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Natural killer (NK) effector functions can be triggered by inflammatory cytokines and engagement of activating receptors. NK cell production of IFN-γ, an important immunoregulatory cytokine, exhibits activation-specific IFN-γ regulation. Resting murine NK cells exhibit activation-specific metabolic requirements for IFN-γ production, which are reversed for activating receptor-mediated stimulation following IL-15 priming. While both cytokine and activating receptor stimulation leads to similar IFN-γ protein production, only cytokine stimulation upregulatesIfngtranscript, suggesting that protein production is translationally regulated after receptor stimulation. Based on these differences in IFN-γ regulation, we hypothesized that ex vivo IL-15 priming of murine NK cells allows a switch to IFN-γ transcription upon activating receptor engagement. Transcriptional analysis of primed NK cells compared to naïve cells or cells cultured with low-dose IL-15 demonstrated that primed cells strongly upregulatedIfngtranscript following activating receptor stimulation. This was not due to chromatin accessibility changes in theIfnglocus or changes in ITAM signaling, but was associated with a distinct transcriptional signature induced by ITAM stimulation of primed compared to naïve NK cells. Transcriptional analyses identified a common signature of c-Myc (Myc) targets associated withIfngtranscription. While Myc marked NK cells capable ofIfngtranscription, Myc itself was not required forIfngtranscription using a genetic model of Myc deletion. This work highlights altered regulatory networks in IL-15 primed cells, resulting in distinct gene expression patterns and IFN-γ regulation in response to activating receptor stimulation.

Summary Natural killer (NK) cell effector functions are dependent on metabolic regulation of cellular function; however, less is known about in vivo metabolic pathways required for NK cell antiviral function. Mice with an inducible NK-specific deletion of Cox10, which encodes a component of electron transport chain complex IV, were generated to investigate the role of oxidative phosphorylation in NK cells during murine cytomegalovirus (MCMV) infection. Ncr1-Cox10Δ/Δ mice had normal numbers of NK cells but impaired expansion of antigen-specific Ly49H+ NK cells and impaired NK cell memory formation. Proliferation in vitro and homeostatic expansion were intact, indicating a specific metabolic requirement for antigen-driven proliferation. Cox10-deficient NK cells upregulated glycolysis, associated with increased AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) activation, although this was insufficient to protect the host. These data demonstrate that oxidative metabolism is required for NK cell antiviral responses in vivo.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with

Natural killer (NK) cells are innate lymphocytes important for the control of infections and tumors. Their effector functions can be triggered by inflammatory cytokines or engagement of ITAM-bearing activating receptors. We previously reported that IFN-γ production through ITAM signaling is metabolism-dependent in naïve NK cells. However, cytokine stimulation of naïve cells or priming with high-dose IL-15 prior to ITAM stimulation leads to metabolism-independent IFN-γ production. While both cytokine and receptor stimulation leads to similar IFN-γ protein production, only cytokines upregulate Ifng transcript, suggesting that protein production is translationally regulated in the receptor condition. Based on these observations, we hypothesized that IL-15 priming leads to transcriptional regulation of IFN-γ production in response to ITAM signaling. Indeed, IL-15 primed NK cells strongly upregulated Ifng transcript after receptor stimulation, rendering this receptor stimulation metabolism-independent. Inhibition of glycolysis with 2DG or mTOR with rapamycin during priming inhibited Ifng upregulation upon receptor stimulation and returned metabolism-dependent IFN-γ production. RNA-seq analysis of cytokine-, receptor-stimulated, and IL-15 cultured NK cells identified an association between glycolysis, mTORC1 signaling, and MYC targets with Ifng upregulation. Ongoing work aims to identify how ITAM signaling differs in IL-15 primed versus naïve NK cells. Our studies demonstrate distinct mechanisms of IFN-γ protein production in response to ITAM-mediated signaling between naïve and IL-15 primed NK cells.

