Brendan E. Russ

Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds

Haloquadratum walsbyi is frequently a dominant member of the microbial communities in hypersaline waters. 16S rRNA gene sequences indicate that divergence within this species is very low but relatively few sites have been examined, particularly in the southern hemisphere. The diversity of Haloquadratum was examined in three coastal, but geographically distant saltern crystallizer ponds in Australia, using both culture-independent and culture-dependent methods. Two 97%-OTU, comprising Haloquadratum- and Halorubrum-related sequences, were shared by all three sites, with the former OTU representing about 40% of the sequences recovered at each site. Sequences 99.5% identical to that of Hqr. walsbyi C23T were present at all three sites and, overall, 98% of the Haloquadratum-related sequences displayed ≤2% divergence from that of the type strain. While haloarchaeal diversity at each site was relatively low (9–16 OTUs), seven phylogroups (clones and/or isolates) and 4 different clones showed ≤90% sequence identity to classified taxa, and appear to represent novel genera. Six of these branched together in phylogenetic tree reconstructions, forming a clade (MSP8-clade) whose members were only distantly related to classified taxa. Such sequences have only rarely been previously detected but were found at all three Australian crystallizers.

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.

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.

T cell immunity as a tool for studying epigenetic regulation of cellular differentiation.

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.

Epigenetic plasticity of Cd8a locus during CD8 + T-cell development and effector differentiation and reprogramming

Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8+ effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8low effector cells respond to interferon-γ by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8+ T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

Defining the molecular blueprint that drives CD8+ T cell differentiation in response to infection

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.

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.

Can T cells be too exhausted to fight back

The transcriptional network becomes less malleable in persistently activated T cells When T cells are persistently activated by antigen, such as during chronic infection or in cancer, they can become functionally incapable of performing their effector activities, a condition called T cell exhaustion. Exhaustion therefore thwarts optimal immune control of infection and tumors. There is a need to learn more about the molecular factors that drive T cell exhaustion and just how malleable T cell immunity is once exhaustion is established. On pages 1165 and 1160 of this issue, Sen et al. (1) and Pauken et al. (2), respectively, demonstrate that T cell exhaustion represents a stable differentiation state, underpinned by the apparently irreversible installation of an exhaustion-specific genetic landscape. This implies that perhaps in a majority of cases of persistent immune activation, T cells are too exhausted to fight back against cancer or pathogens.

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.

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.