Search

The functional specialization of CD4 T lymphocytes into various subtypes, including T H 1 and T FH cells, is crucial for effective immune responses. T FH cells facilitate B cell differentiation within germinal centers, while T H 1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of Integrin α4 in regulating T FH and T H 1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating deletions in the gene. By analyzing the effects of Itga4 deficiency on T FH and T H 1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the T H 1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of Integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wi, Choi, Kim, Choi, Pipkin and Choi.)

Ongoing viral transcription from the reservoir of HIV-1 infected long-lived memory CD4 + T cells presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. Here, we examine the impact of dCA on host immune CD4 + T-cell transcriptional and epigenetic states. We performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA treatment of primary human memory CD4 + T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response, and T-cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4 + T cells during HIV-1 infection. In addition, dCA suppresses the expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets, and reservoirs. dCA's benefits thus extend beyond viral transcription inhibition, modulating the immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV infection., (© 2024 Mori et al.)

CTL differentiation is controlled by the crosstalk of various transcription factors and epigenetic modulators. Uncovering this process is fundamental to improving immunotherapy and designing novel therapeutic approaches. In this study, we show that polycomb repressive complex 1 subunit chromobox (Cbx)4 favors effector CTL differentiation in a murine model. Cbx4 deficiency in CTLs induced a transcriptional signature of memory cells and increased the memory CTL population during acute viral infection. It has previously been shown that besides binding to H3K27me3 through its chromodomain, Cbx4 functions as a small ubiquitin-like modifier (SUMO) E3 ligase in a SUMO-interacting motifs (SIM)-dependent way. Overexpression of Cbx4 mutants in distinct domains showed that this protein regulates CTL differentiation primarily in an SIM-dependent way and partially through its chromodomain. Our data suggest a novel role of a polycomb group protein Cbx4 controlling CTL differentiation and indicated SUMOylation as a key molecular mechanism connected to chromatin modification in this process., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Follicular helper T (Tfh) cells are essential for germinal center (GC) B cell responses. However, it is not clear which PD-1 + CXCR5 + Bcl6 + CD4 + T cells will differentiate into PD-1 hi CXCR5 hi Bcl6 hi GC-Tfh cells and how GC-Tfh cell differentiation is regulated. Here, we report that the sustained Tigit expression in PD-1 + CXCR5 + CD4 + T cells marks the precursor Tfh (pre-Tfh) to GC-Tfh transition, whereas Tigit - PD-1 + CXCR5 + CD4 + T cells upregulate IL-7Rα to become CXCR5 + CD4 + T memory cells with or without CCR7. We demonstrate that pre-Tfh cells undergo substantial further differentiation at the transcriptome and chromatin accessibility levels to become GC-Tfh cells. The transcription factor c-Maf appears critical in governing the pre-Tfh to GC-Tfh transition, and we identify Plekho1 as a stage-specific downstream factor regulating the GC-Tfh competitive fitness. In summary, our work identifies an important marker and regulatory mechanism of PD-1 + CXCR5 + CD4 + T cells during their developmental choice between memory T cell fate and GC-Tfh cell differentiation., (© 2023. The Author(s).)

T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage-defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation., (Copyright © 2023 by The American Association of Immunologists, Inc.)

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8 + tissue-resident memory T cells (T RM ), the developmental origins and transcriptional regulation of CD4 + T RM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4 + T RM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating T H 1 and the progressive acquisition of a mature T RM program. Single-cell RNA sequencing identified heterogeneity among established CD4 + T RM , which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. T H 1-associated Blimp1 and Id2 and T FH -associated Bcl6 were required for early T RM formation and development of a mature T RM population in the SI. These results demonstrate a developmental relationship between T H 1 effector cells and the establishment of early T RM , as well as highlighted differences in CD4 + versus CD8 + T RM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4 + T RM in response to viral infection.