STAT3 is a transcription factor that plays a role in the proliferation, survival, and function of many different cell types. In T cells, STAT3 is required for the polarization and function of Th17 cells and can inhibit development of peripheral Tregs. STAT3 polymorphisms have been linked to several common autoimmune disorders where the balance between Th17 and Tregs has been disrupted. Patients with germline gain-of-function variants in STAT3 present with a syndrome of early-onset poly-autoimmunity. We generated two mouse models of STAT3 GOF by CRISPR/Cas9. In these models, mice have a single point mutation, p.G421R or p.T716M, which are two of the exact amino acid substitutions identified in patients. Heterozygous STAT3 GOF mice do not show spontaneous disease or constitutive activation of STAT3 and instead display delayed STAT3 de-phosphorylation following IL-6 stimulation. We hypothesized that STAT3 GOF would enhance Th17 responses and found increased Th17 polarization from naïve STAT3 GOF T cells in vitro. To test whether this increased disease susceptibility in vivo, we treated WT and STAT3 GOF mice with topical imiquimod. Compared to WT, STAT3 GOF mice have more severe disease as demonstrated by ear swelling and epidermal thickness, as well as increased numbers of Th17 cells in peripheral organs at day 7 and in the skin at day 14. No difference was observed in IL-17+ γδ T cell populations in these organs at either time point. STAT3 GOF mice crossed to Rag 1 −/− or IL-22−/− show decreased disease severity, indicating that this response may be mediated by adaptive lymphocytes and IL-22 signaling. These data indicate that STAT3 GOF can exacerbate disease by enhancing Th17 responses that may drive autoimmunity.

Summary Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages., Graphical Abstract, Highlights • Induced Eomes deletion results in a rapid decrease in NK cell numbers • Eomes-deleted stage III NK cells exhibit increased apoptosis • Eomes-deleted stage II and III NK cells exhibit differentiation defects • Induced Eomes deletion compromises NK cytotoxicity and MHCI−/− rejection in vivo, The transcription factor Eomes is important for early natural killer (NK) cell development. Wagner et al. utilize an inducible, type 1 ILC-specific cre model to demonstrate a stage-specific role for Eomes in NK cell survival and homeostasis as well as a persistent requirement for Eomes in promoting NK cell cytotoxicity.

There has been increasing recognition of the importance of cellular metabolism and metabolic substrates for the function and differentiation of immune cells. In this study, for the first time to our knowledge, we investigate the metabolic requirements for production of IFN-γ by freshly isolated NK cells. Primary murine NK cells mainly use mitochondrial oxidative phosphorylation at rest and with short-term activation. Remarkably, we discovered significant differences in the metabolic requirements of murine NK cell IFN-γ production depending upon the activation signal. Stimulation of NK cell IFN-γ production was independent of glycolysis or mitochondrial oxidative phosphorylation when cells were activated with IL-12 plus IL-18. By contrast, stimulation via activating NK receptors required glucose-driven oxidative phosphorylation. Prolonged treatment with high-dose, but not low-dose, IL-15 eliminated the metabolic requirement for receptor stimulation. In summary, this study demonstrates that metabolism provides an essential second signal for induction of IFN-γ production by activating NK cell receptors that can be reversed with prolonged high-dose IL-15 treatment.

NK cell activation has been shown to be metabolically regulated in vitro; however, the role of metabolism during in vivo NK cell responses to infection is unknown. We examined the role of glycolysis in NK cell function during murine cytomegalovirus (MCMV) infection and the ability of IL-15 to prime NK cells during CMV infection. The glucose metabolism inhibitor 2-deoxy-ᴅ-glucose (2DG) impaired both mouse and human NK cell cytotoxicity following priming in vitro. Similarly, MCMV-infected mice treated with 2DG had impaired clearance of NK-specific targets in vivo, which was associated with higher viral burden and susceptibility to infection on the C57BL/6 background. IL-15 priming is known to alter NK cell metabolism and metabolic requirements for activation. Treatment with the IL-15 superagonist ALT-803 rescued mice from otherwise lethal infection in an NK-dependent manner. Consistent with this, treatment of a patient with ALT-803 for recurrent CMV reactivation after hematopoietic cell transplant was associated with clearance of viremia. These studies demonstrate that NK cell-mediated control of viral infection requires glucose metabolism and that IL-15 treatment in vivo can reduce this requirement and may be effective as an antiviral therapy.

There has been increasing recognition of the importance of metabolism on immune cell differentiation, homeostasis, and function. Recently, our lab and others have begun to investigate the metabolic requirements for NK cell differentiation and activation. Here, we describe approaches for the in vitro assessment of NK cell metabolism. We present methods for using inhibitors to alter cellular metabolism, measurement of intracellular ATP in NK cells, assessment of real-time glycolysis and oxidative phosphorylation by an extracellular flux assay from Seahorse Biosciences, and some basic protocols for stimulation of NK cells via cytokines and receptors.

