DNA architectural protein CTCF facilitates subset-specific chromatin interactions to limit the formation of memory CD8+ T cells.

Although the importance of genome organization for transcriptional regulation of cell-fate decisions and function is clear, the changes in chromatin architecture and how these impact effector and memory CD8+ T cell differentiation remain unknown. Using Hi-C, we studied how genome configuration is integrated with CD8+ T cell differentiation during infection and investigated the role of CTCF, a key chromatin remodeler, in modulating CD8+ T cell fates through CTCF knockdown approaches and perturbation of specific CTCF-binding sites. We observed subset-specific changes in chromatin organization and CTCF binding and revealed that weak-affinity CTCF binding promotes terminal differentiation of CD8+ T cells through the regulation of transcriptional programs. Further, patients with de novo CTCF mutations had reduced expression of the terminal-effector genes in peripheral blood lymphocytes. Therefore, in addition to establishing genome architecture, CTCF regulates effector CD8+ T cell heterogeneity through altering interactions that regulate the transcription factor landscape and transcriptome.

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells.

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation, and function. Here, we examined the impact of the microbiota on CD8+ T cell memory. Antigen-activated CD8+ T cells transferred into germ-free mice failed to transition into long-lived memory cells and had transcriptional impairments in core genes associated with oxidative metabolism. The microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8+ T cells, and SCFAs were required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that butyrate uncoupled the tricarboxylic acid cycle from glycolytic input in CD8+ T cells, which allowed preferential fueling of oxidative phosphorylation through sustained glutamine utilization and fatty acid catabolism. Our findings reveal a role for the microbiota in promoting CD8+ T cell long-term survival as memory cells and suggest that microbial metabolites guide the metabolic rewiring of activated CD8+ T cells to enable this transition.

Distinct epigenetic signatures delineate transcriptional programs during virus-specific CD8(+) T cell differentiation.

The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methylation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene transcription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate transcriptional regulation of CTL genes with related functions is achieved via distinct epigenetic mechanisms.

SATB1 ensures appropriate transcriptional programs within naïve CD8+ T cells.

Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4+ T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8+ T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8+ T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8+ T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1m1Anu/m1Anu ). Satb1m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1m1Anu/m1Anu CD8+ T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1m1Anu/m1Anu and wild-type effector CD8+ T cells converged. While there were no overt differences, primary respiratory infection of Satb1m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8+ effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8+ T cells and ensures appropriate CD8+ T-cell effector gene expression upon activation.

GILZ regulates type I interferon release and sequesters STAT1.

Glucocorticoids remain a mainstay of modern medicine due to their ability to broadly suppress immune activation. However, they cause severe adverse effects that warrant urgent development of a safer alternative. The glucocorticoid-induced leucine zipper (GILZ) gene, TSC22D3, is one of the most highly upregulated genes in response to glucocorticoid treatment, and reduced GILZ mRNA and protein levels are associated with increased severity of inflammation in systemic lupus erythematosus (SLE), Ulcerative Colitis, Psoriasis, and other autoimmune/autoinflammatory diseases. Here, we demonstrate that low GILZ permits expression of a type I interferon (IFN) signature, which is exacerbated in response to TLR7 and TLR9 stimulation. Conversely, overexpression of GILZ prevents IFN-stimulated gene (ISG) up-regulation in response to IFNα. Moreover, GILZ directly binds STAT1 and prevents its nuclear translocation, thereby negatively regulating IFN-induced gene expression and the auto-amplification loop of the IFN response. Thus, GILZ powerfully regulates both the expression and action of type I IFN, suggesting restoration of GILZ as an attractive therapeutic strategy for reducing reliance on glucocorticoids.

CD4+ T help promotes influenza virus-specific CD8+ T cell memory by limiting metabolic dysfunction.

