Fasting reshapes tissue-specific niches to improve NK cell-mediated anti-tumor immunity.

Fasting is associated with improved outcomes in cancer. Here, we investigated the impact of fasting on natural killer (NK) cell anti-tumor immunity. Cyclic fasting improved immunity against solid and metastatic tumors in an NK cell-dependent manner. During fasting, NK cells underwent redistribution from peripheral tissues to the bone marrow (BM). In humans, fasting also reduced circulating NK cell numbers. NK cells in the spleen of fasted mice were metabolically rewired by elevated concentrations of fatty acids and glucocorticoids, augmenting fatty acid metabolism via increased expression of the enzyme CPT1A, and Cpt1a deletion impaired NK cell survival and function in this setting. In parallel, redistribution of NK cells to the BM during fasting required the trafficking mediators S1PR5 and CXCR4. These cells were primed by an increased pool of interleukin (IL)-12-expressing BM myeloid cells, which improved IFN-γ production. Our findings identify a link between dietary restriction and optimized innate immune responses, with the potential to enhance immunotherapy strategies.

Early antigen receptor signaling in natural killer cells alters STAT4-dependent fate decisions via epigenetic remodeling.

Natural Killer (NK) cells are innate cytotoxic lymphocytes that possess features of adaptive immunity, including antigen specificity and clonal expansion. NK cells rapidly respond to cytokines released during the innate phase of viral infection and are thought to migrate from circulation into infected organs to execute their early effector functions. However, recent evidence suggests that tissue-resident NK cells are among the first responders to viral infection. In this study, we observe that antigen receptor signaling precedes substantial proinflammatory cytokine signaling in a population of NK cells during mouse cytomegalovirus infection. Early antigen receptor signals epigenetically prime NK cells for optimal expansion during the later adaptive phase of the antiviral response. Mechanistically, receptor signaling increases chromatin accessibility at STAT4-binding genomic sites within differentiating NK cells. To promote adaptive programming of NK cells during infection, activating receptor-dependent epigenetic remodeling antagonizes IL-12 driven terminal maturation, poises NK cells for proliferation via sustained CDK6 expression, and antagonizes early apoptosis of short-lived effector cells via suppression of Bim. Thus, antigen receptor signaling alters an IL-12 dependent fate decision during the innate-to-adaptive transition of antiviral NK cells.

Cardinal features of immune memory in innate lymphocytes.

The ability of vertebrates to 'remember' previous infections had once been attributed exclusively to adaptive immunity. We now appreciate that innate lymphocytes also possess memory properties akin to those of adaptive immune cells. In this Review, we draw parallels from T cell biology to explore the key features of immune memory in innate lymphocytes, including quantity, quality, and location. We discuss the signals that trigger clonal or clonal-like expansion in innate lymphocytes, and highlight recent studies that shed light on the complex cellular and molecular crosstalk between metabolism, epigenetics, and transcription responsible for differentiating innate lymphocyte responses towards a memory fate. Additionally, we explore emerging evidence that activated innate lymphocytes relocate and establish themselves in specific peripheral tissues during infection, which may facilitate an accelerated response program akin to those of tissue-resident memory T cells.

Cutting Edge: STAT4 Promotes Bhlhe40 Induction to Drive Protective IFN-γ from NK Cells during Viral Infection.

NK cells represent a cellular component of the mammalian innate immune system, and they mount rapid responses against viral infection, including the secretion of the potent antiviral effector cytokine IFN-γ. Following mouse CMV infection, Bhlhe40 was the most highly induced transcription factor in NK cells among the basic helix-loop-helix family. Bhlhe40 upregulation in NK cells depended upon IL-12 and IL-18 signals, with the promoter of Bhlhe40 enriched for STAT4 and the permissive histone H3K4me3, and with STAT4-deficient NK cells showing an impairment of Bhlhe40 induction and diminished H3K4me3. Transcriptomic and protein analysis of Bhlhe40-deficient NK cells revealed a defect in IFN-γ production during mouse CMV infection, resulting in diminished protective immunity following viral challenge. Finally, we provide evidence that Bhlhe40 directly promotes IFN-γ by binding throughout the Ifng loci in activated NK cells. Thus, our study reveals how STAT4-mediated control of Bhlhe40 drives protective IFN-γ secretion by NK cells during viral infection.

