Metabolic requirements of NK cells during the acute response against retroviral infection.

Natural killer (NK) cells are important early responders against viral infections. Changes in metabolism are crucial to fuel NK cell responses, and altered metabolism is linked to NK cell dysfunction in obesity and cancer. However, very little is known about the metabolic requirements of NK cells during acute retroviral infection and their importance for antiviral immunity. Here, using the Friend retrovirus mouse model, we show that following infection NK cells increase nutrient uptake, including amino acids and iron, and reprogram their metabolic machinery by increasing glycolysis and mitochondrial metabolism. Specific deletion of the amino acid transporter Slc7a5 has only discrete effects on NK cells, but iron deficiency profoundly impaires NK cell antiviral functions, leading to increased viral loads. Our study thus shows the requirement of nutrients and metabolism for the antiviral activity of NK cells, and has important implications for viral infections associated with altered iron levels such as HIV and SARS-CoV-2.

Is There Natural Killer Cell Memory and Can It Be Harnessed by Vaccination? Can Natural Killer and CD8 T Cells Switch Jobs?

Natural killer (NK) cells are components of innate immunity mediating defense at early times after viral infections. Their cytokine production and cell-mediated cytotoxicity functions overlap those of CD8 T cells elicited later during primary adaptive immune responses, but the populations are distinguished by their basal states and activating receptors as well as the kinetics of their responses. Demonstration of long-lived NK cells has led to speculation on the potential for inducing these to contribute to immunological memory. Conversely, activated CD8 T cells can acquire responses to innate cytokines and, as a result, have the potential to contribute to innate immunity. These observations beg the question: what is required to be a player in innate and adaptive immunity?

mTORC1-dependent metabolic reprogramming is a prerequisite for NK cell effector function.

The mammalian target of rapamycin 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 NK cells, lymphocytes that play key roles in antiviral and antitumor immunity. This study investigated the hypothesis that mTORC1-controlled metabolism underpins normal NK cell proinflammatory 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 IFN-γ, which is 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, upregulating 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 IFN-γ 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.

TRIM68 negatively regulates IFN-ß production by degrading TRK fused gene, a novel driver of IFN-ß downstream of anti-viral detection systems.

In recent years members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to both positively and negatively regulate viral defence and as such are emerging as compelling targets for modulating the anti-viral immune response. In this study we identify TRIM68, a close homologue of TRIM21, as a novel regulator of Toll-like receptor (TLR)- and RIG-I-like receptor (RLR)-driven type I IFN production. Proteomic analysis of TRIM68-containing complexes identified TRK-fused gene (TFG) as a potential TRIM68 target. Overexpression of TRIM68 and TFG confirmed their ability to associate, with TLR3 stimulation appearing to enhance the interaction. TFG is a known activator of NF-κB via its ability to interact with inhibitor of NF-κB kinase subunit gamma (IKK-γ) and TRAF family member-associated NF-κB activator (TANK). Our data identifies a novel role for TFG as a positive regulator of type I IFN production and suggests that TRIM68 targets TFG for lysosomal degradation, thus turning off TFG-mediated IFN-ß production. Knockdown of TRIM68 in primary human monocytes resulted in enhanced levels of type I IFN and TFG following poly(I:C) treatment. Thus TRIM68 targets TFG, a novel regulator of IFN production, and in doing so turns off and limits type I IFN production in response to anti-viral detection systems.

Mutations in GATA2 cause human NK cell deficiency with specific loss of the CD56(bright) subset.

Mutations in the transcription factor GATA2 underlie the syndrome of monocytopenia and B- and natural killer (NK)-cell lymphopenia associated with opportunistic infections and cancers. In addition, patients have recurrent and severe viral infections. NK cells play a critical role in mediating antiviral immunity. Human NK cells are thought to mature in a linear fashion, with the CD56(bright) stage preceding terminal maturation to the CD56(dim) stage, considered the most enabled for cytotoxicity. Here we report an NK cell functional defect in GATA2-deficient patients and extend this genetic lesion to what is considered to be the original NK cell-deficient patient. In most cases, GATA2 deficiency is accompanied by a severe reduction in peripheral blood NK cells and marked functional impairment. The NK cells detected in peripheral blood of some GATA2-deficient patients are exclusively of the CD56(dim) subset, which is recapitulated on in vitro NK cell differentiation. In vivo, interferon α treatment increased NK cell number and partially restored function but did not correct the paucity of CD56(bright) cells. Thus, GATA2 is required for the maturation of human NK cells and the maintenance of the CD56(bright) pool in the periphery. Defects in GATA2 are a novel cause of profound NK cell dysfunction.

