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OBJECTIVES: Influenza causes significant morbidity and mortality, especially in high-risk populations. Although current vaccination regimens are the best method to combat annual influenza disease, vaccine efficacy can be low in high-risk groups, such as haematopoietic stem cell transplant (HSCT) recipients. METHODS: We comprehensively assessed humoral immunity, antibody landscapes, systems serology and influenza-specific B-cell responses, together with their phenotypes and isotypes, to the inactivated influenza vaccine (IIV) in HSCT recipients in comparison to healthy controls. RESULTS: Inactivated influenza vaccine significantly increased haemagglutination inhibition (HAI) titres in HSCT recipients, similar to healthy controls. Systems serology revealed increased IgG1 and IgG3 antibody levels towards the haemagglutinin (HA) head, but not to neuraminidase, nucleoprotein or HA stem. IIV also increased frequencies of total, IgG class-switched and CD21loCD27+ influenza-specific B cells, determined by HA probes and flow cytometry. Strikingly, 40% of HSCT recipients had markedly higher antibody responses towards A/H3N2 vaccine strain than healthy controls and showed cross-reactivity to antigenically drifted A/H3N2 strains by antibody landscape analysis. These superior humoral responses were associated with a greater time interval after HSCT, while multivariant analyses revealed the importance of pre-existing immune memory. Conversely, in HSCT recipients who did not respond to the first dose, the second IIV dose did not greatly improve their humoral response, although 50% of second-dose patients reached a seroprotective HAI titre for at least one of vaccine strains. CONCLUSIONS: Our study demonstrates efficient, although time-dependent, immune responses to IIV in HSCT recipients, and provides insights into influenza vaccination strategies targeted to immunocompromised high-risk groups.

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.

Glucocorticoids (GCs) are potent anti-inflammatory agents and are broadly used in treating rheumatoid arthritis (RA) patients, albeit with adverse side effects associated with long-term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action. We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CCL17 has a non-redundant role in inflammatory arthritis. Here, we provide molecular evidence that GCs can suppress GM-CSF-mediated upregulation of IRF4 and CCL17 expression via downregulating JMJD3 expression and activity. In mouse models of inflammatory arthritis, GC treatment inhibited CCL17 expression and ameliorated arthritic pain-like behavior and disease. Significantly, GC treatment of RA patient peripheral blood mononuclear cells ex vivo resulted in decreased CCL17 production. This delineated pathway potentially provides new therapeutic options for the treatment of many inflammatory conditions, where GCs are used as an anti-inflammatory drug but without the associated adverse side effects.

Stuttering is a common speech disorder that interrupts speech fluency and tends to cluster in families. Typically, stuttering is characterized by speech sounds, words or syllables which may be repeated or prolonged and speech that may be further interrupted by hesitations or 'blocks'. Rare variants in a small number of genes encoding lysosomal pathway proteins have been linked to stuttering. We studied a large four-generation family in which persistent stuttering was inherited in an autosomal dominant manner with disruption of the cortico-basal-ganglia-thalamo-cortical network found on imaging. Exome sequencing of three affected family members revealed the PPID c.808C>T (p.Pro270Ser) variant that segregated with stuttering in the family. We generated a Ppid p.Pro270Ser knock-in mouse model and performed ex vivo imaging to assess for brain changes. Diffusion-weighted MRI in the mouse revealed significant microstructural changes in the left corticospinal tract, as previously implicated in stuttering. Quantitative susceptibility mapping also detected changes in cortico-striatal-thalamo-cortical loop tissue composition, consistent with findings in affected family members. This is the first report to implicate a chaperone protein in the pathogenesis of stuttering. The humanized Ppid murine model recapitulates network findings observed in affected family members.

Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of up to ∼1 µm per acquisition. This approach however, places high demands on fluorophore brightness and photoswitching resilience meaning that imaging entire cell volumes in 3D using SM super-resolution remains challenging. Here we employ SM astigmatism together with multiplane acquisition to visualize the whole nuclear lamina of COS-7 and T cells in 3D. Nuclear lamina provides structural support to the nuclear envelope and participates in vital nuclear functions including internuclear transport, chromatin organization and gene regulation. Its position at the periphery of the nucleus provides a visible reference of the nuclear boundary and can be used to quantify the spatial distribution of intranuclear components such as histone modifications and transcription factors. We found Alexa Fluor 647, a popular photoswitchable fluorophore, remained viable for over an hour of continuous high laser power exposure, and provided sufficient brightness detectable up to 8 µm deep into a cell, allowing us to capture the entire nuclear lamina in 3D. Our approach provides sufficient super-resolution detail of nuclear lamina morphology to enable quantification of overall nuclear dimensions and local membrane features.

