Impaired T cell protein kinase C delta activation decreases ERK pathway signaling in idiopathic and hydralazine-induced lupus

T cells from patients with lupus or treated with the lupus-inducing drug hydralazine have defective ERK phosphorylation. The reason for the impaired signal transduction is unknown but important to elucidate, because decreased T cell ERK pathway signaling causes a lupus-like disease in animal models by decreasing DNA methyltransferase expression, leading to DNA hypomethylation and overexpression of methylation-sensitive genes with subsequent autoreactivity and autoimmunity. We therefore analyzed the PMA stimulated ERK pathway phosphorylation cascade in CD4(+) T cells from patients with lupus and in hydralazine-treated cells. The defect in these cells localized to protein kinase C (PKC)delta. Pharmacologic inhibition of PKCdelta or transfection with a dominant negative PKCdelta mutant caused demethylation of the TNFSF7 (CD70) promoter and CD70 overexpression similar to lupus and hydralazine-treated T cells. These results suggest that defective T cell PKCdelta activation may contribute to the development of idiopathic and hydralazine-induced lupus through effects on T cell DNA methylation.     

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.     

Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs

Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4(+) lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

A Proof-of-Principle Demonstration of a Novel Microarray-Based Method for Quantifying DNA Methylation Levels

Demethylation of CD11a (ITGAL; GeneID:3683; HGNC: 6148) and CD70 (TNFSF7; GeneID:970; HGNC:11937) regulatory regions in CD4⁺ T cells contributes to the development of autoreactivity and autoantibody overstimulation in systemic lupus erythematosus (SLE). In this study, we present a novel approach for measuring the methylation status of CD11a and CD70 promoter sequences. The procedure combines the standard method of bisulfite conversion of methylated CpG pairs with high-throughput oligonucleotide microarray-based technology that allows for rapid quantification of deoxycytosine and deoxymethylcytosine content in bisulfite-treated DNA samples. The microarrays were first used to generate a standard curve from fully methylated and fully unmethylated DNA samples using a one-dimensional linear regression equation that calculated fluorescence emission as a function of methylation levels. The methylation status of the CD70 and CD11a promoters in SLE and control CD4⁺ T cell samples were measured, and the microarray prediction was found to be highly accurate when compared to bisulfite sequencing. Furthermore, the microarrays were able to detect differences in the methylation status between SLE patient and healthy control samples. These results indicate that our new microarray-based assay could prove to be a highly reliable, rapid, and cost effective diagnostic and prognostic test for SLE.     

CpG methylation of the IFNG gene as a mechanism to induce immunosuppression [correction of immunosupression] in tumor-infiltrating lymphocytes

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4(+) T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19(+) B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4(+) T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4(+) T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4(+) T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4(+) cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosuppression.     

Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.     

Regulatory defects in Cbl and mitogen-activated protein kinase (extracellular signal-related kinase) pathways cause persistent hyperexpression of CD40 ligand in human lupus T cells

To identify intrinsic defects in lupus, we studied short-term, CD4(+) T cell lines that were established from 16 lupus patients (active or inactive) and 15 normal subjects by stimulating once with anti-CD3, anti-CD28, and IL-2. After resting, the pure CD4(+) T cells were exposed to anergy-inducing stimulation with plate-bound anti-CD3 mAb in the absence of APC. Lupus T cells showed prolonged high level expression of CD40 ligand (CD40L, CD154) even in the face of anergy protocol, which shut down CD40L expression in normal T cells. The sustained CD40L expression in lupus T cells did not correlate with memory status or Th deviation, and was relatively independent of IL-2 or other autocrine or paracrine signals via CD28 or CTLA-4. Cyclosporin A could block CD40L expression by lupus T cells when added early during the anti-CD3 stimulation period, but only partially when added later, indicating that another mechanism regulates the prolonged hyperexpression of CD40L besides the Ca(2+) --> calcineurin-dependent NF-AT pathway. When exposed to the anergy protocol, lupus T cells, in marked contrast to normal T cells, did not phosphorylate Cbl/Cbl-b but continued to express strongly phosphorylated extracellular signal-regulated kinase (ERK); U0126, a specific inhibitor of mitogen-activated protein kinase kinase --> ERK, could block both the early and the prolonged hyperexpression of CD40L. Thus, pathways regulating the activities of Cbl and one particular mitogen-activated protein kinase, ERK, are involved in the prolonged hyperexpression of CD40L in lupus T cells.     