Natural killer cell responses during viral infections: flexibility and conditioning of innate immunity by experience.

Natural killer (NK) cells mediate innate defense against viral infections, but the mechanisms in place to access their functions as needed during diverse challenges while limiting collateral damage are poorly understood. Recent molecular characterization of effects mediated through infection-induced inhibitory/activating NK receptor-ligand pairs and cytokines are providing new insights into pathways regulating their responses and revealing unexpected consequences for NK cell subset effects, maintenance, proliferation and function through times overlapping with adaptive and long-lived immunity. The observations define flexible pathways for experience-induced 'conditioning' and challenge narrowly defined roles for NK cells and innate immunity as first responders with prescribed functions. They suggest that individual experiences as well as genes influence the innate immune resources available to fight off an infection.

Here today--not gone tomorrow: roles for activating receptors in sustaining NK cells during viral infections.

The conclusive evidence supporting a role for NK cells in defense against viruses has been obtained under conditions of NK cell deficiencies prior to infections. NK cell proliferation can be induced during infections, but the advantages of resulting expansion have been unclear because NK cell basal frequency is already high. However, NK cell decreases are also observed during certain conditions of viral infection. Given the range of potent antiviral and immunoregulatory functions of NK cells, such "disappearance" dramatically changes the resources available to the host. New studies demonstrate that proliferation dependent on activating receptors for virus-induced ligands is key for NK cell maintenance, and allows their continued availability for control of adaptive immune responses and immunopathology. This pathway for sustaining NK cells may represent a system used generally to select subsets for rescue during homeostatic purging. In the case of NK cells, though, nonselection limits continued access to the many beneficial functions of NK cells. The observations resolve the long-standing conundrum of reported NK cell increases and decreases during viral infections. Moreover, they demonstrate a previously unappreciated role for activating receptors, i.e. to keep NK cells here today and also tomorrow.

On immunologists and microbiologists: ground zero in the battle for interdisciplinary knowledge.

The individual disciplines of microbiology and immunology are exploding with new information necessary for understanding host-pathogen relationships, infectious diseases, cancer, and autoimmunity. Because of overlapping scientific interests, immunologists and microbiologists often share common academic affiliations. The coexistence is uneasy. Significant problems arise because the groups have evolved different intellectual traditions. Pressures are intensified by sporadic changes in perceptions of their relative worth. As the mixing of microbiologists and immunologists can be likened to ground zero in the fight for interdisciplinary knowledge, it is useful, at this time of escalating data acquisition and growing appreciation for multidisciplinary research, to examine their histories, the challenges to amalgamation, and the advantages of their association for the advancement of knowledge and the delivery of protection against disease. The exploration supports a recommitment to integration of the disciplines and a proposal to facilitate this by inclusion of expertise bridging the areas.

A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA.

CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified HMGB1 as a CpG-ODN-binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9's redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFalpha secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFalpha, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.

Keeping NK cells in highly regulated antiviral warfare.

Natural killer (NK) cells use multiple mechanisms to defend against viral infections, and different stimuli can activate these antiviral effects. When engaged, receptors for innate cytokines produced during infections and for ligands on target cells can both induce NK cell cytotoxicity and the production of cytokines. These stimuli use different classes of intracellular signaling pathways to elicit the overlapping responses. What is the advantage of using different roads to the same ends? One answer might be in the nature of the alternative regulatory pathways that are in place to control the respective stimuli. A model of flexibility in accessing NK cell function, in the context of negative regulation of particular intracellular signaling pathways, is proposed here.

Ikaros is required for plasmacytoid dendritic cell differentiation.

Plasmacytoid dendritic cells (pDCs) are specialized DCs that produce high levels of type I IFN upon viral infection. Despite their key immunoregulatory role, little is known about pDC ontogeny or how developmental events regulate their function. We show that mice expressing low levels of the transcription factor Ikaros (Ik(L/L)) lack peripheral pDCs, but not other DC subsets. Loss of pDCs is associated with an inability to produce type I IFN after challenge with Toll-like receptor-7 and -9 ligands, or murine cytomegalovirus (MCMV) infection. In contrast, conventional DCs are present in normal numbers and exhibit normal responses in vivo after challenge with MCMV or inactivated toxoplasma antigen. Interestingly, Ik(L/L) bone marrow (BM) cells contain a pDC population that appears blocked at the Ly-49Q- stage of differentiation and fails to terminally differentiate in response to Flt-3L, a cytokine required for pDC differentiation. This differentiation block is strictly dependent on a cell-intrinsic requirement for Ikaros in pDC-committed precursors. Global gene expression profiling of Ik(L/L) BM pDCs reveals an up-regulation of genes not normally expressed, or expressed at low levels, in WT pDCs. These studies suggest that Ikaros controls pDC differentiation by silencing a large array of genes.