There has been increasing recognition of the importance of metabolism on immune cell differentiation, homeostasis, and function. Recently, our lab and others have begun to investigate the metabolic requirements for NK cell differentiation and activation. Here, we describe approaches for the in vitro assessment of NK cell metabolism. We present methods for using inhibitors to alter cellular metabolism, measurement of intracellular ATP in NK cells, assessment of real-time glycolysis and oxidative phosphorylation by an extracellular flux assay from Seahorse Biosciences, and some basic protocols for stimulation of NK cells via cytokines and receptors.

Studies in diverse cell types have rediscovered the importance of basic cellular metabolism in regulating immune responses. For example, glycolytic and oxidative metabolism are counter-regulated in T cells to develop inflammatory effectors vs. memory and regulatory cells, respectively. NK cells, which are first responders to viral infection and malignant transformation, may be similarly regulated. We recently showed that NK cell cytotoxic activity during infection with murine cytomegalovirus (MCMV) required glucose metabolism, whereas other NK cell responses such as cytokine production did not. We hypothesized that oxidative metabolism would be required for some of these glycolysis-independent responses. To test this, we examined responses to MCMV in mice with an NK-specific deletion of Cox10, which is required for assembly of electron transport chain complex IV. These “Ncr1-Cox10” mice had normal NK cell development and numbers, and stimulated Cox10-deficient NK cells survived and proliferated normally in vitro. However, Ncr1-Cox10 mice were more susceptible to MCMV infection, with impaired expansion of MCMV-specific Ly49H+ NK cells and increased apoptosis of splenic NK cells. Thus, we find that Cox10 is required by NK cells to expand and survive in response to MCMV infection. These deficiencies were not found in vitro, emphasizing the importance of using NK-specific genetic models to investigate metabolism in physiological contexts.

Autoimmune diseases are thought to be caused by a loss of self-tolerance of the immune system. One candidate marker of immune dysregulation in autoimmune disease is the presence of increased double negative T cells (DNTs) in the periphery. DNTs are characteristically elevated in autoimmune lymphoproliferative syndrome, a systemic autoimmune disease caused by defective lymphocyte apoptosis due to Fas pathway defects. DNTs have also been found in the peripheral blood of adult patients with systemic lupus erythematosus (SLE), where they may be pathogenic. DNTs in children with autoimmune disease have not been investigated.We evaluated DNTs in pediatric patients with SLE, mixed connective tissue disease (MCTD), juvenile idiopathic arthritis (JIA), or elevated antinuclear antibody (ANA) but no systemic disease. DNTs (CD3(+)CD56(-)TCRαβ(+)CD4(-)CD8(-)) from peripheral blood mononuclear cells were analyzed by flow cytometry from 54 pediatric patients including: 23 SLE, 15 JIA, 11 ANA and 5 MCTD compared to 28 healthy controls.Sixteen cases (29.6 %) had elevated DNTs (≥2.5 % of CD3(+)CD56(-)TCRαβ(+) cells) compared to 1 (3.6 %) control. Medication usage including cytotoxic drugs and absolute lymphocyte count were not associated with DNT levels, and percentages of DNTs were stable over time. Analysis of multiple phenotypic and activation markers showed increased CD45RA expression on DNTs from patients with autoimmune disease compared to controls.DNTs are elevated in a subset of pediatric patients with autoimmune disease and additional investigations are required to determine their precise role in autoimmunity.

Several recent studies have demonstrated that innate immune NK cells exhibit memory-like properties with enhanced nonspecific and specific recall responses. Cytokine activation alone of murine NK cells induces the differentiation of memory-like cells that are more likely to produce IFN-γ, a key NK cell cytokine important for activation of the immune response. Using an adoptive cotransfer system, we first show that cytokine-induced memory-like responses are NK intrinsic. However, engraftment of donor NK cells in NK-competent hosts is poor because of homeostatic control mechanisms. Therefore, we used alymphoid Rag- and common γ-chain–deficient mice as recipients and observed homeostatic expansion of cotransferred cytokine-activated and control donor NK cells. Despite proliferation of all cells, NK cells derived from those cells originally activated by cytokines retained an intrinsic enhanced capacity to produce IFN-γ when restimulated in vitro with cytokines or target cells. These NK cell memory-like responses persisted for at least 4 wk in alymphoid hosts and 12 wk in NK-competent hosts. These findings indicate that memory-like NK cells can readily self-renew and maintain enhanced function in a lymphopenic host for at least a month.