Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Suppression of adaptive NK cell expansion by macrophage-mediated phagocytosis inhibited by 2B4-CD48.

Infection of mice by mouse cytomegalovirus (MCMV) triggers activation and expansion of Ly49H+ natural killer (NK) cells, which are virus specific and considered to be "adaptive" or "memory" NK cells. Here, we find that signaling lymphocytic activation molecule family receptors (SFRs), a group of hematopoietic cell-restricted receptors, are essential for the expansion of Ly49H+ NK cells after MCMV infection. This activity is largely mediated by CD48, an SFR broadly expressed on NK cells and displaying augmented expression after MCMV infection. It is also dependent on the CD48 counter-receptor, 2B4, expressed on host macrophages. The 2B4-CD48 axis promotes expansion of Ly49H+ NK cells by repressing their phagocytosis by virus-activated macrophages through inhibition of the pro-phagocytic integrin lymphocyte function-associated antigen-1 (LFA-1) on macrophages. These data identify key roles of macrophages and the 2B4-CD48 pathway in controlling the expansion of adaptive NK cells following MCMV infection. Stimulation of the 2B4-CD48 axis may be helpful in enhancing adaptive NK cell responses for therapeutic purposes.

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.

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.

Infection induces tissue-resident memory NK cells that safeguard tissue health.

Tissue health is dictated by the capacity to respond to perturbations and then return to homeostasis. Mechanisms that initiate, maintain, and regulate immune responses in tissues are therefore essential. Adaptive immunity plays a key role in these responses, with memory and tissue residency being cardinal features. A corresponding role for innate cells is unknown. Here, we have identified a population of innate lymphocytes that we term tissue-resident memory-like natural killer (NKRM) cells. In response to murine cytomegalovirus infection, we show that circulating NK cells were recruited in a CX3CR1-dependent manner to the salivary glands where they formed NKRM cells, a long-lived, tissue-resident population that prevented autoimmunity via TRAIL-dependent elimination of CD4+ T cells. Thus, NK cells develop adaptive-like features, including long-term residency in non-lymphoid tissues, to modulate inflammation, restore immune equilibrium, and preserve tissue health. Modulating the functions of NKRM cells may provide additional strategies to treat inflammatory and autoimmune diseases.

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.

ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment.

In inflamed tissues, monocytes differentiate into macrophages (mo-Macs) or dendritic cells (mo-DCs). In chronic nonresolving inflammation, mo-DCs are major drivers of pathogenic events. Manipulating monocyte differentiation would therefore be an attractive therapeutic strategy. However, how the balance of mo-DC versus mo-Mac fate commitment is regulated is not clear. In the present study, we show that the transcriptional repressors ETV3 and ETV6 control human monocyte differentiation into mo-DCs. ETV3 and ETV6 inhibit interferon (IFN)-stimulated genes; however, their action on monocyte differentiation is independent of IFN signaling. Instead, we find that ETV3 and ETV6 directly repress mo-Mac development by controlling MAFB expression. Mice deficient for Etv6 in monocytes have spontaneous expression of IFN-stimulated genes, confirming that Etv6 regulates IFN responses in vivo. Furthermore, these mice have impaired mo-DC differentiation during inflammation and reduced pathology in an experimental autoimmune encephalomyelitis model. These findings provide information about the molecular control of monocyte fate decision and identify ETV6 as a therapeutic target to redirect monocyte differentiation in inflammatory disorders.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells.

Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.

Epigenetic regulation of natural killer cell memory.

Immunological memory is the underlying mechanism by which the immune system remembers previous encounters with pathogens to produce an enhanced secondary response upon re-encounter. It stands as the hallmark feature of the adaptive immune system and the cornerstone of vaccine development. Classic recall responses are executed by conventional T and B cells, which undergo somatic recombination and modify their receptor repertoire to ensure recognition of a vast number of antigens. However, recent evidence has challenged the dogma that memory responses are restricted to the adaptive immune system, which has prompted a reevaluation of what delineates "immune memory." Natural killer (NK) cells of the innate immune system have been at the forefront of these pushed boundaries, and have proved to be more "adaptable" than previously thought. Like T cells, we now appreciate that their "natural" abilities actually require a myriad of signals for optimal responses. In this review, we discuss the many signals required for effector and memory NK cell responses and the epigenetic mechanisms that ultimately endow their enhanced features.