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., Competing Interests: Declaration of interests A.W.G. is a member of the scientific advisory board of ArsenalBio. S.J.T. is a member of the scientific advisory board for Medicago, Inc., QC, Canada. No funding from Medicago or ArsenalBio was provided for this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4 + T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat's basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.

T follicular helper (T FH ) cells are a specialized subset of CD4 T cells that deliver critical help signals to B cells for the production of high-affinity Abs. Understanding the genetic program regulating T FH differentiation is critical if one wants to manipulate T FH cells during vaccination. A large number of transcription factor (TFs) involved in the regulation of T FH differentiation have been characterized. However, there are likely additional unknown TFs required for this process. To identify new TFs, we screened a large short hairpin RNA library targeting 353 TFs in mice using an in vivo RNA interference screen. Yin Yang 1 (YY-1) was identified as a novel positive regulator of T FH differentiation. Ablation of YY-1 severely impaired T FH differentiation following acute viral infection and protein immunization. We found that the zinc fingers of YY-1 are critical to support T FH differentiation. Thus, we discovered a novel TF involved in the regulation of T FH cells., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Robust immunity to intracellular infections is mediated by antigen-specific naive CD8 T cells that become activated and differentiate into phenotypically and functionally diverse subsets of effector cells, some of which terminally differentiate and others that give rise to memory cells that provide long-lived protection. This developmental system is an outstanding model with which to elucidate how regulation of chromatin structure and transcriptional control establish gene expression programs that govern cell fate determination, insights from which are likely to be useful for informing the design of immunotherapeutic approaches to engineer durable immunity to infections and tumors. A unifying framework that describes how naive CD8 T cells develop into memory cells is still outstanding. We propose a model that incorporates a common early linear path followed by divergent paths that slowly lose capacity to interconvert and discuss classical and contemporary observations that support these notions, focusing on insights from transcriptional control and chromatin regulation., (Copyright © 2022 Cold Spring Harbor Laboratory Press; all rights reserved.)

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability., Competing Interests: A.W. Goldrath reported “other” from Pandion Therapeutics and “other” from ArsenalBio outside the submitted work, and is on the SAB of Pandion Therapeutics and ArsenalBio. J. Qi reported “other” from Epiphanes outside the submitted work. No other disclosures were reported., (© 2021 Milner et al.)

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine 1 . As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues 2 . Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes 3 , but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4 + T effector (T eff ) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation 4 . Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in T eff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP T eff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1 -/- or Rag2 - / -  mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

The transcriptional and epigenetic regulation of CD8 + T cell differentiation is critical for balancing pathogen eradication and long-term immunity by effector and memory CTLs, respectively. In this study, we demonstrate that the lysine demethylase 6b (Kdm6b) is essential for the proper generation and function of effector CD8 + T cells during acute infection and tumor eradication. We found that cells lacking Kdm6b (by either T cell-specific knockout mice or knockdown using short hairpin RNA strategies) show an enhanced generation of memory precursor and early effector cells upon acute viral infection in a cell-intrinsic manner. We also demonstrate that Kdm6b is indispensable for proper effector functions and tumor protection, and that memory CD8 + T cells lacking Kdm6b displayed a defective recall response. Mechanistically, we identified that Kdm6b, through induction of chromatin accessibility in key effector-associated gene loci, allows for the proper generation of effector CTLs. Our results pinpoint the essential function of Kdm6b in allowing chromatin accessibility in effector-associated genes, and identify Kdm6b as a potential target for therapeutics in diseases with dysregulated effector responses., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Adaptive immunity to intracellular pathogens and tumors is mediated by antigen-experienced CD8 T cells. Individual naive CD8 T cells have the potential to differentiate into a diverse array of antigen-experienced subsets that exhibit distinct effector functions, life spans, anatomic positioning, and potential for regenerating an entirely new immune response during iterative pathogenic exposures. The developmental process by which activated naive cells undergo diversification involves regulation of chromatin structure and transcription but is not entirely understood. This review examines how alterations in chromatin structure, transcription factor binding, extracellular signals, and single-cell gene expression explain the differential development of distinct effector (T EFF ) and memory (T MEM ) CD8 T cell subsets. Special emphasis is placed on how Runx proteins function with additional transcription factors to pioneer changes in chromatin accessibility and drive transcriptional programs that establish the core attributes of cytotoxic T lymphocytes, subdivide circulating and non-circulating T MEM cell subsets, and govern terminal differentiation. The discussion integrates the roles of specific cytokine signals, transcriptional circuits and how regulation of individual nucleosomes and RNA polymerase II activity can contribute to the process of differentiation. A model that integrates many of these features is discussed to conceptualize how activated CD8 T cells arrive at their fates., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