Developmental stuttering is a condition of speech dysfluency, characterized by pauses, blocks, prolongations and sound or syllable repetitions. It affects around 1% of the population, with potential detrimental effects on mental health and long-term employment. Accumulating evidence points to a genetic aetiology, yet gene-brain associations remain poorly understood due to a lack of MRI studies in affected families. Here we report the first neuroimaging study of developmental stuttering in a family with autosomal dominant inheritance of persistent stuttering. We studied a four-generation family, 16 family members were included in genotyping analysis. T1-weighted and diffusion-weighted MRI scans were conducted on seven family members (six male; aged 9-63 years) with two age and sex matched controls without stuttering (n = 14). Using Freesurfer, we analysed cortical morphology (cortical thickness, surface area and local gyrification index) and basal ganglia volumes. White matter integrity in key speech and language tracts (i.e. frontal aslant tract and arcuate fasciculus) was also analysed using MRtrix and probabilistic tractography. We identified a significant age by group interaction effect for cortical thickness in the left hemisphere pars opercularis (Broca's area). In affected family members this region failed to follow the typical trajectory of age-related thinning observed in controls. Surface area analysis revealed the middle frontal gyrus region was reduced bilaterally in the family (all cortical morphometry significance levels set at a vertex-wise threshold of P < 0.01, corrected for multiple comparisons). Both the left and right globus pallidus were larger in the family than in the control group (left P = 0.017; right P = 0.037), and a larger right globus pallidus was associated with more severe stuttering (rho = 0.86, P = 0.01). No white matter differences were identified. Genotyping identified novel loci on chromosomes 1 and 4 that map with the stuttering

Latent HIV-1 provirus in infected individuals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3' LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo samples. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5' LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.

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.

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.

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.

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape.

CD8+ T cells play a pivotal role in clearing intracellular pathogens and combatting tumours. Upon infection, naïve CD8+ T cells differentiate into effector and memory cells, and this program is underscored by large-scale and coordinated changes in the chromatin architecture and gene expression. Importantly, recent evidence demonstrates that the epigenetic mechanisms that regulate the capacity for rapid effector function of memory T cells are shared by innate immune cells such as natural killer (NK) cells. Thus, it appears that the crucial difference between innate and adaptive immunity is the presence of the naïve state. This important distinction raises an intriguing new hypothesis, that the naïve state was evolutionary installed to restrain a default program of effector and memory differentiation in response to antigen recognition. We argue that the hallmark of adaptive T immunity is therefore the naïve program, which actively maintains CD8+ T cell quiescence until receipt of appropriate activation signals. In this review, we examine the mechanistic control of naïve CD8+ T cell quiescence and summarise the multiple levels of restraint imposed in naïve cells in to limit spontaneous and inappropriate activation. This includes epigenetic mechanisms and transcription factor (TF) regulation of gene expression, in addition to novel inhibitory receptors, abundance of RNA, and protein degradation.

Special AT-rich binding protein-1 (SATB1) is a global chromatin organizer capable of activating or repressing gene transcription in mice and humans. The role of SATB1 is pivotal for T-cell development, with SATB1-knockout mice being neonatally lethal, although the exact mechanism is unknown. Moreover, SATB1 is dysregulated in T-cell lymphoma and proposed to suppress transcription of the Pdcd1 gene, encoding the immune checkpoint programmed cell death protein 1 (PD-1). Thus, SATB1 expression in T-cell subsets across different tissue compartments in humans is of potential importance for targeting PD-1. Here, we comprehensively analyzed SATB1 expression across different human tissues and immune compartments by flow cytometry and correlated this with PD-1 expression. We investigated SATB1 protein levels in pediatric and adult donors and assessed expression dynamics of this chromatin organizer across different immune cell subsets in human organs, as well as in antigen-specific T cells directed against acute and chronic viral infections. Our data demonstrate that SATB1 expression in humans is the highest in T-cell progenitors in the thymus, and then becomes downregulated in mature T cells in the periphery. Importantly, SATB1 expression in peripheral mature T cells is not static and follows fine-tuned expression dynamics, which appear to be tissue- and antigen-dependent. Furthermore, SATB1 expression negatively correlates with PD-1 expression in virus-specific CD8+ T cells. Our study has implications for understanding the role of SATB1 in human health and disease and suggests an approach for modulating PD-1 in T cells, highly relevant to human malignancies or chronic viral infections.

