Francisco García-Cózar

Transcriptional Mechanisms Underlying Lymphocyte Tolerance

In lymphocytes, integration of Ca2+ and other signaling pathways results in productive activation, while unopposed Ca2+ signaling leads to tolerance or anergy. We show that the Ca2+-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. Ca2+/calcineurin signaling induces a limited set of anergy-associated genes, distinct from genes induced in the productive immune response; these genes are upregulated in vivo in tolerant T cells and are largely NFAT dependent. T cells lacking NFAT1 are resistant to anergy induction; conversely, NFAT1 induces T cell anergy if prevented from interacting with its transcriptional partner AP-1 (Fos/Jun). Thus, in the absence of AP-1, NFAT imposes a genetic program of lymphocyte anergy that counters the program of productive activation mediated by the cooperative NFAT:AP-1 complex.

Selective Inhibition of NFAT Activation by a Peptide Spanning the Calcineurin Targeting Site of NFAT

NFAT transcription factors play a key role in the immune response. The activation of NFAT proteins is controlled by calcineurin, the calmodulin-dependent phosphatase that is inhibited by the immunosuppressive drugs cyclosporin A and FK506. Here we identify a short conserved sequence in NFAT proteins that targets calcineurin to NFAT. Mutation of a single residue in this sequence impairs the calcineurin-mediated dephosphorylation and nuclear translocation of NFAT1. Peptides spanning the region inhibit the ability of calcineurin to bind to and dephosphorylate NFAT proteins, without affecting the phosphatase activity of calcineurin against other substrates. When expressed intracellularly, a corresponding peptide inhibits NFAT dephosphorylation, nuclear translocation, and NFAT-mediated expression in response to stimulation. Thus, disruption of the enzyme-substrate docking interaction that directs calcineurin to NFAT can effectively block NFAT-dependent functions.

Two-site Interaction of Nuclear Factor of Activated T Cells with Activated Calcineurin

Transcription factors belonging to the nuclear factor of activated T cells (NFAT) family regulate the expression of cytokine genes and other inducible genes during the immune response. The functions of NFAT proteins are directly controlled by the calcium- and calmodulin-dependent phosphatase calcineurin. Here we show that the binding of calcineurin to NFAT is substantially increased when calcineurin is activated with calmodulin and calcium. FK506·FKBP12 drug-immunophilin complexes inhibited the interaction of NFAT with activated calcineurin much more effectively than they inhibited the interaction with inactive calcineurin, suggesting that part of the interaction with activated calcineurin involved the enzyme active site. We have previously shown that NFAT is targeted to inactive calcineurin at a region distinct from the calcineurin active site (Aramburu, J., Garcia-Cozar, F. J., Raghavan, A., Okamura, H., Rao, A., and Hogan, P. G. (1998)Mol. Cell 1, 627–637); this region is also involved in NFAT binding to activated calcineurin, since binding is inhibited by an NFAT peptide spanning the calcineurin docking site on NFAT. The interacting surfaces are located on the catalytic domain of the calcineurin A chain and on an 86-amino acid fragment of the NFAT regulatory domain. NFAT binding to the calcineurin catalytic domain was inhibited by the calcineurin autoinhibitory domain and the RII substrate peptide, which bind in the calcineurin active site, as well as by the NFAT docking site peptide, which binds to a region of calcineurin distinct from the active site. We propose that, in resting cells, NFAT is targeted to a region of the calcineurin catalytic domain that does not overlap the calcineurin active site. Upon cell activation, displacement of the autoinhibitory domain by calmodulin binding allows NFAT to bind additionally to the calcineurin active site, thus positioning NFAT for immediate dephosphorylation at functional phosphoserine residues.

Hepatitis C virus core protein up-regulates anergy-related genes and a new set of genes, which affects T cell homeostasis

Hepatitis C virus (HCV) infection is the main cause for chronic hepatitis, leading to cirrhosis and hepatic carcinoma. Virally induced immune dysfunction has been called as the cause for viral persistence. Previous results demonstrate that CD4 Jurkat cells stably expressing the HCV core protein show an increased activation of NFAT transcription factor and an impaired IL‐2 promoter activity, affecting intracellular signaling pathways in a manner that mimics clonal anergy. We had shown previously that NFAT activates a transcriptional program, ensuing in immunological tolerance. In the present work, we have engineered lentiviral vectors expressing the HCV core to analyze the events, which unfold in the initial phase of HCV core‐induced anergy. We show that genes initially described to be up‐regulated by ionomycin‐induced anergy in mice are also up‐regulated in humans, not only by ionomycin but also by HCV core expression. We also show that HCV core is sufficient to cause NFAT nuclear translocation and a slow‐down in cell‐cycle progression, and using whole genome microarrays, we identify novel genes up‐regulated in Jurkat cells expressing HCV core. The relevance of our results is highlighted by the presence of HCV in CD4 T cells from HCV chronically infected patients.

CD4+ primary T cells expressing HCV-core protein upregulate Foxp3 and IL-10, suppressing CD4 and CD8 T cells.