There is continued interest in developing novel vaccine strategies that induce establish optimal CD8+ cytotoxic T lymphocyte (CTL) memory for pathogens like the influenza A viruses (IAVs), where the recall of IAV-specific T cell immunity is able to protect against serologically distinct IAV infection. While it is well established that CD4+ T cell help is required for optimal CTL responses and the establishment of memory, when and how CD4+ T cell help contributes to determining the ideal memory phenotype remains unclear. We assessed the quality of IAV-specific CD8+ T cell memory established in the presence or absence of a concurrent CD4+ T cell response. We demonstrate that CD4+ T cell help appears to be required at the initial priming phase of infection for the maintenance of IAV-specific CTL memory, with "unhelped" memory CTL exhibiting intrinsic dysfunction. High-throughput RNA-sequencing established that distinct transcriptional signatures characterize the helped vs. unhelped IAV-specific memory CTL phenotype, with the unhelped set showing a more "exhausted T cell" transcriptional profile. Moreover, we identify that unhelped memory CTLs exhibit defects in a variety of energetic pathways, leading to diminished spare respiratory capacity and diminished capacity to engage glycolysis upon reactivation. Hence, CD4+ T help at the time of initial priming promotes molecular pathways that limit exhaustion by channeling metabolic processes essential for the rapid recall of memory CD8+ T cells.

UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation.

Over 50% of the world's population is infected with Human Cytomegalovirus (HCMV). HCMV is responsible for serious complications in the immuno-compromised and is a leading cause of congenital birth defects. The molecular function of many HCMV proteins remains unknown, and a deeper understanding of the viral effectors that modulate virion maturation is required. In this study, we observed that UL34 is a viral protein expressed with leaky late kinetics that localises to the nucleus during infection. Deletion of UL34 from the HCMV genome (ΔUL34) did not abolish the spread of HCMV. Instead, over >100-fold fewer infectious virions were produced, so we report that UL34 is an augmenting gene. We found that ΔUL34 is dispensable for viral DNA replication, and its absence did not alter the expression of IE1, MCP, gB, UL26, UL83, or UL99 proteins. In addition, ΔUL34 infections were able to progress through the replication cycle to form a viral assembly compartment; however, virion maturation in the cytoplasm was abrogated. Further examination of the nucleus in ΔUL34 infections revealed replication compartments with aberrant morphology, containing significantly less assembled capsids, with almost none undergoing subsequent maturation. Therefore, this work lays the foundation for UL34 to be further investigated in the context of nuclear organization and capsid maturation during HCMV infection.

Dynamic regulation of permissive histone modifications and GATA3 binding underpin acquisition of granzyme A expression by virus-specific CD8(+) T cells.

Numerous studies have focused on the molecular regulation of perforin (PFP) and granzyme B (GZMB) expression by activated cytotoxic T lymphocytes (CTLs), but little is known about the molecular factors that underpin granzyme A (GZMA) expression. In vitro activation of naïve CD8(+) T cells, in the presence of IL-4, enhanced STAT6-dependent GZMA expression and was associated with GATA3 binding and enrichment of transcriptionally permissive histone posttranslational modifications (PTMs) across the Gzma gene locus. While GZMA expression by effector influenza A virus specific CTLs was also associated with a similar permissive epigenetic signature, memory CTL lacked enrichment of permissive histone PTMs at the Gzma locus, although this was restored within recalled secondary effector CTLs. Importantly, GZMA expression by virus-specific CTLs was associated with GATA3 binding at the Gzma locus, and independent of STAT6-mediated signaling. This suggests regulation of GZMA expression is underpinned by differentiation-dependent regulation of chromatin composition at the Gzma locus and that, given GATA3 is key for CTL differentiation in response to infection, GATA3 expression is regulated by a distinct, IL-4 independent, signaling pathway. Overall, this study provides insights into the molecular mechanisms that control transcription of Gzma during virus-induced CD8(+) T-cell differentiation.

Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids. METHODS: We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice. RESULTS: Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli. CONCLUSIONS: Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

Avenues to autoimmune arthritis triggered by diverse remote inflammatory challenges.