Glucocorticoids (GCs) are potent anti-inflammatory drugs whose mode of action is complex and still debatable. One likely cellular target of GCs are monocytes/macrophages. The role of GCs in monocyte survival is also debated. Although both granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) are important regulators of macrophage lineage functions including their survival, the former is often associated with proinflammatory functions while the latter is important in lineage homeostasis. We report here that the GC, dexamethasone, induces apoptosis in GM-CSF-treated human monocytes while having no impact on M-CSF-induced monocyte survival. To understand how GCs, GM-CSF, and M-CSF are regulating monocyte survival and other functions during inflammation, we firstly examined the transcriptomic changes elicited by these three agents in human monocytes, either acting alone or in combination. Transcriptomic and Ingenuity pathway analyses found that dexamethasone differentially modulated dendritic cell maturation and TREM1 signaling pathways in GM-CSF-treated and M-CSF-treated monocytes, two pathways known to be regulated by ERK1/2 activity. These analyses led us to provide evidence that the GC inhibits ERK1/2 activity selectively in GM-CSF-treated monocytes to induce apoptosis. It is proposed that this inhibition of ERK1/2 activity leads to inactivation of p90 ribosomal-S6 kinase and Bad dephosphorylation leading in turn to enhanced caspase-3 activity and subsequent apoptosis. Furthermore, pharmacological inhibition of GC receptor activity restored the ERK1/2 signaling and prevented the GC-induced apoptosis in GM-CSF-treated monocytes. Increased tissue macrophage numbers, possibly from enhanced survival due to mediators such as GM-CSF, can correlate with inflammatory disease severity; also reduction in these numbers can correlate with the therapeutic benefit of a number of agents, including GCs. We propose that the ERK1/2 signaling pathway pro

TNF is a pro-inflammatory cytokine produced by both lymphoid and non-lymphoid cells. As a consequence of the widespread expression of its receptors (TNFR1 and 2), TNF plays a role in many important biological processes. In the context of influenza A virus (IAV) infection, TNF has variably been implicated in mediating immunopathology as well as suppression of the immune response. Although a number of cell types are able to produce TNF, the ability of CD8+ T cells to produce TNF following viral infection is a hallmark of their effector function. As such, the regulation and role of CD8+ T cell-derived TNF following viral infection is of great interest. Here, we show that the biphasic production of TNF by CD8+ T cells following in vitro stimulation corresponds to distinct patterns of epigenetic modifications. Further, we show that a global loss of TNF during IAV infection results in an augmentation of the peripheral virus-specific CD8+ T cell response. Subsequent adoptive transfer experiments demonstrated that this attenuation of the CD8+ T cell response was largely, but not exclusively, conferred by extrinsic TNF, with intrinsically-derived TNF making only modest contributions. In conclusion, TNF exerts an immunoregulatory role on CD8+ T cell responses following IAV infection, an effect that is largely mediated by extrinsically-derived TNF.

FOXP2 is the major gene associated with severe, persistent, developmental speech and language disorders. While studies in the original family in which a FOXP2 mutation was found showed volume reduction and reduced activation in core language and speech networks, there have been no imaging studies of different FOXP2 mutations. We conducted a multimodal MRI study in an eight-year-old boy (A-II) with a de novo FOXP2 intragenic deletion. A-II showed marked bilateral volume reductions in the hippocampus, thalamus, globus pallidus, and caudate nucleus compared with 26 control males (effect sizes from -1 to -3). He showed no detectable functional MRI activity when repeating nonsense words. The hippocampus is implicated for the first time in FOXP2 diseases. We conclude that FOXP2 anomaly is either directly or indirectly associated with atypical development of widespread subcortical networks early in life.

Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4-dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.

CD8(+) T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8(+) T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8(+) T cell responses and the establishment of immunological CD8(+) T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8(+) T cell responses. Importantly, functional memory CD8(+) T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4(+) T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8(+) T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.

UNLABELLED: The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity. IMPORTANCE: The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system resp

Although CD4(+) T cell help (Th) is critical for inducing optimal B cell and CD8(+) T cell responses, it remains unclear whether induction of CD4(+) Th responses postinfection are also dependent on CD4(+) T cell help. In this study, we show that activation of adoptively transferred Th cells during primary influenza A virus (IAV) infection enhances both the magnitude and functional breadth of endogenous primary IAV-specific CD4(+) T cell responses. This enhancement was dependent on CD154-CD40-dependent dendritic cell licensing and resulted in a greater recall capacity of IAV-specific CD4(+) and CD8(+) T memory responses after heterologous IAV infection. These data suggest that engaging pre-existing CD4 responses at the time of priming may be a strategy for improving cellular immunity after vaccination.

Epilepsy-aphasia syndromes (EAS) are a group of rare, severe epileptic encephalopathies of unknown etiology with a characteristic electroencephalogram (EEG) pattern and developmental regression particularly affecting language. Rare pathogenic deletions that include GRIN2A have been implicated in neurodevelopmental disorders. We sought to delineate the pathogenic role of GRIN2A in 519 probands with epileptic encephalopathies with diverse epilepsy syndromes. We identified four probands with GRIN2A variants that segregated with the disorder in their families. Notably, all four families presented with EAS, accounting for 9% of epilepsy-aphasia cases. We did not detect pathogenic variants in GRIN2A in other epileptic encephalopathies (n = 475) nor in probands with benign childhood epilepsy with centrotemporal spikes (n = 81). We report the first monogenic cause, to our knowledge, for EAS. GRIN2A mutations are restricted to this group of cases, which has important ramifications for diagnostic testing and treatment and provides new insights into the pathogenesis of this debilitating group of conditions.

