FURIN regulates cytotoxic T-lymphocyte effector function and memory cell transition in mice.

The proprotein convertase subtilisin/kexins (PCSKs) regulate biological actions by cleaving immature substrate proteins. The archetype PCSK, FURIN, promotes the pathogenicity of viruses by proteolytically processing viral proteins. FURIN has also important regulatory functions in both innate and adaptive immune responses but its role in the CD8+ CTLs remains enigmatic. We used a T-cell-specific FURIN deletion in vivo to demonstrate that FURIN promotes host response against the CTL-dependent lymphocytic choriomeningitis virus by virtue of restricting viral burden and augmenting interferon gamma (IFNG) production. We also characterized Furin KO CD8+ T cells ex vivo, including after their activation with FURIN regulating cytokines IL12 or TGFB1. Furin KO CD8+ T cells show an inherently activated phenotype characterized by the upregulation of effector genes and increased frequencies of CD44+ , TNF+ , and IFNG+ cells. In the activated CTLs, FURIN regulates the productions of IL2, TNF, and GZMB and the genes associated with the TGFBR-signaling pathway. FURIN also controls the expression of Eomes, Foxo1, and Bcl6 and the levels of ITGAE and CD62L, which implies a role in the development of CTL memory. Collectively, our data suggest that the T-cell expressed FURIN is important for host responses in viral infections, CTL homeostasis/activation, and memory development.

More things in heaven and earth: defining innate and adaptive immunity.

Natural killer cells have emerged as key components of innate immunity with critical antimicrobial functions. New work showing that they can also be accessed by vaccination to deliver antigen-specific memory responses and protect against subsequent viral infections challenges the traditional distinctions made between 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.

Proliferation conditions promote intrinsic changes in NK cells for an IL-10 response.

Constitutively found at high frequencies, the role for NK cell proliferation remains unclear. In this study, a shift in NK cell function from predominantly producing IFN-γ, a cytokine with proinflammatory and antimicrobial functions, to producing the immunoregulatory cytokine IL-10 was defined during extended murine CMV infection. The response occurred at times subsequent to IL-12 production, but the NK cells elicited acquired responsiveness to IL-12 and IL-21 for IL-10 production. Because neither IL-12 nor IL-21 was required in vivo, however, additional pathways appeared to be available to promote NK cell IL-10 expression. In vitro studies with IL-2 to support proliferation and in vivo adoptive transfers into murine CMV-infected mice demonstrated that NK cell proliferation and further division enhanced the change. In contrast to the sustained open profile of the IFN-γ gene, NK cells responding to infection acquired histone modifications in the IL-10 gene indicative of changing from a closed to an open state. The IL-10 response to IL-12 was proliferation dependent ex vivo if the NK cells had not yet expanded in vivo but independent if they had. Thus, a novel role for proliferation in supporting changing innate cell function is reported.

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.

Immunoregulatory cytokine networks: 60 years of learning from murine cytomegalovirus.

Innate immunity defends against infection but also mediates immunoregulatory effects shaping innate and adaptive responses. Studies of murine cytomegalovirus (MCMV) infections have helped elucidate the mechanisms inducing, as well as the elicited soluble and cellular networks contributing to, innate immunity. Specialized receptors are engaged by infection-induced structures to stimulate production of key innate cytokines. These then stimulate cytokine and cellular responses such as activation of natural killer (NK) cells to mediate elevated killing by type 1 interferon (IFN) and/or to produce the pro-inflammatory and antiviral cytokine IFN-γ by interleukin 12 (IL-12). An inter-systemic loop, with IL-6 inducing glucocorticoid release, negatively regulates these early cytokine responses. As infections advance into periods of overlapping innate and adaptive responses, however, the cells are intrinsically conditioned to modify the biological effects of exposure to individual cytokines. Some pathways are turned off to inhibit an existing, whereas others are broadened for acquisition of a new, response function. Remarkably, extended NK cell proliferation during MCMV infection is associated with epigenetic modifications shifting the state of the inhibitory cytokine IL-10 gene from closed to open and results in their becoming equipped to produce this cytokine. When induced, NK cell IL-10 negatively regulates the magnitude of adaptive responses to protect against immune pathology. Thus, innate immunoregulatory cytokine networks are integral to pro-inflammatory and defense functions, but responding cells have the flexibility to undergo cell intrinsic conditioning with changing network characteristics to result in a new negative immunoregulatory function, and consequently, both promote beneficial and limit detrimental immune responses.

Regulating type 1 IFN effects in CD8 T cells during viral infections: changing STAT4 and STAT1 expression for function.