Environmental factors contribute to development of autoimmune diseases. For instance, human autoimmune arthritis can associate with intestinal inflammation, cigarette smoking, periodontal disease, and various infections. The cellular and, molecular pathways whereby such remote challenges might precipitate arthritis or flares remain unclear. Here, we used a transfer model of self-reactive arthritis-inducing CD4(+) cells from KRNtg mice that, upon transfer, induce a very mild form of autoinflammatory arthritis in recipient animals. This model enabled us to identify external factors that greatly aggravated disease. We show that several distinct challenges precipitated full-blown arthritis, including intestinal inflammation through DSS-induced colitis, and bronchial stress through Influenza infection. Both triggers induced strong IL-17 expression primarily in self-reactive CD4(+) cells in lymph nodes draining the site of inflammation. Moreover, treatment of mice with IL-1ß greatly exacerbated arthritis, while transfer of KRNtg CD4(+) cells lacking IL-1R significantly reduced disease and IL-17 expression. Thus, IL-1ß enhances the autoaggressive potential of self-reactive CD4(+) cells, through increased Th17 differentiation, and this influences inflammatory events in the joints. We propose that diverse challenges that cause remote inflammation (lung infection or colitis, etc.) result in IL-1ß-driven Th17 differentiation, and this precipitates arthritis in genetically susceptible individuals. Thus the etiology of autoimmune inflammatory arthritis likely relates to diverse triggers that converge to a common pathway involving IL-1ß production and Th17 cell distribution.

GILZ regulates Th17 responses and restrains IL-17-mediated skin inflammation.

Patients with inflammatory autoimmune diseases are routinely treated with synthetic glucocorticoids to suppress immunopathology. A crucial outcome of glucocorticoid exposure is induction of glucocorticoid-induced leucine zipper (GILZ), a protein with multiple functions that include inhibition of key immune cell signalling pathways. Here we report that GILZ maintains a threshold for activation of Th17 responses and IL-17-dependent pathology. GILZ expression was deficient in lesional skin of psoriasis patients and was negatively correlated with the pro-inflammatory cytokines IL-23, IL-17A and IL-22, and with STAT3 expression. Deficiency of GILZ in mice resulted in excessive inflammation and pro-inflammatory cytokine expression in the imiquimod model of psoriasis, and dendritic cells lacking GILZ produced greater IL-1, IL-23 and IL-6 in response to imiquimod stimulation in vitro. These cytokines stimulate Th17 cell differentiation, and we found unchallenged GILZ-deficient mice to have spontaneous production of IL-17A and IL-22 in vivo. We also identified a T cell-intrinsic role for GILZ in limiting Th17 cell formation in vitro in response to Th17-promoting cytokines IL-1ß and IL-23. Addition of IL-6 under these conditions suppressed GILZ, allowing T cell proliferation and expression of Th17 genes, whereas exogenous delivery of GILZ using a cell-permeable fusion protein restored regulation of Th17 cell proliferation. Thus, GILZ has a non-redundant function to constrain pathogenic Th17 responses, with clinical implications for psoriasis.

Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+ T cells.

Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8(+) T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8(+) T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8(+) T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture.

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.

Active maintenance of CD8+ T cell naïvety through regulation of global genome architecture.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

UL49 is an essential subunit of the viral pre-initiation complex that regulates human cytomegalovirus gene transcription.

More than half the world's population is infected with human cytomegalovirus (HCMV), causing congenital birth defects and impacting the immuno-compromised. Many of the >170 HCMV genes remain uncharacterized, and this gap in knowledge limits the development of novel antivirals. In this study, we investigated the essential viral protein UL49 and found it displayed leaky late expression kinetics, and localized to nuclear replication compartments. Cells infected with mutant UL49 virus were unable to produce infectious virions and phenocopied other beta-gamma viral pre-initiation complex (vPIC) subunit (UL79, UL87, UL91, UL92, and UL95) mutant infections. RNA-seq analysis of vPIC mutant infections revealed a consistent diminution of genes encoding capsid subunits, including TRX2/UL85 and MCP/UL86, envelope glycoproteins gM, gL and gO, and egress-associated tegument proteins UL99 and UL103. Therefore, as a member of the vPIC, UL49 serves as a fundamental HCMV effector that governs viral gene transcription required to complete the replication cycle.

