Matthew E. Pipkin

Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells.

Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs

Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) γ expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-γ, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

DNA Methylation and Chromatin Structure Regulate T Cell Perforin Gene Expression

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4+ and CD8+ T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4+ and CD8+ cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at −1 kb and the distal promoter at −0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5′ flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.

MicroRNA-221–222 Regulate the Cell Cycle in Mast Cells

MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that have emerged as key posttranscriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development and differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells and found that miR-221–222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors, and potential cis-regulatory regions that control miR-221–222 transcription. Overexpression of miR-221–222 in a model mast cell line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221–222 partially counteracted expression of the cell-cycle inhibitor p27kip1, we found that in the mouse alternative splicing results in two p27kip1 mRNA isoforms that differ in their 3′ untranslated region, only one of which is subject to miR-221–222 regulation. Additionally, transgenic expression of miR-221–222 from bacterial artificial chromosome clones in embryonic stem cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides further insights on miR-221–222 transcriptional regulation as well as evidences that miR-221–222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli.

MicroRNA-directed program of cytotoxic CD8+ T-cell differentiation

Significance Development of cytotoxic T lymphocytes (CTLs) from activated CD8+ T cells is a key step of the antiviral immune response and is marked by the up-regulation of lytic molecules (perforin, granzymes). How this process is regulated at the posttranscriptional level is still largely unknown. Here we report that Dicer and microRNAs (miRNAs) restrict the expression of lytic molecules in mouse and human CTLs, and describe a unique signaling network that controls the expression of perforin, eomesodermin, and the IL-2Rα chain (CD25) downstream of IL-2 and inflammatory signals through miR-139 and miR-150 in differentiating CTLs. Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTLs, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes up-regulation of perforin, granzymes, and effector cytokines. Genome-wide analysis of microRNA (miR, miRNA) changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of an miRNA network that controls perforin, eomesodermin, and IL-2Rα expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation, and antigenic stimulation. Overall, our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation.

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.

The transcriptional control of the perforin locus.

Summary:  Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) use cytotoxic granules containing perforin and granzymes to lyse infected or malignant host cells, thereby providing immunity to intracellular microbes and tumors. Perforin is essential for cytotoxic granule‐mediated killing. Perforin expression is regulated transcriptionally and correlates tightly with the development of cells that can exhibit cytotoxic activity. Although a number of genes transcribed by T cells and NK cells have been studied, the cell‐specificity of perforin gene expression makes it an ideal model system in which to clarify the transcriptional mechanisms that guide the development and activation of cytotoxic lymphocytes. In this review, we discuss what is known about perforin expression and its regulation, then elaborate on recent studies that utilized chromosome transfer and bacterial artificial chromosome transgenics to define a comprehensive set of cis‐regulatory regions that control transcription of the human PRF1 gene in a near‐physiologic context. In addition, we compare the human and murine Prf1 loci and discuss how transcription factors known to be important for driving CTL differentiation might also directly regulate the cis‐acting domains that control Prf1. Our review emphasizes how studies of PRF1/Prf1 gene transcription can illuminate not only the mechanisms of cytotoxic lymphocyte differentiation but also some basic principles of transcriptional regulation.

A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes

The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of ∼20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes.

High-resolution nucleosome mapping of targeted regions using BAC-based enrichment

We report a target enrichment method to map nucleosomes of large genomes at unprecedented coverage and resolution by deeply sequencing locus-specific mononucleosomal DNA enriched via hybridization with bacterial artificial chromosomes. We achieved ∼10 000-fold enrichment of specific loci, which enabled sequencing nucleosomes at up to ∼500-fold higher coverage than has been reported in a mammalian genome. We demonstrate the advantages of generating high-sequencing coverage for mapping the center of discrete nucleosomes, and we show the use of the method by mapping nucleosomes during T cell differentiation using nuclei from effector T-cells differentiated from clonal, isogenic, naïve, primary murine CD4 and CD8 T lymphocytes. The analysis reveals that discrete nucleosomes exhibit cell type-specific occupancy and positioning depending on differentiation status and transcription. This method is widely applicable to mapping many features of chromatin and discerning its landscape in large genomes at unprecedented resolution.

SnapShot: effector and memory T cell differentiation.

The differentiation of T cells is an ideal system to study the molecular basis of lineage specification in mammalian cells. Upon stimulation with antigen during infection or inflammation, naive peripheral T cells differentiate into various types of effector T cells with specific immune functions. Naive CD4+ T cells differentiate into at least four subsets (lineages) of T helper (Th) cells: Th1, Th2, Th17, or “induced” regulatory T cells (iTregs). Each subset is distinguished by the cytokines that they produce (Ansel et al., 2006; Lee et al., 2006; Zhou et al., 2009). Naive CD8+ T cells differentiate into effector cytolytic T lymphocytes (CTLEff) that kill infected host cells using the poreforming protein perforin and serine esterases called granzymes (Cruz-Guilloty et al., 2009). Alternatively, naive CD8+ T cells can differentiate into memory CTLs (CTLMem) that survive long-term and protect the host from reinfection (Kaech and Wherry, 2007). T cell differentiation is in large part determined by signals from the environment and is shaped by numerous feedback and feed-forward loops (bold arrows) that modulate and reinforce the direction in which differentiation proceeds (Singh, 2007). Transcription factors (boxes) play a key role in this process by forming networks in which they reinforce or oppose each other’s actions. This SnapShot illustrates the differentiation pathways for several of the best characterized T cell subsets.