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127lowPD-1highTIM-3high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.

Up-regulation of FOXP3 and induction of suppressive function in CD4 + Jurkat T-cells expressing hepatitis C virus core protein

HCV (hepatitis C virus) infection is a serious health care problem that affects more than 170 million people worldwide. Viral clearance depends on the development of a successful cellular immune response against the virus. Interestingly, such a response is altered in chronically infected patients, leading to chronic hepatitis that can result in liver fibrosis, cirrhosis and hepatocellular carcinoma. Among the mechanisms that have been described as being responsible for the immune suppression caused by the virus, Treg-cells (regulatory T-cells) are emerging as an essential component. In the present work we aim to study the effect of HCV-core protein in the development of T-cells with regulatory-like function. Using a third-generation lentiviral system to express HCV-core in CD4+ Jurkat T-cells, we describe that HCV-core-expressing Jurkat cells show an up-regulation of FOXP3 (forkhead box P3) and CTLA-4 (cytotoxic T-lymphocyte antigen-4). Moreover, we show that HCV-core-transduced Jurkat cells are able to suppress CD4+ and CD8+ T-cell responses to anti-CD3 plus anti-CD28 stimulation.

Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction

The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine‐phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine‐based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL‐2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC‐γ1 and SLP‐76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine‐based motifs of LAT for the binding of important partners. The functional role of LAT serine‐based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP‐70 shows enhanced phosphorylation. Therefore, the LAT serine‐based motifs likely regulate signaling pathways that are essential for T cell physiology.

The membrane adaptor LAT is proteolytically cleaved following Fas engagement in a tyrosine phosphorylation-dependent fashion.

Engagement of the TCR (T-cell receptor) induces tyrosine phosphorylation of the LAT (linker for the activation of T-cells) adaptor, and thereby it recruits several cytosolic mediators for downstream signalling pathways. The Fas protein is essential for T-lymphocyte apoptosis, and following Fas engagement, many proteins are proteolytically cleaved, including several molecules that are important for the transduction of TCR intracellular signals. In the present study, we demonstrate that the adaptor LAT is also subject to a proteolytic cleavage in mature T-lymphocytes and thymocytes in response to Fas engagement, and also on TCR stimulation, and we identify three aspartic acid residues at which LAT is cleaved. Interestingly, these aspartic acid residues are located in proximity to several functionally important tyrosine residues of LAT, raising the possibility that their phosphorylation could modulate LAT cleavage. Consistent with that hypothesis, we show that induction of phosphorylation by pervanadate or H2O2 in Jurkat cells and thymocytes inhibits Fas-mediated cleavage of LAT. Moreover, we show that LAT proteolysis is also enhanced during anergy induction of primary human T-cells, suggesting that LAT cleavage may act as a regulator of TCR-mediated activation of T-cells and not only as a transducer of cell death promoting stimuli.

Genomic structure and chromosome location of the human gene encoding the zinc finger autoantigen ZNF330

We have recently described a novel zinc finger cDNA, ZNF330, which was immunologicaly characterized as a new human autoantigen, highly conserved during evolution from nematodes to humans. The protein was found at the nucleolus and the cytoplasm in interphase and transiently associates with centromeres in mitosis as determined by immunofluorescence analysis. We now describe that the association of ZNF330 with the nucleolus but not with the cytoplasm is RNA-dependent as shown by RNAse treatment of fixed culture cells, since ZNF330 localization was unaffected by DNAse treatment. We also report the cloning, structural organization and chromosome location of the human ZNF330 gene. The gene is comprised of 10 exons and spans approximately 16 kb of genomic DNA. The conserved residues forming nine CXXC motifs are contained in exons 3 to 9. Several major transcription initiation sites were located 126, 124 and 121 bp upstream of the translation initiation codon ATG, as determined by primer extension analysis. The human ZNF330 gene was mapped by FISH to chromosome 4q31.1→q31.2, the site of the FRA4C locus previously described as a common fragile site for acquired chromosome instability in humans.

Sesquiterpenes as immunosuppressants.

Background. The search for new immunosuppressive drugs is a high priority. Sesquiterpenes constitute a family of compounds with a great variety of biological activities due to their reactive moieties. Methods. Human tumor cell lines and murine primary cells and human peripheral blood mononuclear cells or primary CD4+ cells from healthy individuals were stimulated in the presence of sesquiterpenes. Cell division was analyzed by 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester, cell cycle progression by Hoecht, and cell death by Anexin-V and propidium iodine staining. Cytokine secretion was analyzed by means of a bioplex assay. Results. Two sesquiterpene derivatives of the 18 previously shown to inhibit vegetal cell growth are shown to block cell division and cell cycle progression in human and murine cell lines and primary cells. Cytokine secretion is also impaired on stimulation in the presence of sesquiterpenes. Conclusions. Here, we show that sesquiterpenes heliannuols constitute a novel family of molecules with potential use as immunosuppressants. Moreover, we show that an assay based on the allelopathic effect of plant leads can be used as a cost-effective screening previous to studies in mammalian cells.