KDM6B-dependent chromatin remodeling underpins effective virus-specific CD8+ T cell differentiation.

Naive CD8+ T cell activation results in an autonomous program of cellular proliferation and differentiation. However, the mechanisms that underpin this process are unclear. Here, we profile genome-wide changes in chromatin accessibility, gene transcription, and the deposition of a key chromatin modification (H3K27me3) early after naive CD8+ T cell activation. Rapid upregulation of the histone demethylase KDM6B prior to the first cell division is required for initiating H3K27me3 removal at genes essential for subsequent T cell differentiation and proliferation. Inhibition of KDM6B-dependent H3K27me3 demethylation limits the magnitude of an effective primary virus-specific CD8+ T cell response and the formation of memory CD8+ T cell populations. Accordingly, we define the early spatiotemporal events underpinning early lineage-specific chromatin reprogramming that are necessary for autonomous CD8+ T cell proliferation and differentiation.

The role of epigenetics in the acquisition and maintenance of effector function in virus-specific CD8 T cells.

CD8(+) T cells are critical for protecting the body from infectious disease. To achieve this protection, CD8(+) T cells must undergo a highly involved process of differentiation that involves the activation of naïve/quiescent cells followed by robust rounds of cell division and the acquisition of effector functions that mediate viral clearance. After the pathogen is eliminated, a small number of these cells survive into long-lived memory and maintain the capacity to respond rapidly and reacquire effector function after secondary exposure to their cognate antigen. This review focuses on how CD8(+) T cells acquire and regulate effector functions and how the capacity to produce effector molecules is maintained into memory.

Virus-host interactions in salt lakes.

Natural hypersaline waters are widely distributed around the globe, as both continental surface waters and sea floor lakes, the latter being maintained by the large density difference between the hypersaline and overlying marine water. Owing to the extreme salt concentrations, close to or at saturation (approximately 35%, w/v), such waters might be expected to be devoid of life but, in fact, maintain dense populations of microbes. The majority of these microorganisms are halophilic prokaryotes belonging to the Domain Archaea, 'haloarchaea'. Viruses infecting haloarchaea are a vital part of hypersaline ecosystems, in many circumstances outnumbering cells by 10-100-fold. However, few of these 'haloviruses' have been isolated and even fewer have been characterised in molecular detail. In this review, we explore the methods used by haloviruses to replicate within their hosts and consider the implications of haloviral-haloarchaeal interactions for salt lake ecology.

Rare variants in non-coding regulatory regions of the genome that affect gene expression in systemic lupus erythematosus.

Personalized medicine approaches are increasingly sought for diseases with a heritable component. Systemic lupus erythematosus (SLE) is the prototypic autoimmune disease resulting from loss of immunologic tolerance, but the genetic basis of SLE remains incompletely understood. Genome wide association studies (GWAS) identify regions associated with disease, based on common single nucleotide polymorphisms (SNPs) within them, but these SNPs may simply be markers in linkage disequilibrium with other, causative mutations. Here we use an hierarchical screening approach for prediction and testing of true functional variants within regions identified in GWAS; this involved bioinformatic identification of putative regulatory elements within close proximity to SLE SNPs, screening those regions for potentially causative mutations by high resolution melt analysis, and functional validation using reporter assays. Using this approach, we screened 15 SLE associated loci in 143 SLE patients, identifying 7 new variants including 5 SNPs and 2 insertions. Reporter assays revealed that the 5 SNPs were functional, altering enhancer activity. One novel variant was linked to the relatively well characterized rs9888739 SNP at the ITGAM locus, and may explain some of the SLE heritability at this site. Our study demonstrates that non-coding regulatory elements can contain private sequence variants affecting gene expression, which may explain part of the heritability of SLE.