PubMed Health⌕ Search

Biomedical subjects

Vigo Heissmeyer

Publications and source records attributed to Vigo Heissmeyer.

11 recordsLinked to original sources
PubMed2015

The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells.

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.

Animals↗
PubMed2006

Rapid conditional knock-down-knock-in system for mammalian cells.

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down-knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein.

Cell Cycle Proteins↗
PubMed2006

FOXP3 controls regulatory T cell function through cooperation with NFAT.

Antigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.

Amino Acid Sequence↗
PubMed2006

Essential role for IkappaB kinase beta in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation.

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Transcriptional basis of lymphocyte tolerance.

Signaling through lymphocyte antigen receptors has the potential to initiate several distinct outcomes: proliferation, differentiation, apoptosis, or functional unresponsiveness. Expansion and differentiation of effector T cells is required for defense against foreign antigens, whereas functional unresponsiveness, termed anergy, is a cell-intrinsic mechanism that contributes to peripheral self-tolerance. Other mechanisms of peripheral tolerance include the 'dominant' tolerance imposed by regulatory T cells and immunosuppression mediated by interleukin-10 and transforming growth factor-beta. T- and B-cell antigen receptor ligation induces an increase in intracellular calcium levels as well as activating additional signaling pathways that are further potentiated by costimulatory receptors. In this review, we argue that cell-intrinsic programs of peripheral anergy and tolerance are imposed by sustained calcium signaling in lymphocytes. We address in particular the role of the calcium-dependent transcription factor nuclear factor for activation of T cells, which is activated by antigen receptor stimulation and, depending on the presence or absence of input from its transcriptional partner, activator protein-1, dictates two distinct transcriptional programs: activation or tolerance.

Animals↗
PubMed2005

Efficiency of RNA interference in the mouse hematopoietic system varies between cell types and developmental stages.

RNA interference (RNAi) is a naturally occurring posttranscriptional gene-silencing mechanism that has been adapted as a genetic tool for loss-of-function studies of a variety of organisms. It is more widely applicable than classical gene targeting and allows for the simultaneous inactivation of several homologous genes with a single transgene. Recently, RNAi has been used for conditional and conventional gene inactivation in mice. Unlike gene targeting, RNAi is a dynamic process, and its efficiency may vary both between cell types and throughout development. Here we demonstrate that RNAi can be used to target three separately encoded isoforms of the bcl-2 family gene bfl-1/A1 in a conditional manner in mice. The extent of gene inactivation varies between different cell types and is least efficient in mature lymphocytes. Our data suggest that RNAi is affected by factors beyond small interfering RNA-mRNA stoichiometry.

Animals↗
PubMed2005

A molecular dissection of lymphocyte unresponsiveness induced by sustained calcium signalling.

In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells.

Calcium Signaling↗
PubMed2004

E3 ligases in T cell anergy--turning immune responses into tolerance.

Peripheral tolerance is an important strategy used by the immune system to prevent self-reactive lymphocytes from attacking host tissues. A variety of mechanisms contribute to peripheral tolerance, among them activation-induced cell death, suppression by regulatory T cells, and T cell anergy or unresponsiveness. Recent work has led to a better understanding of the cell-intrinsic program that establishes T cell anergy. A major insight is that during the induction phase of anergy, incomplete stimulation (T cell receptor stimulation without costimulation) leads via calcium influx to an altered gene expression program that includes up-regulation of several E3 ubiquitin ligases. When the anergic T cells contact antigen-presenting cells, intracellular signaling proteins are monoubiquitinated and targeted for lysosomal degradation, thus decreasing intracellular signaling and also resulting in decreased stability of the T cell-antigen-presenting cell contact. We propose a molecular program leading to T cell anergy and discuss other proteins that may play a role.

Animals↗
PubMed2004

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins.

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Animals↗
PubMed2004

Degradation of Bcl10 induced by T-cell activation negatively regulates NF-kappa B signaling.

Bcl10 is a critical regulator of NF-kappa B activity in T and B cells, coupling antigen receptor signaling to NF-kappa B activation via protein kinase C (PKC). Here we show that PKC or T-cell receptor (TCR)/CD28 signaling results in downregulation of Bcl10 protein levels, thereby attenuating NF-kappa B transcriptional activity. Bcl10 degradation requires an intact caspase recruitment domain and is not observed after stimulation with tumor necrosis factor alpha or lipopolysaccharides. Bcl10 downregulation is not affected by proteasome inhibitors but is accompanied by transient localization to lysosomal vesicles, suggesting involvement of the lysosomal pathway rather than the proteasome. The HECT domain ubiquitin ligases NEDD4 and Itch promote ubiquitination and degradation of Bcl10, thus downmodulating NF-kappa B activation. Since CD3/CD28-induced activation of JNK is not affected by the decline of Bcl10, degradation of Bcl10 selectively terminates IKK/NF-kappa B signaling in response to TCR stimulation. Together, these results suggest a new mechanism of negative signaling in which TCR/PKC signaling initially activates Bcl10 but later promotes its degradation.

Adaptor Proteins, Signal Transducing↗