Rapid demethylation of the IFN-gamma gene occurs in memory but not naive CD8 T cells

DNA methylation is an epigenetic mechanism of gene regulation. We have determined that specific modifications in DNA methylation at the IFN-gamma locus occur during memory CD8 T cell differentiation in vivo. Expression of the antiviral cytokine IFN-gamma in CD8 T cells is highly developmental stage specific. Most naive cells must divide before they express IFN-gamma, while memory cells vigorously express IFN-gamma before cell division. Ag-specific CD8 T cells were obtained during viral infection of mice and examined directly ex vivo. Naive cells had an IFN-gamma locus with extensive methylation at three specific CpG sites. An inhibitor of methylation increased the amount of IFN-gamma in naive cells, indicating that methylation contributes to the slow and meager production of IFN-gamma. Effectors were unmethylated and produced large amounts of IFN-gamma. Interestingly, while memory cells were also able to produce large amounts of IFN-gamma, the gene was partially methylated at the three CpG sites. Within 5 h of antigenic stimulation, however, the gene was rapidly demethylated in memory cells. This was independent of DNA synthesis and cell division, suggesting a yet unidentified demethylase. Rapid demethylation of the IFN-gamma promoter by an enzymatic factor only in memory cells would be a novel mechanism of differential gene regulation. This differentiation stage-specific mechanism reflects a basic immunologic principle: naive cells need to expand before becoming an effective defense factor, whereas memory cells with already increased precursor frequency can rapidly mount effector functions to eliminate reinfecting pathogens in a strictly Ag-dependent fashion.     

Methylation status of CpG-rich islands on active and inactive mouse X chromosomes

Single copy probes derived from CpG-rich island clones fromEag I andNot I linking libraries and nine rare-cutter restriction endonucleases were used to investigate the methylation status of CpG-rich islands on the inactive and active X chromosomes (Chr) of the mouse. Thirteen of the 14 probes used detected CpG-rich islands in genomic DNA. The majority of island CpGs detected by rare-cutter restriction endonucleases were methylated on the inactive X Chr and unmethylated on the active X Chr, but some heterogeneity within the cell population used to make genomic DNA was detected. The CpG-rich islands detected by two putative pseudoautosomal probes remained unmethylated on both the active and inactive X Chrs. Otherwise, distance from the X Chr inactivation center did not affect the methylation profile of CpG-rich islands. We conclude that methylation of CpG-rich islands is a general feature of X Chr inactivation.     

Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation

DNA sequences have previously been identified in the first intron of the mouse Hprt gene that are methylated on the inactive but not the active X chromosome. The temporal relationship between methylation of these sequences and X-inactivation was studied in teratocarcinoma cells and postimplantation mouse embryos: the sequences are unmethylated prior to X-inactivation and do not become methylated on the inactive X in most fetal cells until several days postinactivation. Such inactive X-specific methylation occurs in a significantly smaller proportion of the cells in the extra-embryonic tissues, yolk sac mesoderm and endoderm, than in the fetus. These data suggest that the inactive X-specific methylation of sequences such as those in the first intron of the Hprt gene does not play any role in the primary events of X-inactivation, but may function as part of a secondary, tissue-specific mechanism for maintaining the inactive state.     

On CD28/CD40 ligand costimulation, common gamma-chain signals, and the alloimmune response

Activation and robust expansion of naive T cells often require T cell costimulatory signals and T cell growth factors. However, the precise growth and costimulation requirements for activation and expansion of CD4(+) and CD8(+) T cells in vivo in allograft response are still not clearly defined. In the present study, we critically examined the role of CD28/CD40 ligand (CD40L) costimulation and the common gamma-chain (gamma(c)) signals, a shared signaling component by receptors for all known T cell growth factors (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21), in activation and expansion of CD4(+) and CD8(+) T cells in the allogeneic hosts. We found that CD28/CD40L costimulation and the gamma(c) signals are differentially involved in proliferation and clonal expansion of CD4(+) and CD8(+) T cells in response to alloantigen stimulation. CD8(+) T cells are highly dependent on the gamma(c) signals for survival, expansion, and functional maturation, whereas in vivo expansion of alloreactive CD4(+) T cells is largely gamma(c) independent. T cell costimulation via CD28 and CD40L, however, is necessary and sufficient for activation and expansion of CD4(+) T cells in vivo. In a skin transplant model, blocking both CD28/CD40L and the gamma(c) pathways induced prolonged skin allograft survival. Our study provides critical insights that the CD4 and CD8 compartments are most likely governed by distinct mechanisms in vivo, and targeting both costimulatory and gamma(c) signals may be highly effective in certain cytopathic conditions involving activation of both CD4(+) and CD8(+) T cells.     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

Deleterious effect of HIV-1 plasma viremia on B cell costimulatory function

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4(+) T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4(+) T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4(+) T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4(+) T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4(+) T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4(+) T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4(+) T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4(+) T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.     

The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4⁺ T, CD19⁺ B, and splenic CD4^-CD19^- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19⁺ and CD4^-CD19^- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.