T follicular helper (T FH ) cells are a distinct type of CD4 + T cells that are essential for most antibody and B lymphocyte responses. T FH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of T FH cells and its activity is essential for T FH cell differentiation and function. However, how Bcl-6 controls T FH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of T FH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits.

The Hippo pathway regulates cell proliferation and organ size through control of the transcriptional regulators YAP (yes-associated protein) and TAZ. Upon extracellular stimuli such as cell-cell contact, the pathway negatively regulates YAP through cytoplasmic sequestration. Under conditions of low cell density, YAP is nuclear and associates with enhancer regions and gene promoters. YAP is mainly described as a transcriptional activator of genes involved in cell proliferation and survival. Using a genome-wide approach, we show here that, in addition to its known function as a transcriptional activator, YAP functions as a transcriptional repressor by interacting with the multifunctional transcription factor Yin Yang 1 (YY1) and Polycomb repressive complex member enhancer of zeste homologue 2 (EZH2). YAP colocalized with YY1 and EZH2 on the genome to transcriptionally repress a broad network of genes mediating a host of cellular functions, including repression of the cell-cycle kinase inhibitor p27, whose role is to functionally promote contact inhibition. This work unveils a broad and underappreciated aspect of YAP nuclear function as a transcriptional repressor and highlights how loss of contact inhibition in cancer is mediated in part through YAP repressive function. SIGNIFICANCE: This study provides new insights into YAP as a broad transcriptional repressor of key regulators of the cell cycle, in turn influencing contact inhibition and tumorigenesis., (©2020 American Association for Cancer Research.)

Multidrug resistance-1 (MDR1) acts as a chemotherapeutic drug efflux pump in tumor cells, although its physiological functions remain enigmatic. Using a recently developed MDR1-knockin reporter allele (Abcb1aAME), we found that constitutive MDR1 expression among hematopoietic cells was observed in cytolytic lymphocytes-including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer cells-and regulated by Runt-related (Runx) transcription factors. Whereas MDR1 was dispensable for naive CD8+ T cell development, it was required for both the normal accumulation of effector CTLs following acute viral infection and the protective function of memory CTLs following challenge with an intracellular bacterium. MDR1 acted early after naive CD8+ T cell activation to suppress oxidative stress, enforce survival, and safeguard mitochondrial function in nascent CTLs. These data highlight an important endogenous function of MDR1 in cell-mediated immune responses and suggest that ongoing efforts to intentionally inhibit MDR1 in cancer patients could be counterproductive., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Chen et al.)

T cell receptor (TCR) stimulation of naive CD8 + T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This corrects the article DOI: 10.1038/nature24993.

Tissue-resident memory CD8 + T (T RM ) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control T RM cell differentiation and homeostasis are not fully understood. Here we show that mouse T RM precursor cells represent a unique CD8 + T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of T RM cell differentiation and homeostasis. Runx3 was required to establish T RM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with T RM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8 + tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8 + T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of T RM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8 + T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8 + T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8 + T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8 + T cell differentiation.