Dynamic variability in SHP-1 abundance determines natural killer cell responsiveness.

Interactions between human leukocyte antigen (HLA) molecules on target cells and the inhibitory killer cell immunoglobulin-like receptors (KIRs) and heterodimeric inhibitory receptor CD94-NKG2A on human natural killer (NK) cells shape and program various response capacities. A functionally orthologous system exists in mice, consisting of major histocompatibility complex (MHC) molecules on target cells and the inhibitory Ly49 and CD94-NKG2A receptors on NK cells. Here, we found that the abundance of Src homology 2 domain­containing phosphatase-1 (SHP-1) in NK cells was established by interactions between MHCs and NK cell inhibitory receptors, although phenotypically identical NK cell populations still showed substantial variability in endogenous SHP-1 abundance and NK cell response potential. Human and mouse NK cell populations with high responsiveness had low SHP-1 abundance, and a reduction in SHP-1 abundance in NK cells enhanced their responsiveness. Computational modeling of NK cell activation by membrane-proximal signaling events identified SHP-1 as a negative amplitude regulator, which was validated by single-cell analysis of human NK cell responsiveness. The amount of mRNA and protein varied among responsive NK cells despite their similar chromatin accessibility to that of unresponsive cells, suggesting dynamic regulation of SHP-1 abundance. Low intracellular SHP-1 abundance was a biomarker of responsive NK cells. Together, these data suggest that enhancing NK cell function through the acute loss of SHP-1 abundance or activity may enhance the tumoricidal capacity of NK cells.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8ß, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rß, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Human cytomegalovirus expands a CD8+ T cell population with loss of BCL11B expression and gain of NK cell identity.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C+TCRαß+CD8+ T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C+CD8+ T cells are oligoclonal and do not up-regulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C+CD8+ T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is down-regulated in NKG2C+CD8+ T cells when compared with conventional NKG2C−CD8+ T cells. BCL11B deletion in conventional CD8+ T cells resulted in the emergence of a similar innate-like CD56+CD94+DAP12+NKG2C+CD45RA+CCR7−PD-1−/low T cell population with activity against HLA-E+ targets. On the basis of their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C+CD8+ T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T cell­based therapies.

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.

The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation.

Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.

Divergent Role for STAT5 in the Adaptive Responses of Natural Killer Cells.

Natural killer (NK) cells are innate lymphocytes with the capacity to elicit adaptive features, including clonal expansion and immunological memory. Because signal transducer and activator of transcription 5 (STAT5) is essential for NK cell development, the roles of this transcription factor and its upstream cytokines interleukin-2 (IL-2) and IL-15 during infection have not been carefully investigated. In this study, we investigate how STAT5 regulates transcription during viral infection. We demonstrate that STAT5 is induced in NK cells by IL-12 and STAT4 early after infection and that partial STAT5 deficiency results in a defective capacity of NK cells to generate long-lived memory cells. Furthermore, we find a functional dichotomy of IL-2 and IL-15 signaling outputs during viral infection, whereby both cytokines drive clonal expansion, but only IL-15 is required for memory NK cell survival. We thus highlight a role for STAT5 signaling in promoting an optimal anti-viral NK cell response.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization.

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

Cutting Edge: STAT1-Mediated Epigenetic Control of Rsad2 Promotes Clonal Expansion of Antiviral NK Cells.

NK cells represent a cellular component of innate immunity but possess features of adaptive immunity, including clonal expansion and establishment of long-lived memory following infection. During mouse CMV (MCMV) infection, we observed Rsad2 (which encodes Viperin) to be among the most highly induced IFN stimulatory genes in activated NK cells, correlating with increased chromatin accessibility at the Rsad2 gene locus. Furthermore, in NK cells stimulated with IFN-α, the promoter region of Rsad2 was enriched for STAT1 binding and the permissive histone mark H3K4me3. IFN-αR- and STAT1-deficient NK cells showed an impairment of Rsad2 induction and chromatin accessibility during MCMV infection. Finally, Rsad2-deficient NK cells were defective in clonal expansion and memory formation following exposure to MCMV, in part because of greater apoptosis. Thus, our study reveals a critical mechanism of STAT1-mediated epigenetic control of Rsad2 to promote the adaptive behavior of NK cells during viral infection.