ETS2 repressor factor (ERF) is involved in T lymphocyte maturation acting as regulator of thymocyte lineage commitment.

Thymocyte differentiation and lineage commitment is regulated by an extensive network of transcription factors and signaling molecules among which Erk plays a central role. However, Erk effectors as well as the molecular mechanisms underlying this network are not well understood. Erf is a ubiquitously expressed transcriptional repressor regulated by Erk-dependent phosphorylation. Here, we investigated the role of Erf in T cell maturation and lineage commitment, using a double-fluorescent Erf-floxed mouse to produce thymus-specific Erf knockouts. We observed significant accumulation of thymocytes in the CD4/CD8 DP stage, followed by a significant reduction in CD4SP cells, a trend for lower CD8SP cell frequency, and an elevated percentage of γδ expressing thymocytes in Erf-deficient mice. Also, an elevated number of CD69+ TCRß+ cells indicates that thymocytes undergoing positive selection accumulate at this stage. The expression of transcription factors Gata3, ThPOK, and Socs1 that promote CD4+ cell commitment was significantly decreased in Erf-deficient mice. These findings suggest that Erf is involved in T cell maturation, acting as a positive regulator during CD4 and eventually CD8 lineage commitment, while negatively regulates the production of γδ T cells. In addition, Erf-deficient mice displayed decreased percentages of CD4+ and CD8+ splenocytes and elevated levels of IL-4 indicating that Erf may have an additional role in the homeostasis, differentiation, and immunologic response of helper and cytotoxic T cells in the periphery. Overall, our results show, for the first time, Erf's involvement in T cell biology suggesting that Erf acts as a potential regulator during thymocyte maturation and thymocyte lineage commitment, in γδ T cell generation, as well as in Th cell differentiation.

IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development.

Although interferon-ß is used as first-line therapy for multiple sclerosis, the cell type-specific activity of type I interferons in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis, remains obscure. In this study, we have elucidated the in vivo immunomodulatory role of type I interferon signaling in T cells during experimental autoimmune encephalomyelitis by use of a novel transgenic mouse, carrying a cd2-ifnar1 transgene on a interferon-α/ß receptor 1 null genetic background, thus allowing expression of the interferon-α/ß receptor 1 and hence, a functional type I interferon receptor exclusively on T cells. These transgenic mice exhibited milder experimental autoimmune encephalomyelitis with reduced T cell infiltration, demyelination, and axonal damage in the central nervous system. It is noteworthy that interferon-ß administration in transgenic mice generated a more pronounced, protective effect against experimental autoimmune encephalomyelitis compared with untreated littermates. In vivo studies demonstrated that before experimental autoimmune encephalomyelitis onset, endogenous type I interferon receptor signaling in T cells led to impaired T-helper 17 responses, with a reduced fraction of CCR6(+) CD4(+) T cells in the periphery. At the acute phase, an increased proportion of interleukin-10- and interferon-γ-producing CD4(+) T cells was detected in the periphery of the transgenic mice, accompanied by up-regulation of the interferon-γ-induced gene Irgm1 in peripheral T cells. Together, these results reveal a hitherto unknown T cell-associated protective role of type I interferon in experimental autoimmune encephalomyelitis that may provide valuable clues for designing novel therapeutic strategies for multiple sclerosis.

Analysis of protein interaction networks for the detection of candidate hepatitis B and C biomarkers.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are the major causes of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC). The resolution or chronicity of acute infection is dependent on a complex interplay between virus and innate/adaptive immunity. The mechanisms that lead a significant proportion of patients to more severe liver disease are not clearly defined and involve virus induced host gene/protein alterations. The utilization of protein interaction networks (PINs) is expected to identify novel aspects of the disease concerning the patients' immune response to virus as well as the main pathways that are involved in the development of fibrosis and HCC. In this study, we designed several PINs for HBV and HCV and employed topological, modular, and functional analysis techniques in order to determine significant network nodes that correspond to prominent candidate biomarkers. The networks were built using data from various interaction databases. When the overall PINs of HBV and HCV were compared, 48 nodes were found in common. The implementation of a statistical ranking procedure indicated that three of them are of higher importance.

Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: a distinct target in the calcium dependent pathway.

Calcium (Ca2+) plays an essential role in lymphocyte activation and differentiation by affecting signaling pathways leading to cytokine production. Among the enzymes responding to calcium increase, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been involved in anergy with a still poorly characterized role. IL-10 produced by different T lymphocyte subpopulations is critical mediator of tolerance. We tested the hypothesis that CaMKII may be involved in IL-10 production. We report that CaMKII upregulates IL-10 production by primary human T lymphocytes stimulated through the antigen receptor or bypassing that. Overexpression of constitutively active mutant forms of Calcineurin or CaMKII specifically increase IL-10 protein product and IL-10 mRNA accumulation in T lymphocytes. By cotransfecting constitutively active CaMKII with luciferase reporter plasmids carrying specific fragments or the whole IL-10 promoter, we show that CaMKII specifically activates IL-10 promoter activity, whereas it inhibits IL-2 and IL-4 promoter. This effect is mediated by the first 500 bp fragment, which contains binding sites for Myocyte Enhancer Factor-2 (MEF2). A constitutively active mutant of CaMKII activated a luciferase reporter plasmid under the control of MEF2, when cotransfected in T lymphocytes stimulated by Ionomycin and PMA, whereas its inhibitor KN-62 inhibited MEF2 binding in cell lysates of the same cells. Moreover, overexpression of MEF2 enhanced by 2.5-fold IL-10 promoter activity. Our data for the first time suggest a distinct role of CaMKII in the induction of anergy in T lymphocytes, by differential regulation of IL-10 and IL-2 gene transcription suggest MEF2 as a molecular target which can integrate different calcium signals.

Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase.

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response.

cAMP regulates IL-10 production by normal human T lymphocytes at multiple levels: a potential role for MEF2.

Signal transduction by the cAMP/cAMP-dependent protein kinase A (PKA) pathway is triggered through multiple receptors and is important for many processes in a variety of cells. In T cells, the engagement of the TCR-CD3 complex induces cAMP, a second messenger that controls immune response. IL-10, produced by a variety of lymphocyte subpopulations, is an important regulator of this response exerting a wide range of immunomodulatory actions. Elevation of cAMP has been shown to increase IL-10 production by monocytes. However, the mechanism of cAMP mediated regulation of IL-10 production by T lymphocytes remains unclear. In this study using normal peripheral T lymphocytes stimulated either through the TCR-CD3 complex or the TCR-CD3 and the CD28 molecule, we show that IL-10 is produced mainly by memory T lymphocytes after either way of stimulation and is drastically inhibited (70-90%) by cAMP elevating agents. cAMP mediated inhibition was reversed by the use of the specific PKA inhibitor Rp-8-Br-cAMP but not by the addition of exogenous rhIL-2, indicating that the inhibitory effect depends on PKA activation and is not secondary to IL-2 inhibition. Inhibition is taking place at both transcriptional and posttranscriptional level. Transfection of a luciferase reporter plasmid carrying the IL-10 promoter in T cells, revealed that TCR/CD28-induced activation was inhibited by 60% by cAMP elevation. The most sensitive part to cAMP mediated inhibition was a fragment of 135 bp upstream of TATA box, which contains multiple binding sites for MEF-2. Overexpression of MEF-2 in the same cells increased IL-10 promoter activity by 2.5-fold. Stimulation through TCR/CD28 increased MEF-2 binding in its corresponding binding sites which was inhibited by 80% in the presence of cAMP elevating agents. These results suggest that the inhibitory effect of cAMP on IL-10 production by normal peripheral T lymphocytes is cell type and stimulus specific, exerted on multiple levels and involves MEF2 transcription factor.