MyD88-dependent and -independent murine cytomegalovirus sensing for IFN-alpha release and initiation of immune responses in vivo.

Antiviral immunity requires early and late mechanisms in which IFN-alpha and IL-12 play major roles. However, the initial events leading to their production remain largely unclear. Given the crucial role of TLR in innate recognition, we investigated their role in antiviral immunity in vivo. Upon murine CMV (MCMV) infection, both MyD88-/- and TLR9-/- mice were more susceptible and presented increased viral loads compared with C57BL/6, TLR2-/-, TLR3-/-, or TLR4-/- mice. However, in terms of resistance to infection, IFN-alpha production and in many other parameters of early inflammatory responses, the MyD88-/- mice showed a more defective response than TLR9-/- mice. In the absence of the TLR9/MyD88 signaling pathway, cytokine production was dramatically impaired with a complete abolition of bioactive IL-12p70 serum release contrasting with a high flexibility for IFN-alpha release, which is initially (36 h) plasmacytoid dendritic cell- and MyD88-dependent, and subsequently (44 h) PDC-, MyD88-independent and, most likely, TLR-independent. NK cells from MCMV-infected MyD88-/- and TLR9-/- mice displayed a severely impaired IFN-gamma production, yet retained enhanced cytotoxic activity. In addition, dendritic cell activation and critical inflammatory cell trafficking toward the liver were still effective. In the long term, except for isotype switching to MCMV-specific IgG1, the establishment of Ab responses was not significantly altered. Thus, our results demonstrate a critical requirement of TLR9 in the process of MCMV sensing to assure rapid antiviral responses, coordinated with other TLR-dependent and -independent events that are sufficient to establish adaptive immunity.

Immune modulation of the hypothalamic-pituitary-adrenal (HPA) axis during viral infection.

Compelling data has been amassed indicating that soluble factors, or cytokines, emanating from the immune system can have profound effects on the neuroendocrine system, in particular the hypothalamic- pituitary-adrenal (HPA) axis. HPA activation by cytokines (via the release of glucocorticoids), in turn, has been found to play a critical role in restraining and shaping immune responses. Thus, cytokine-HPA interactions represent a fundamental consideration regarding the maintenance of homeostasis and the development of disease during viral infection. Although reviews exist that focus on the bi-directional communication between the immune system and the HPA axis during viral infection (188,235), others have focused on the immunomodulatory effects of glucocorticoids during viral infection (14,225). This review, however, concentrates on the other side of the bi-directional loop of neuroendocrine-immune interactions, namely, the characterization of HPA axis activity during viral infection and the mechanisms employed by cytokines to stimulate glucocorticoid release.

Defective B cell responses in the absence of SH2D1A.

More than half of patients with X-linked lympho-proliferative disease, which is caused by a defect in the intracellular adapter protein SH2D1A, suffer from an extreme susceptibility to Epstein-Barr virus. One-third of these patients, however, develop dysgammaglobulenemia without an episode of severe mononucleosis. Here we show that in SH2D1A(-/-) mice, both primary and secondary responses of all Ig subclasses are severely impaired in response to specific antigens. Because germinal centers were absent in SH2D1A(-/-) mice upon primary immunization, and because SH2D1A was detectable in wt germinal center B cells, we examined whether SH2D1A(-/-) B cell functions were impaired. Using the adoptive cotransfer of B lymphocytes from hapten-primed SH2D1A(-/-) mice with CD4(+) T cells from primed wt mice into irradiated wt mice provided evidence that signal transduction events controlled by SH2D1A are essential for B cell activities resulting in antigen specific IgG production. Defects in naive SH2D1A(-/-) B cells became evident upon cotransfer with non-primed wt CD4(+) cells into Rag2(-/-) recipients. Thus, both defective T and B cells exist in the absence of SH2D1A, which may explain the progressive dysgammaglobulinemia in a subset of X-linked lympho-proliferative disease patients without involvement of Epstein-Barr virus.

T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells.