Control of nutrient uptake by IRF4 orchestrates innate immune memory.

Natural killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen specificity, clonal expansion and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified that the transcription factor IRF4 integrates signals to coordinate the NK cell response during mouse cytomegalovirus infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest that IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

3D Chromatin Dynamics during Innate and Adaptive Immune Memory Acquisition.

Immune cells responding to pathogens undergo molecular changes that are intimately linked to genome organization. Recent work has demonstrated that natural killer (NK) and CD8 + T cells experience substantial transcriptomic and epigenetic rewiring during their differentiation from naïve to effector to memory cells. Whether these molecular adaptations are accompanied by changes in three-dimensional (3D) chromatin architecture is unknown. In this study, we combine histone profiling, ATAC-seq, RNA-seq and high-throughput chromatin capture (HiC) assay to investigate the dynamics of one-dimensional (1D) and 3D chromatin during the differentiation of innate and adaptive lymphocytes. To this end, we discovered a coordinated 1D and 3D epigenetic remodeling during innate immune memory differentiation, and demonstrate that effector CD8 + T cells adopt an NK-like architectural program that is maintained in memory cells. Altogether, our study reveals the dynamic nature of the 1D and 3D genome during the formation of innate and adaptive immunological memory.

Lactate dehydrogenase A-dependent aerobic glycolysis promotes natural killer cell anti-viral and anti-tumor function.

Natural killer (NK) cells are cytotoxic lymphocytes capable of rapid cytotoxicity, cytokine secretion, and clonal expansion. To sustain such energetically demanding processes, NK cells must increase their metabolic capacity upon activation. However, little is known about the metabolic requirements specific to NK cells in vivo. To gain greater insight, we investigated the role of aerobic glycolysis in NK cell function and demonstrate that their glycolytic rate increases rapidly following viral infection and inflammation, prior to that of CD8+ T cells. NK cell-specific deletion of lactate dehydrogenase A (LDHA) reveals that activated NK cells rely on this enzyme for both effector function and clonal proliferation, with the latter being shared with T cells. As a result, LDHA-deficient NK cells are defective in their anti-viral and anti-tumor protection. These findings suggest that aerobic glycolysis is a hallmark of NK cell activation that is key to their function.

Deconvoluting global cytokine signaling networks in natural killer cells.

Cytokine signaling via signal transducer and activator of transcription (STAT) proteins is crucial for optimal antiviral responses of natural killer (NK) cells. However, the pleiotropic effects of both cytokine and STAT signaling preclude the ability to precisely attribute molecular changes to specific cytokine-STAT modules. Here, we employed a multi-omics approach to deconstruct and rebuild the complex interaction of multiple cytokine signaling pathways in NK cells. Proinflammatory cytokines and homeostatic cytokines formed a cooperative axis to commonly regulate global gene expression and to further repress expression induced by type I interferon signaling. These cytokines mediated distinct modes of epigenetic regulation via STAT proteins, and collective signaling best recapitulated global antiviral responses. The most dynamically responsive genes were conserved across humans and mice, which included a cytokine-STAT-induced cross-regulatory program. Thus, an intricate crosstalk exists between cytokine signaling pathways, which governs NK cell responses.

Cytomegalovirus Infection Drives Avidity Selection of Natural Killer Cells.

The process of affinity maturation, whereby T and B cells bearing antigen receptors with optimal affinity to the relevant antigen undergo preferential expansion, is a key feature of adaptive immunity. Natural killer (NK) cells are innate lymphocytes capable of "adaptive" responses after cytomegalovirus (CMV) infection. However, whether NK cells are similarly selected on the basis of their avidity for cognate ligand is unknown. Here, we showed that NK cells with the highest avidity for the mouse CMV glycoprotein m157 were preferentially selected to expand and comprise the memory NK cell pool, whereas low-avidity NK cells possessed greater capacity for interferon-γ (IFN-γ) production. Moreover, we provide evidence for avidity selection occurring in human NK cells during human CMV infection. These results delineate how heterogeneity in NK cell avidity diversifies NK cell effector function during antiviral immunity, and how avidity selection might serve to produce the most potent memory NK cells.

Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (Treg) cells4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain Treg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce Treg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1ß. Macrophages in the small intestine produce IL-1ß, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining Treg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of Treg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1ß-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.

Identification and editing of stem-like cells in methylcholanthrene-induced sarcomas.

The cancer stem cell (CSC) paradigm posits that specific cells within a tumor, so-called CSC-like cells, have differing levels of tumorigenicity and chemoresistance. Original studies of CSCs identified them in human cancers and utilized mouse xenograft models to define the cancer initiating properties of these cells, thereby hampering the understanding of how immunity could affect CSCs. Indeed, few studies have characterized CSCs in the context of cancer immunoediting, and it is currently not clear how immunity could impact on the levels or stem-like behavior of CSCs. Using the well-studied 3'methylcholanthrene (MCA) model of primary sarcoma formation, we have defined a CSC-like population within MCA-induced sarcomas as expressing high levels of stem cell antigen-1 (Sca-1) and low levels of CD90. These Sca-1+CD90- CSC-like cells had higher tumor initiating ability, could spontaneously give rise to Sca-1-negative cells, and formed more sarcospheres than corresponding non-CSC-like cells. Moreover, when examining MCA-induced sarcomas that were in the equilibrium phase of cancer growth, higher levels of CSC-like cells were found compared to MCA-induced sarcomas in the escape phase of cancer progression. Notably, CSC-like cells also emerged during escape from anti-PD-1 or anti-CTLA4 therapy, thus suggesting that CSC-like cells could evade immune therapy. Finally, we demonstrate that paradoxically, interferon (IFN)-γ produced in vivo by immune cells could promote the emergence of CSC-like cells. Our findings define the existence of a Sca1+CD90- CSC-like population in the MCA-sarcoma model capable of differentiation, tumorsphere formation, and increased tumor initiation in vivo. These cells may also act as mediators of immune resistance during cancer immunoediting and immune therapy.

Cell-cell fusion as a mechanism of DNA exchange in cancer.

Cell-cell fusion describes the process by which two cells combine their plasma membranes and become a single cell, possessing and retaining certain genetic information from each parent cell. Here, using a Cre-loxP-based method initially developed to investigate extracellular vesicle targeting, we found that cancer cells spontaneously and rapidly deliver DNA to non-cancer cells in vitro via a cell-cell fusion event. The resulting hybrid cells were aneuploid and possessed enhanced clonal diversity and chemoresistance compared to non-hybrid cancer cells. We also observed cell-cell fusion to occur in vivo between melanoma cells and non-cancer cells of both hematopoietic and non-hematopoietic lineages. These findings suggest that cell-cell fusion occurs during the natural progression of cancer and show that this mechanism has the potential to cause massive genomic alterations that are observed in cancer. Furthermore, these findings somewhat contradict recent publications suggesting that the Cre-loxP method measures only extracellular vesicle-mediated intercellular communication.

Nrf2 Induces IL-17D to Mediate Tumor and Virus Surveillance.

Cells undergoing xenobiotic or oxidative stress activate the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2), which initiates an intrinsic "stress surveillance" pathway. We recently found that the cytokine IL-17D effects a form of extrinsic stress surveillance by inducing antitumor immunity, but how IL-17D is regulated remains unknown. Here, we show that Nrf2 induced IL-17D in cancer cell lines. Moreover, both Nrf2 and IL-17D were induced in primary tumors as well as during viral infection in vivo. Expression of IL-17D in tumors and virally infected cells is essential for optimal protection of the host as il17d(-/-) mice experienced a higher incidence of tumors and exacerbated viral infections compared to wild-type (WT) animals. Moreover, activating Nrf2 to induce IL-17D in established tumors led to natural killer cell-dependent tumor regression. These data demonstrate that Nrf2 can initiate both intrinsic and extrinsic stress surveillance pathways and highlight the use of Nrf2 agonists as immune therapies for cancer and infection.