Adjuvant activity of type I interferons.

Type I interferons (IFNs) produced primarily by plasmacytoid dendritic cells (pDCs) as part of the innate immune response to infectious agents induce the maturation of myeloid DCs and enhance antigen presentation. Type I IFNs also enhance apoptosis of virus-infected cells, stimulate cross priming and enhanced presentation of viral peptides. Type I IFNs are powerful polyclonal B-cell activators that induce a strong primary humoral immune response characterized by isotype switching and protection against virus challenge. Type I IFNs stimulate an IgG2a antibody response characteristic of Th1 immunity when ad-mixed with influenza virus vaccine and injected intramuscurarly (i.m.) or administered intranasally. The adjuvant activity of type I IFNs has been shown to involve direct effects of IFN on B-cells, effects on T-cells, as well as effects on antigen presentation. Oromucosal administration of type I IFNs concomitantly with i.m. injection of vaccine alone can also enhance the antibody response to influenza vaccination by enhancing trafficking of antigen-presenting cells towards the site of vaccination. Recombinant IFNs are potent adjuvants that may find application in both parenterally and mucosally administered vaccines.

Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erk1/2 activity.

The p38 mitogen-activated protein kinase regulates many cellular processes in almost all eukaryotic cell types. In T cells, p38 was shown to regulate thymic development and cytokine production. Here, the role of p38 on interleukin-2 (IL-2) production by human peripheral blood CD4+ T cells was examined. When T cells were stimulated under weak stimulation conditions, pharmaceutical and molecular p38 inhibitors induced a dramatic increase of IL-2 production. In contrast, IL-2 levels were not affected significantly when strong stimulation was provided to T cells. The increase in IL-2 production, following p38 inhibition, was associated with a strong up-regulation of extracellular signal-regulated kinase (Erk)1/2 activity. Furthermore the Erk inhibitor U0126 was able to counteract the effect of p38 inhibition on IL-2 production, supporting the conclusion that p38 mediates its effect through Erk. These results suggest that the p38 kinase, through its ability to control Erk activation levels, acts as a gatekeeper, which prevents inappropriate IL-2 production. Also, the finding that p38 acts in a strength-of-stimulation-dependent way provides an explanation for previously reported, contradictory results regarding the role of this kinase in IL-2 expression.

Boosting the immune response: an alternative combination therapy for cancer patients.

Transformation of normal cells into malignant cancer involves a number of changes in the genome. These changes include chromosomal translocations, exon deletions and gene mutations, to name a few, that result in deregulation of the regulatory circuits of the cell and consequently in profound changes in their antigenic composition, including expression of mutated proteins, overexpression of proteins that are produced at much lesser levels in normal tissue and expression of aberrantly glycosylated proteins. It is well established that these new antigenic entities, referred to as tumor-associated antigens (TAA), are recognized by the immune system and elicit immune reactivity. However, the immune reaction is overpowered by the cancer potential to grow and to metastasize. Cancer immunotherapy aims at boosting the naturally occurring immune response at a level that will prevail over the ability of cancer cells to escape immune attack. After more than 20 years of intense research and a growing number of clinical trials, most of which gave marginal results, it is now clear that the task is not an easy one. However, during the same period of time, we have gained a much better understanding of the mechanisms of tumor growth, the mechanisms by which the immune system is activated or becomes tolerant and of the natural relationship between cancers and the immune system. Also, even though few, some immunotherapy strategies have demonstrated positive clinical responses and are already used as standard therapies. These developments give the impetus to explore and devise new strategies that will by more effective in strengthening the immune reactivity at a level that will counteract the tumor potential. In this paper we review the knowledge gained from basic research related to cancer immunity and from clinical trials. We propose that, based on this knowledge, improved clinical protocols can be elaborated.

IL-2 and IL-10 production by human CD4+T cells is differentially regulated by p38: mode of stimulation-dependent regulation of IL-2.

