Burkitt's lymphoma and Hodgkin's lymphoma) and epithelial cells (e.g., nasopharyngeal carcinoma). In immunosuppressed or immunocompromised individuals, EBV can cause (fatal) lymphoproliferative disease. In contrast, in healthy individuals, EBV is well controlled by the immune system. The widespread and mostly asymptomatic persistent EBV infections in adults reflect the balance between viral replication and host immune control.EBV dedicates part of its genome to immune evasion functions (15). Modulation of T-cell recognition is an important target for EBV, as the virus resides intracellularly for most of its life cycle. Fragments of viral proteins can be displayed at the cell surface by human leukocyte antigen (HLA) class I and class II molecules for the activation of T cells carrying receptors of the appropriate specificity. Activated T cells may then lead to elimination of the pathogen, among other ways through destruction of the infeced cell. To escape from antiviral immunity, EBV should interfere with both CD8 ϩ and CD4 ϩ T-cell responses, particularly as the virus infects B lymphocytes expressing both classes of HLA molecules. The EBV nuclear protein EBNA1 contains a glycine-alanine repeat domain that renders the protein resistant to proteasomal degradation and inhibits...

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus found in more than 90% of adults in all populations worldwide. Following primary infection in immunocompetent individuals, the virus generally establishes lifelong persistence in B lymphocytes. Most EBV-infected B cells in the healthy host show a resting phenotype with very limited expression of the viral genome (34,41,58). However, expression of a broader panel of 11 "latent" viral genes leads to growth transformation of B cells (26,27). This growth transformation is thought to be a mechanism for expansion of the pool of EBV in the infected host. In addition, the lytic replicative cycle, which produces infectious virions, is likely to be important for periodic expansion of the pool of virus-infected cells within the host and for horizontal transmission of the virus. Latently infected, growthtransformed B-lymphoblastoid cells are good targets for EBVspecific cytotoxic T lymphocytes (CTL). This limits the potentially pathogenic consequences of uncontrolled proliferation of virus-infected cells (43). It is unclear whether lytically infected cells are subjected to the same immune controls. CD8 ϩ CTL are a major line of defense in the immune response to viral infection (23,43,44). The T-cell receptors on virus-specific CTL interact with major histocompatibility complex (MHC) class I molecules presenting viral peptides on the surface of the infected cell. Presentation of viral peptides involves several components of the endogenous antigen-processing pathway, including cleavage of antigen by proteasomes, translocation of peptides into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP), and loading onto heterodimers of newly synthesized MHC class I heavy chain and ␤ 2 -microglobulin. This stabilized trimeric complex is then transported to the plasma membrane (33, 40). In EBV-transformed lymphoblastoid cells, the virally encoded latent membrane protein-1 (LMP1) enhances antigen-processing functions at several levels. LMP1 alters proteasome subunit expression and upregulates expression of TAP subunit proteins, MHC class I and class II molecules (51, 62), and cell surface adhesion molecules (20,59). Conversely, the EBNA1 protein of EBV contains an unusual glycine-alanine repeat sequence which, while not interfering with antigen processing of other viral and cell genes, prevents processing of antigenic EBNA1 peptides and their presentation to CTL (29).While it is crucial for the host-virus relationship that proliferation of growth-transformed EBV-infected B cells be controlled by virus-specific CTL immunosurveillance, efficient virus production in the lytic cycle would benefit from effective evasion of CTL responses. As reviewed elsewhere (5, 23, 40), the alpha-and betaherpesviruses utilize numerous immune evasion strategies in order to achieve persistence, and with rare exceptions, the viral genes demonstrated to interfere with immune recognition are expressed during the lytic cycle of these herpesviruses. Only r...