Natural killer (NK) and CD1d-restricted Valpha14i natural killer T (NKT) cells play a critical early role in host defense. Here we show that mice with a targeted deletion of T-bet, a T-box transcription factor required for Th1 cell differentiation, have a profound, stem cell-intrinsic defect in their ability to generate mature NK and Valpha14i NKT cells. Both cell types fail to complete normal terminal maturation and are present in decreased numbers in peripheral lymphoid organs of T-bet(-/-) mice. T-bet expression is regulated during NK cell differentiation by NK-activating receptors and cytokines known to control NK development and effector function. Our results identify T-bet as a key factor in the terminal maturation and peripheral homeostasis of NK and Valpha14i NKT cells.

Characterization of an interleukin-6- and adrenocorticotropin-dependent, immune-to-adrenal pathway during viral infection.

There has been longstanding interest in the capacity of the immune system to access immunomodulatory glucocorticoid responses without invoking upstream neuroendocrine secretagogues, including CRH and ACTH. Here, we investigate the role of CRH and ACTH in adrenal glucocorticoid responses to murine cytomegalovirus (MCMV). Mice infected with MCMV exhibit IL-6-dependent glucocorticoid responses that peak at 36 h post infection and protect against cytokine (TNFalpha)-mediated lethality. Acute administration of a CRH-antibody (Ab) completely eliminated ACTH responses to both low- and high-dose MCMV. However, corticosterone responses in CRH-Ab-treated animals remained apparent in mice infected with low-dose MCMV and were robust in mice infected with high-dose MCMV. CRH-knockout (KO) mice exhibited robust corticosterone responses to both MCMV doses, despite reduced baseline and MCMV-induced ACTH. Interestingly, robust corticosterone responses in CRH-Ab-treated and CRH-KO mice were associated with exaggerated IL-6 levels, and IL-6 and corticosterone concentrations in infected CRH-Ab-treated animals were significantly correlated. Neutralization of IL-6 responses in infected CRH-KO mice reduced corticosterone responses by approximately 70%. Finally, MCMV-infected mice deprived of ACTH by hypophysectomy failed to elicit glucocorticoid responses, despite elevated plasma IL-6 concentrations. Taken together, these results suggest that a greater than normal induction of IL-6 compensates for the absence of a normal CRH-dependent ACTH surge during viral infection. This enhanced IL-6 response, in turn, may mediate a direct immune-adrenal pathway that can become a predominant driving force for glucocorticoid induction in the absence of CRH. However, the presence of ACTH appears to serve as a necessary permissive factor, enabling direct cytokine actions on the adrenal gland.

TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection.

Dendritic cells (DCs) present microbial antigens to T cells and provide inflammatory signals that modulate T cell differentiation. While the role of DCs in adaptive immunity is well established, their involvement in innate immune defenses is less well defined. We have identified a TNF/iNOS-producing (Tip)-DC subset in spleens of Listeria monocytogenes-infected mice that is absent from CCR2-deficient mice. The absence of Tip-DCs results in profound TNF and iNOS deficiencies and an inability to clear primary bacterial infection. CD8 and CD4 T cell responses to L. monocytogenes antigens are preserved in CCR2-deficient mice, indicating that Tip-DCs are not essential for T cell priming. Tip-DCs, as the predominant source of TNF and iNOS during L. monocytogenes infection, orchestrate and mediate innate immune defense against this intracellular bacterial pathogen.

Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection.

NK cell cytotoxicity, IFN-gamma expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-gamma expression, whereas IFN-alphabeta/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-alphabeta/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-alphabeta, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors' nonredundant functions under in vivo physiological conditions.

Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1alpha delivery to the liver.

Macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) is critical for liver NK cell inflammation and delivery of IFN-gamma to mediate downstream protective responses against murine cytomegalovirus (MCMV) infections. This system was used to evaluate the upstream contribution of the type 1 IFNs, IFN-alpha/beta, in promotion of MIP-1alpha production. Mice deficient in IFN-alpha/beta functions, as a result of mutation in the receptor for these cytokines (IFN-alpha/betaR(-)), were profoundly deficient in MIP-1alpha expression and accumulation of NK cells and macrophages in the liver and had increased sensitivity to MCMV infection. The cytokines themselves were responsible for the immunoregulatory effects, since administration of recombinant IFN-alpha (rIFN-alpha) to immunocompetent mice also induced these changes. IFN-alpha/beta was required for NK cell accumulation during infection, and MIP-1alpha was required for NK cell accumulation in response to administered rIFN-alpha. In vivo trafficking assays demonstrated a requirement for IFN-alpha/betaR signaling for leukocyte localization in, and delivery of MIP-1alpha-producing macrophages to, the liver. These results extend characterization of the cytokine and chemokine cascade required for protection against viral infections in tissues by defining IFN-alpha/beta-dependent mechanisms promoting MIP-1alpha production and the resulting hepatic accumulation of NK cells.