Antigenic stimulation of T cells initiates a complex series of intracellular signaling pathways that target and activate different cytokine genes. The participation of mitogen-activated protein kinases (MAPKs) in these processes has not been studied thoroughly and in some instances conflicting results have been reported. Here we have examined the role of p38 MAPK on IL-2 and IL-10 production following activation of human CD4+ T cells or of the leukemic cell line Hut-78, with either plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 (plCD3, plCD3/sCD28), or with cross-linked anti-CD3 and anti-CD28 (crsCD3+CD28), or with PMA plus ionomycin. Pharmacological inhibition of the p38 pathway with either SB203580, SB202190, or SKF86002 strongly downregulated IL-10 production by T cells stimulated with any of the above treatments. In contrast the effect of p38 inhibition on IL-2 was stimulus dependent. Thus, p38 inhibition strongly upregulated IL-2 production (up to 10-fold) in the plCD3- and plCD3/sCD28-stimulated cultures while it had minimal or no effect in the other two stimulation protocols. Intracellular and mRNA levels of IL-2 and IL-10 were also upregulated and downregulated, respectively, by p38 inhibitors in the plCD3/sCD28-stimulated CD4+ T cells. Also, the induction of IL-2 and the parallel suppression of IL-10 by p38 inhibitors were independent of the balance between these two cytokines, as demonstrated by the addition of exogenous IL-10 or blocking anti-IL-10 antibody in CD4+ and Hut-78 cell cultures. These results show that p38 acts as a molecular switch that changes the balance between IL-2 and IL-10. This is especially important considering the opposing role of these cytokines in peripheral immune tolerance.

Low expression of interferon regulatory factor-1 and identification of novel exons skipping in patients with chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN-alpha) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK-STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor-1 (IRF-1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF-1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF-1 in CML is currently unknown. Therefore, mutational analysis of IRF-1 was performed and its expression pattern was also studied in CML patients. We studied IRF-1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full-length IRF-1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full-length IRF-1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full-length IRF-1 mRNA was observed. These findings demonstrate that, in CML patients, IRF-1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full-length IRF-1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease.

Characterization of the cysteine-rich secretory protein 3 gene as an early-transcribed gene with a putative role in the pathophysiology of Sjögren's syndrome.

OBJECTIVE: To identify genes that may participate in the pathophysiology of Sjögren's syndrome (SS), the technique of differential display was applied to labial minor salivary gland (MSG) biopsy samples. METHODS: Total RNA was isolated from MSG biopsy samples from a woman with primary SS and a control subject, and the differential display protocol with 8 different random oligonucleotide primers was performed. One particular differentially expressed fragment showed 98% homology with the cysteine-rich secretory protein 3 (CRISP-3) gene. The result was verified by reverse transcription-polymerase chain reaction (RT-PCR) with messenger RNA (mRNA) samples from MSG biopsy tissues obtained from 4 women with primary SS. A CRISP-3 RNA probe was synthesized for in situ hybridization of 7 MSG biopsy samples from patients with primary SS. In an attempt to interpret the expression of CRISP-3, normal peripheral blood lymphocytes (PBLs) were activated in vitro at different time points and assayed for CRISP-3 expression. Finally, B cells were transfected with the coding region of CRISP-3 and monitored for the up-regulation of different B cell activation markers. RESULTS: The CRISP-3 gene was detected by RT-PCR in all SS patients tested. Mainly the mononuclear cells infiltrating the MSGs of patients expressed CRISP-3 mRNA. In addition, CRISP-3 was detected by RT-PCR between 30 minutes and 6 hours in phorbol myristate acetate-activated normal PBLs, while staurosporine inhibited this expression. CRISP-3-transfected B cells exhibited an up-regulation in CD25 surface expression. CONCLUSION: The CRISP-3 gene is identified as a novel early response gene that may participate in the pathophysiology of the autoimmune lesions of SS.