Type 1 IFNs can conditionally activate all of the signal transducers and activators of transcription molecules (STATs), including STAT4. The best-characterized signaling pathways use STAT1, however, and type 1 IFN inhibition of cell proliferation is STAT1 dependent. We report that type 1 IFNs can basally stimulate STAT1- and STAT4-dependent effects in CD8 T cells, but that CD8 T cells responding to infections of mice with lymphocytic choriomenigitis virus have elevated STAT4 and lower STAT1 expression with significant consequences for modifying the effects of type 1 IFN exposure. The phenotype was associated with preferential type 1 IFN activation of STAT4 compared with STAT1. Stimulation through the TCR induced elevated STAT4 expression, and STAT4 was required for peak expansion of antigen-specific CD8 T cells, low STAT1 levels, and resistance to type 1 IFN-mediated inhibition of proliferation. Thus, a mechanism is discovered for regulating the consequences of type 1 IFN exposure in CD8 T cells, with STAT4 acting as a key molecule in driving optimal antigen-specific responses and overcoming STAT1-dependent inhibition of proliferation.

Cutting edge: a novel mechanism bridging innate and adaptive immunity: IL-12 induction of CD25 to form high-affinity IL-2 receptors on NK cells.

NK cell expression and use of the IL-2Rα-chain (CD25), required for the high-affinity IL-2R, remain poorly understood. The studies reported in this article demonstrate that infections with murine CMV (MCMV), but not with lymphocytic choriomeningitis virus, induce CD25 on NK cells, along with high levels of IL-12 and IL-18. The cytokines act ex vivo to increase CD25 levels, and IL-12, IL-12R, and STAT4, but not the NK activating receptor Ly49H, are required for peak induction in vivo. All examined NK cell populations are driven into proliferation and incorporate BrdU in response to high ex vivo concentrations of IL-2, but only those from MCMV infection respond to low ex vivo concentrations of IL-2. The numbers of NK cells elicited during MCMV infection are reduced by IL-2 neutralization. Thus, a link between innate and adaptive immunity is established by which composition of innate cytokine responses sets up to promote NK cell use of a factor supporting adaptive responses.

High basal STAT4 balanced by STAT1 induction to control type 1 interferon effects in natural killer cells.

The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-gamma induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN-mediated STAT4 activation for IFN-gamma production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-gamma, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-gamma production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.

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.

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.

Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta.

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.

Interferon alpha/beta and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo.

Interferon (IFN)-alpha/beta and interleukin (IL)-12 are cytokines critical in defense against viruses, but their cellular sources and mechanisms of regulation for in vivo expression remain poorly characterized. The studies presented here identified a novel subset of dendritic cells (DCs) as major producers of the cytokines during murine cytomegalovirus (MCMV) but not lymphocytic choriomeningitis virus (LCMV) infections. These DCs differed from those activated by Toxoplasma antigen but were related to plasmacytoid cells, as assessed by their CD8alpha(+)Ly6G/C(+)CD11b(-) phenotype. Another DC subset (CD8alpha(2)Ly6G/C(-)CD11b(+)) also contributed to IL-12 production in MCMV-infected immunocompetent mice, modestly. However, it dramatically increased IL-12 expression in the absence of IFN-alpha/beta functions. Conversely, IFN-alpha/beta production was greatly reduced under these conditions. Thus, a cross-regulation of DC subset cytokine responses was defined, whereby secretion of type I IFNs by CD8alpha(+) DCs resulted in responses limiting IL-12 expression by CD11b(+) DCs but enhancing overall IFN-alpha/beta production. Taken together, these data indicate that CD8alpha(+)Ly6G/C(+)CD11b(-) DCs play important roles in limiting viral replication and regulating immune responses, through cytokine production, in some but not all viral infections. They also illustrate the plasticity of cellular sources for innate cytokines in vivo and provide new insights into the roles of IFNs in shaping immune responses to viruses.

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex.

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.

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?

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.

The neuronal chemokine CX3CL1/fractalkine selectively recruits NK cells that modify experimental autoimmune encephalomyelitis within the central nervous system.

Leukocyte trafficking to the central nervous system (CNS), regulated in part by chemokines, determines severity of the demyelinating diseases multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To examine chemokine receptor CX3CR1 in EAE, we studied CX3CR1(GFP/GFP) mice, in which CX3CR1 targeting by insertion of Green Fluorescent Protein (GFP) allowed tracking of CX3CR1+ cells in CX3CR1(+/GFP) animals and cells destined to express CX3CR1 in CX3CR1(GFP/GFP) knockouts. NK cells were markedly reduced in the inflamed CNS of CX3CR1-deficient mice with EAE, whereas recruitment of T cells, NKT cells and monocyte/macrophages to the CNS during EAE did not require CX3CR1. Impaired recruitment of NK cells in CX3CR1(GFP/GFP) mice was associated with increased EAE-related mortality, nonremitting spastic paraplegia and hemorrhagic inflammatory lesions. The absence of CD1d did not affect the severity of EAE in CX3CR1(GFP/GFP) mice, arguing against a role for NKT cells. Accumulation of NK cells in livers of wild-type (WT) and CX3CR1(GFP/GFP) mice with cytomegalovirus hepatitis was equivalent, indicating that CX3CL1 mediated chemoattraction of NK cells was relatively specific for the CNS. These results are the first to define a chemokine that governs NK cell migration to the CNS, and the findings suggest novel therapeutic manipulation of CX3CR1+ NK cells.