Cbx3/HP1γ is a histone reader whose function in the immune system is not completely understood. Here, we demonstrate that in CD8 + T cells, Cbx3/HP1γ insufficiency leads to chromatin remodeling accompanied by enhanced Prf1, Gzmb and Ifng expression. In tumors obtained from Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8 + T cells, there is an increase of CD8 + effector T cells expressing the stimulatory receptor Klrk1/NKG2D, a decrease in CD4 + CD25 + FOXP3 + regulatory T cells (Treg cells) as well as CD25 + CD4 + T cells expressing the inhibitory receptor CTLA4. Together these changes in the tumor immune environment may have mitigated tumor burden in Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8 + T cells. These findings suggest that targeting Cbx3/HP1γ can represent a rational therapeutic approach to control growth of solid tumors.

Functional genomics approaches that leverage the RNAi pathway have been applied in vivo to examine the roles of hundreds or thousands of genes; mainly in the context of cancer. Here, we discuss principles guiding the design of RNAi screens, parameters that determine success and recent developments that have improved accuracy and expanded the applicability of these approaches to other in vivo settings, including the immune system. We review recent studies that have applied in vivo RNAi screens in T cells to examine genes that regulate T cell differentiation during viral infection, and that control their accumulation in tumors in a model of adoptive T cell therapy. In this context, we put forward an argument as to why RNAi approaches in vivo are likely to provide particularly salient insight into immunology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Classical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time consuming. Here we developed a pooled approach to screen 30-100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4(+) and CD8(+) T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper 1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTLs). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL and enhanced Tfh cell and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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.

Under the cover of snow in February 2011, immunologists convened in Banff, Alberta, Canada, for the Keystone symposium 'Immunologic Memory, Persisting Microbes and Chronic Disease'. These are wide-ranging topics that are typically addressed in separate experimental settings. However, a theme that emerged was the way in which these subjects are inextricably linked, and the importance of addressing them together and deciphering their molecular determinants.

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.

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

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) gamma 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-gamma, 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.

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 p27(kip1), we found that in the mouse alternative splicing results in two p27(kip1) 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.

While the hereditary information encoded in the Watson-Crick base pairing of genomes is largely static within a given individual, access to this information is controlled by dynamic mechanisms. The human genome is pervasively transcribed, but the roles played by the majority of the non-protein-coding genome sequences are still largely unknown. In this review we focus on insights to gene transcriptional regulation by placing special emphasis on genome-wide approaches, and on how non-coding RNAs, which derive from global transcription of the genome, in turn control gene expression. We review recent progress in the field with highlights on the immune system.

Killer lymphocytes release perforin and granzymes from cytotoxic granules into the immunological synapse to destroy target cells as a critical mechanism in the defense against viruses and cancer. Perforin, a Ca(2+)-dependent pore-forming protein that multimerizes in membranes, delivers granzymes into the target cell cytosol. The original model for perforin (acting by forming a cell membrane channel through which granzymes pass) does not fit the experimental data. Recently, an alternative model has been proposed that involves active target cell collaboration with perforin to deliver granzymes and direct the target cell to an apoptotic, rather than necrotic, death.

Perforin gene (PRF1) transcription regulates perforin expression in NK cells and CTL. Here we identified the locus-wide ensemble of cis-acting sequences that drives PRF1 transcription physiologically. By using chromosome transfer, we revealed that de novo activation of a silent PRF1 locus was controlled by a 150 kb domain comprised of 16 DNase I hypersensitive sites (DHSs). These cis-acting sequences included a locus control region (LCR) and conferred developmentally appropriate and lineage-specific expression of human perforin from BAC transgenes. The LCR included four distal DHSs that were required for perforin expression from its natural locus, and their engineered deletion from the PRF1 BAC transgene abolished LCR function and led to rapid gene silencing. Thus, LCR function is central for regulating the developmental and activation-specific PRF1 promoter activity characteristic of NK cells and CTL.

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 approximately 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.