PURPOSE: To characterize the frequency and nature of the family history of seizures in probands with epilepsy falling within the epilepsy-aphasia spectrum (EAS) in order to understand the genetic architecture of this group of disorders. METHODS: Patients with epileptic encephalopathy with continuous spike-and-wave during sleep (ECSWS), Landau-Kleffner syndrome (LKS), atypical benign partial epilepsy (ABPE), and intermediate epilepsy-aphasia disorders (IEAD) were recruited. All affected and available unaffected relatives up to three degrees of relatedness underwent phenotyping using a validated seizure questionnaire. Pedigrees were constructed for all families. The proportion of affected relatives according to each degree of relatedness was calculated. The epilepsy phenotypes in close relatives were analyzed. The data were compared to the families of probands with benign childhood epilepsy with centrotemporal spikes (BECTS) using the same methodology. KEY FINDINGS: Thirty-one probands, including five ECSWS, three LKS, one ABPE, and 22 IEAD were recruited. The mean age of seizure onset was 3.9 (range 0.5-7) years. A male predominance was seen (68%, 21/31) . Sixteen (51.6%) of 31 had a positive family history of seizures. Among 1,254 relatives, 30 (2.4%) had a history of seizures: 13 (10.2%) of 128 first-degree relatives, 5 (1.7%) of 291 second-degree relatives, and 12 (1.4%) of 835 third-degree relatives. Thirteen had febrile seizures, including two who had both febrile seizures and epilepsy. Of the 19 relatives with epilepsy, 4 had BECTS, 4 epilepsies with focal seizures of unknown cause, 3 IEAD, and 7 unclassified. One had genetic generalized epilepsy. In the families of the BECTS probands, 9.8% of first-degree, 3% of second-degree, and 1.5% of third-degree relatives had seizures, which was not significantly different from the EAS cohort families. SIGNIFICANCE: The frequencies of seizures in relatives of probands with EAS suggest that the underlying genetic influenc

The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.

Cellular differentiation is regulated by the strict spatial and temporal control of gene expression. This is achieved, in part, by regulating changes in histone post-translational modifications (PTMs) and DNA methylation that in turn, impact transcriptional activity. Further, histone PTMs and DNA methylation are often propagated faithfully at cell division (termed epigenetic propagation), and thus contribute to maintaining cellular identity in the absence of signals driving differentiation. Cardinal features of adaptive T cell immunity include the ability to differentiate in response to infection, resulting in acquisition of immune functions required for pathogen clearance; and the ability to maintain this functional capacity in the long-term, allowing more rapid and effective pathogen elimination following re-infection. These characteristics underpin vaccination strategies by effectively establishing a long-lived T cell population that contributes to an immunologically protective state (termed immunological memory). As we discuss in this review, epigenetic mechanisms provide attractive and powerful explanations for key aspects of T cell-mediated immunity - most obviously and notably, immunological memory, because of the capacity of epigenetic circuits to perpetuate cellular identities in the absence of the initial signals that drive differentiation. Indeed, T cell responses to infection are an ideal model system for studying how epigenetic factors shape cellular differentiation and development generally. This review will examine how epigenetic mechanisms regulate T cell function and differentiation, and how these model systems are providing general insights into the epigenetic regulation of gene transcription during cellular differentiation.

A cardinal feature of adaptive, cytotoxic T lymphocyte (CTL)-mediated immunity is the ability of naïve CTLs to undergo a program of differentiation and proliferation upon activation resulting in the acquisition of lineage-specific T cell functions and eventual establishment of immunological memory. In this review, we examine the molecular factors that shape both the acquisition and maintenance of lineage-specific effector function in virus-specific CTL during both the effector and memory phases of immunity.

Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.

The importance of costimulation on CD4(+) T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4(+) T-cell help. The role of costimulation under such "helpless" circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44(+) BrdU(+) influenza-specific CD8(+) T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8(+) T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8(+) T cells was affected by the absence of CD4(+) T cells alone. In contrast, both were markedly impaired by CD28 blockade of "helpless" CD8(+) T cells. We suggest that direct CD28 costimulation of CD8(+) T cells is more critical in their priming during primary influenza infection than previously appreciated.

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8(+) T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP(336-374) peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8(+) T cell response and selected a narrowed T cell receptor (TCR) repertoire within two V beta regions (V beta 8.3 and V beta 9). This can be partially explained by the H-2D(b)NPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D(b), including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8(+) responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8(+) T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected