GITR-GITRL System, A Novel Player in Shock and Inflammation

Glucocorticoid-induced TNFR-Related (GITR) protein is a member of the tumor necrosis factor receptor superfamily that modulates acquired and natural immune response. It is expressed in several cells and tissues, including T cells, natural killer cells, and, at lower levels, in cells of innate immunity. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting and endothelial cells. Recent evidence suggests that the GITR/GITRL system participates in the development of inflammatory responses, including shock, either due to early response of neutrophils and macrophages, or together with autoimmune/allergic pathogenesis. The pro-inflammatory role of the GITR/GITRL system is due to: 1) modulation of the extravasation process, 2) activation of innate immunity cells, 3) activation of effector T cells also favored by partial inhibition of suppressor T cells and modulation of dendritic function. This review summarizes the in vivo role of the GITR/GITRL system in inflammation and shock, explaining the mechanisms responsible for their effects, considering the interplay among the different cells of the immune system and transduction pathways activated by GITR and GITRL triggering. The hidden aspects about GITR/GITRL function, crucial for treatment planning of inflammatory diseases and shock by modulation of this system is stressed.

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title: A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis.
abstract: By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.

title: Cloning and characterization of GITR ligand
abstract: The gene encoding the natural ligand of murine glucocorticoid-induced tumor necrosis factor receptor (GITR) was cloned and characterized. The putative GITR ligand (GITRL) is composed of 173 amino acids with features resembling those of type II membrane proteins and is 51% identical to the human activation-inducible TNF receptor (AITR) ligand, TL6. Expression of the GITRL is restricted to immature and mature splenic dendritic cells. GITRL binds GITR expressed on HEK 293 cells and triggers NF-κB activation. Functional studies reveal that soluble CD8-GITRL prevents CD4+CD25+ regulatory T-cell-mediated suppressive activities.

title: Identification of a Novel Activation-inducible Protein of the Tumor Necrosis Factor Receptor Superfamily and Its Ligand*
abstract: Among members of the tumor necrosis factor receptor (TNFR) superfamily, 4-1BB, CD27, and glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) share a striking homology in the cytoplasmic domain. Here we report the identification of a new member, activation-inducible TNFR family member (AITR), which belongs to this subfamily, and its ligand. The receptor is expressed in lymph node and peripheral blood leukocytes, and its expression is up-regulated in human peripheral mononuclear cells mainly after stimulation with anti-CD3/CD28 monoclonal antibodies or phorbol 12-myristate 13-acetate/ionomycin. AITR associates with TRAF1 (TNF receptor-associated factor 1), TRAF2, and TRAF3, and induces nuclear factor (NF)-κB activation via TRAF2. The ligand for AITR (AITRL) was found to be an undescribed member of the TNF family, which is expressed in endothelial cells. Thus, AITR and AITRL seem to be important for interactions between activated T lymphocytes and endothelial cells.

title: Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR
abstract: The tumor necrosis factor (TNF) and TNF receptor (TNFR) gene superfamilies regulate diverse biological functions, including cell proliferation, differentiation, and survival   . We have identified a new TNF-related ligand, designated human GITR ligand (hGITRL), and its human receptor (hGITR), an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related (mGITR) protein . The hGITRL gene mapped to chromosome 1q23, near the gene for the TNF homolog Fas/CD95 ligand . The hGITR gene mapped to chromosome 1p36, near a cluster of five genes encoding TNFR homologs  . We found hGITRL mRNA in several peripheral tissues, and detected hGITRL protein on cultured vascular endothelial cells. The levels of hGITR mRNA in tissues were generally low; in peripheral blood T cells, however, antigen-receptor stimulation led to a substantial induction of hGITR transcripts. Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB, via a pathway that appeared to involve TNFR-associated factor 2 (TRAF2)  and NF-kappaB-inducing kinase (NIK) . Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death. Thus, hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues.

title: Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells.
abstract: Glucocorticoid-induced tumor necrosis receptor (GITR) has been implicated in regulation of T cell suppression by CD25(+)CD4(+) regulatory T cells (Tregs). We isolated a cDNA encoding GITR ligand (GITRL) from mouse endothelioma cells. When stably expressed in HEK293 cells, its specific interaction with GITR was confirmed by flow cytometry with the use of GITR-Fc. The interaction was greatly diminished by the addition of soluble GITRL. Consistent with this, soluble GITRL bound to the cell surface of the GITR-expressing HEK293 cells. Coexpression of GITR with GITRL or stimulation of the GITR-expressing cells with soluble GITRL led to activation of NF-kappaB, which was significantly reduced by anti-GITR. More importantly, GITRL was expressed by both immature and mature dendritic cells, suggesting that the interaction between GITR and GITRL may contribute to immune regulation of Tregs by dendritic cells. This isolated TNFRL represents a bona fide GITRL whose presence has been elusive until this time.

title: Frontline: GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations
abstract: GITR (glucocorticoid‐induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8+ and CD4+ peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti‐CD3 stimulus. Furthermore, expression of the main activation‐induced receptor (IL‐2Rα) and production of IL‐2 and IFN‐γ were increased more with a GITR costimulus than with anti‐CD3 alone. GITR stimulation also enhanced anti‐CD3‐induced ERK phosphorylation, suggesting that GITR is involved in MAPK‐pathway activation. Interestingly, CD4+CD25+ regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR–/– CD4+CD25+ and GITR+/+ CD4+CD25+ cells were equally able to exert suppressor activity on CD4+CD25– responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.

title: Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells
abstract: Recently, agonist antibodies to glucocorticoid-induced tumor necrosis factor receptor (GITR) (tumor necrosis factor receptor superfamily 18) have been shown to neutralize the suppressive activity of CD4+CD25+ regulatory T cells. It was anticipated that this would be the role of the physiological ligand. We have identified and expressed the gene for mouse GITR ligand and have confirmed that its interaction with GITR reverses suppression by CD4+CD25+ T cells. It also, however, provides a costimulatory signal for the antigen-driven proliferation of naïve T cells and polarized T helper 1 and T helper 2 clones. RT-PCR and mAb staining revealed mouse GITR ligand expression in dendritic cells, macrophages, and B cells. Expression was controlled by the transcription factor NF-1 and potentially by alternative splicing of mRNA destabilization sequences.

title: Interactions of Tumor Necrosis Factor (TNF) and TNF Receptor Family Members in the Mouse and Human*
abstract: Ligands of the tumor necrosis factor superfamily (TNFSF) (4–1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin α, lymphotoxin αβ, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxinα andαβ are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

title: GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily
abstract: Glucocorticoid‐induced TNFR‐related gene (GITR; TNFRSF18), a receptor belonging to the TNFR superfamily (TNFRSF), is activated by GITRL. GITR is expressed at low levels on resting responder T lymphocytes and is up‐regulated in T regulatory cells (Treg cells) and in activated T cells. GITRL is expressed in endothelial and antigen‐presenting cells. The cytoplasmic region of GITR has a striking homology with other TNFRSF members (4‐1BB, CD27, OX40) and binds TRAF molecules and Siva. Over recent years, the role of GITR in the development and in the pathophysiology of the immune system has been actively explored by several groups. GITR triggering induces both pro‐ and anti‐apoptotic effects, abrogates the suppressive activity of Treg cells and co‐stimulates responder T cells, with the latter activities over‐stimulating the immune system. In vivo, GITR activation causes development of autoimmune diseases and restores immune responses in a persistent retroviral infection model and in a tumor model. Intriguingly, GITR knockout mice demonstrate lower mortality in an ischemia model. The GITR‐GITRL system appears crucial in regulating immunity and warrants further study.

title: Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: regulation at the interface between regulatory T cells and immune effector cells.
abstract: Mammals and other higher vertebrates have developed an adaptive immune system to defy effectively countless pathogens and cancerous cells encountered during the lifetime of an individual. B and T lymphocytes, which are essential in orchestrating adaptive immune responses, express surface receptors specific for foreign and abnormal self-antigens. Genesis of this antigen receptor repertoire poses significant risks for autoimmunity caused by self-reactive lymphocytes. Therefore, organisms with adaptive immune systems have evolved central and peripheral tolerance mechanisms. In peripheral tissues, regulatory T (Treg) cells function in a dominant, cell-extrinsic manner to limit inflammatory responses and autoimmune disorders. To tap the potential clinical utility of these specialized lymphocytes, advances have been made in understanding how Treg cell-mediated suppression of immune effector cells is achieved and regulated. Importantly, signaling induced by a recently identified member of the tumor necrosis factor receptor (TNFR) family, termed glucocorticoid-induced TNFR family-related gene (GITR), abrogates the suppressive effects of Treg cells. GITR plays a pivotal role in controlling T cell-mediated responses in experimental models of organ-specific autoimmunity, chronic infection, and anti-tumor immunity. These findings highlight the importance of elucidating the molecular underpinnings of GITR-induced signaling. We propose that GITR employs adapter proteins, including TNFR-associated factors (TRAFs), to regulate diverse signaling pathways and transcriptional programs that control the interplay between Treg cells and immune effector cells.

title: The GITR–GITRL interaction: co-stimulation or contrasuppression of regulatory activity?
abstract: Stimulation of T cells through GITR (glucocorticoid-induced tumour-necrosis-factor-receptor-related protein) has been shown to enhance immunity to tumours and viral pathogens, and to exacerbate autoimmune disease. The effects of stimulation through GITR are generally thought to be caused by attenuation of the effector activity of immunosuppressive CD4+CD25+ regulatory T (TReg) cells. Here we propose a model in which GITR–GITR-ligand interactions co-stimulate both responder T-cell functions and the suppressive functions of TReg cells.

title: Human plasmacytoid predendritic cells activate NK cells through glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL).
abstract: Plasmacytoid dendritic cell precursors (pDCs) are professional type I interferon-producing cells, a critical cell type in regulating innate and adaptive immune responses. By microarray gene expression analysis, we found that pDCs activated by virus or CpG-ODN preferentially express the ligand for the glucocorticoid-induced tumor necrosis factor receptor (GITRL), which was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry analysis. Using the same approaches, we found GITR is expressed by activated natural killer (NK) cells and T cells. We show that pDCs activated by CpG-ODN promote NK cell cytotoxicity and interferon (IFN)-gamma production through type I IFNs and GITRL. Using a GITRL-transfected cell line, we further demonstrate that GITRL promotes NK cell cytotoxicity and IFN-gamma production in synergy with interleukin-2 (IL-2), IFN-alpha, and NKG2D triggering. We also demonstrated that pDCs localized in close contact to NK cells in T-cell areas of the tonsils, and a subpopulation of the pDCs expressed GITRL. This study reveals a novel function of GITR/GITRL in pDC-mediated coactivation of NK cells.

title: Role of GITR in activation response of T lymphocytes.
abstract: In this study, we describe the generation and characterization of mice in which GITR gene (TNFRSF18 [tumor necrosis factor receptor superfamily 18]), a member of the TNFRSF expressed mainly on T lymphocytes, has been ablated (GITR(-/-) mice). Results indicate that GITR inactivation does not impair the normal development of the lymphoid organs but modulates T-cell activation. In fact, when GITR(-/-) T lymphocytes are activated by treatment with an anti-CD3 monoclonal antibody they proliferate more than wild-type cells. Moreover, activated GITR(-/-) T lymphocytes express higher levels of interleukin-2 receptor, produce larger amounts of interleukin-2, and are more sensitive to activation-induced cell death than controls. These results suggest that GITR is involved in the regulation of T-cell receptor/CD3-driven T-cell activation and programmed cell death.

title: CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.
abstract: CD4(+)CD25(+) immunoregulatory T cells represent a unique lineage of thymic-derived cells that potently suppress both in vitro and in vivo effector T cell function. We analyzed CD4(+)CD25(+) and CD4(+)CD25(-) T cells by DNA microarray, identifying 29 genes differentially expressed in the resting subpopulations, and 77 that were differentially expressed following activation. Most of these genes were elevated in the CD4(+)CD25(+) population, suggesting a previously activated phenotype. Among these were a number of genes that antagonize signaling, including members of the SOCS family, which may contribute to their anergic phenotype. Multiple cell surface receptors also had increased expression in CD4(+)CD25(+) cells, including GITR, a member of the TNF receptor superfamily. Importantly, antibodies to GITR abrogated suppression, demonstrating a functional role for this receptor in regulating the CD4(+)CD25(+) T cell subset.

title: Cutting Edge: The Natural Ligand for Glucocorticoid-Induced TNF Receptor-Related Protein Abrogates Regulatory T Cell Suppression 1
abstract: CD4+25+ regulatory T (Treg) cells maintain immunological self-tolerance through mechanisms that are only in part understood. Previous studies suggest that the glucocorticoid-induced TNFR-related protein (GITR), which is preferentially expressed on the surface of Treg cells, potentially provides a signal that abrogates Treg suppression. In this study, we show that a soluble form of mouse GITR ligand (sGITR-L) induces GITR-dependent NF-κB activation and blocks in vitro suppression mediated by both resting and preactivated polyclonal and Ag-specific Treg cells. Since sGITR-L along with rIL-2 induces proliferation of CD4+25+ cells, it appears that sGITR-L can break the anergic state of Treg cells. Because sGITR-L also up-regulates IL-2 secretion by activated CD4+25 −T cells, these two sGITR-L induced signals synergize to interfere with suppressor activity by CD4+25+ Treg cells.

title: Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance
abstract: CD25+CD4+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance. We show here that glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR, also known as TNFRSF18)—a member of the tumor necrosis factor–nerve growth factor (TNF-NGF) receptor gene superfamily—is predominantly expressed on CD25+CD4+ T cells and on CD25+CD4+CD8− thymocytes in normal naïve mice. We found that stimulation of GITR abrogated CD25+CD4+ T cell–mediated suppression. In addition, removal of GITR-expressing T cells or administration of a monoclonal antibody to GITR produced organ-specific autoimmune disease in otherwise normal mice. Thus, GITR plays a key role in dominant immunological self-tolerance maintained by CD25+CD4+ regulatory T cells and could be a suitable molecular target for preventing or treating autoimmune disease.

title: Engagement of Glucocorticoid-Induced TNF Receptor Costimulates NKT Cell Activation In Vitro and In Vivo1
abstract: Glucocorticoid-induced TNF receptor (GITR) is known to provide costimulatory signals to CD4+CD25− and CD4+CD25+ T cells during immune responses in vivo. However, the functional roles of GITR expressed on NKT cells have not been well characterized. In this study, we have explored the functions of GITR as a costimulatory factor on NKT cells. GITR was found to be constitutively expressed on NKT cells and its expression was enhanced by TCR signals. GITR engagement using DTA-1, an agonistic mAb against GITR, in the presence of TCR signals, augmented IL-2 production, the expression of activation markers, cell cycle progression, and the nuclear translocations of NF-κB p50 and p65. Furthermore, GITR engagement enhanced the production of IL-4, IL-10, IL-13, and IFN-γ by NKT cells and the expression level of phosphorylated p65 in NKT cells in the presence of TCR engagement, indicating that GITR provides costimulatory signals to NKT cells. The costimulatory effects of GITR on NKT cells were comparable to those of CD28 in terms of cytokine production. Moreover, the coinjection of DTA-1 and α-galactosylceramide into B6 mice induced more IL-4 and IFN-γ production than the coinjection of control mAbs and α-galactosylceramide. In addition, the adoptive transfer of DTA-1-pretreated NKT cells into CD1d−/− mice attenuated hypersensitivity pneumonitis more than control IgG pretreated NKT cells in these mice. These findings demonstrate that GITR engagement on NKT cells modulates immune responses in hypersensitivity pneumonitis in vivo. Taken together, our findings suggest that GITR engagement costimulates NKT cells and contributes to the regulation of immune-associated disease processes in vivo.

title: Recombinant glucocorticoid induced tumour necrosis factor receptor (rGITR) induced COX-2 activity in murine macrophage Raw 264.7 cells.
abstract: Stimulation of murine macrophages with recombinant soluble glucocorticoid induced tumour necrosis factor receptor (GITR) induced cyclooxygenase (COX)-2 protein and generated significant amounts of PGE(2). Previous result demonstrated that macrophages express GITR and GITR ligand constitutively. Induction of COX-2 was synergistic with interferon (INF)-gamma. GITR/ligand system could deliver an activation signal to macrophages in inflammatory processes.

title: Immune responses to p53 in patients with cancer:enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites
abstract: A majority of human cancers, including head and neck cancer (HNC), “overexpress” p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of “regulatory” T cells (Treg) to tumor sites in HNC has not been investigated to date.  Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined.  The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low Ζ expression and produced little IFN-γ after ex vivo stimulation relative to autologous PBMC or PBMC from NC.  Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.

title: Mast Cells Enhance T Cell Activation: Importance of Mast Cell Costimulatory Molecules and Secreted TNF1
abstract: We recently reported that mast cells stimulated via FcεRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3+ T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.

title: Glucocorticoid-induced Tumor Necrosis Factor Receptor Is a p21Cip1/WAF1 Transcriptional Target Conferring Resistance of Keratinocytes to UV Light-induced Apoptosis*
abstract: Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily, is expressed in T lymphocytes, and exerts an anti-apoptotic function in these cells. We reported that GITR is also highly expressed in the skin, specifically in keratinocytes, and that it is under negative transcriptional control of p21Cip1/WAF1, independently from the cell cycle. Although GITR expression is higher in p21-deficient keratinocytes and skin, it is down-modulated with differentiation and in response to UVB. The combined analysis of keratinocytes with increased GITR expression versus normal keratinocytes and skin of mice with a disruption of the GITR gene indicates that this protein protects keratinocytes from UVB-induced apoptosis both in vitro and in vivo.

title: Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) stimulates osteoclast differentiation in response to receptor activator of NF-kappaB ligand (RANKL) in osteoclast cells.
abstract: We found that treatment of osteoclast (OC) precursors with soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) promoted osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF) and receptor for activation of nuclear factor-kappaB ligand (RANKL). Low levels of GITR and its ligand were expressed on the surface of OC precursor cells after incubation with RANKL. Stimulation of osteoclastogenesis by sGITR was blocked by neutralization with anti-GITR ligand antibody (Ab), whereas endogenous GITR did not affect osteoclastogenesis, indicating that enhancement of osteoclastogenesis by sGITR involves signaling via GITR ligand. The addition of sGITR decreased the level of interferon (IFN)-beta, and blockade of endogenous IFN-beta did not affect osteoclastogenesis stimulated by sGITR. We conclude that sGITR enhances osteoclastogenesis by acting on OC precursor cells to lower the level of IFN-beta.

title: Constitutive and cytokine-induced GITR ligand expression on human retinal pigment epithelium and photoreceptors.
abstract: The glucocorticoid-induced TNF-related receptor (GITR) plays a pivotal role in regulating the suppressive function of CD4+CD25+ regulatory T cells. GITR is also involved in the inhibition of T-cell receptor-induced apoptosis and the upregulation of inducible nitric oxide synthase (iNOS). GITR expression on CD4+ T cells has been shown to correlate with the disease course of noninfectious uveitis. The current study was conducted to examine the expression of glucocorticoid-induced TNF-related receptor ligand (GITRL) and its regulation by ocular tissue.



Immunohistochemistry and confocal immunofluorescence microscopy were performed on human ocular tissue to examine the in vivo protein expression of GITRL. The regulation of GITRL was investigated by culturing retinal pigment epithelium (RPE) with proinflammatory cytokines and performing immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The in vivo mRNA expression of GITRL was studied by RT-PCR on RNA from microdissected tissue sections.



Both immunohistochemistry and confocal immunofluorescence microscopy demonstrated that GITRL was expressed constitutively on RPE and photoreceptor inner segments. Immunocytochemistry demonstrated that in vitro stimulated RPE cells expressed GITRL at the protein level, and RT-PCR showed that GITRL was upregulated at 24 hours by proinflammatory cytokines. Constitutive GITRL mRNA expression in vivo was confirmed by RT-PCR analysis of microdissected tissue.



GITRL is expressed constitutively on RPE and in high levels on photoreceptor inner segments. The upregulation of GITRL by proinflammatory cytokines suggests that GITRL may play an important role in ocular immunity. The high level of constitutive GITRL expression on photoreceptor inner segments suggests that photoreceptors participate in the regulation of ocular inflammation.

title: Expression of three members of the TNF-R family of receptors (4-1BB, lymphotoxin-beta receptor, and Fas) in human lung.
abstract: Receptors belonging to the tumour necrosis factor receptor (TNF-R) superfamily are implicated in a variety of important biological processes. Although messenger ribonucleic acid coding for many of these receptors has been detected in lung, little is known about their expression in this organ. In this study, immunohistochemical techniques were used to evaluate the expression of three receptors of this family (4-1BB, lymphotoxin-beta receptor (LTbeta-R), and Fas) in normal human lung, lung carcinomas and by tuberculous and sarcoid granulomas. The 4-1BB receptor was uniformly expressed by endothelial cells of small vessels and by basal epithelial cells within pseudostratified bronchial epithelium. LTbeta-R expression by parenchymal cells was limited to those of epithelial origin (bronchial and bronchiolar epithelium, hyperplastic alveolar epithelium and lung carcinomas). Fas was present on both fibroblasts and epithelial cells (bronchial and alveolar epithelium and most, but not all, carcinomas), but not on endothelial cells. A subpopulation of T-lymphocytes expressed these receptors, but only Fas was detected on normal alveolar macrophages and epithelioid cells. Thus, 4-1BB, LTbeta-R, and Fas have characteristic and only partially overlapping patterns of expression in the lung. The findings should facilitate further evaluation of their role in lung homeostasis and pathology.

title: Expression of glucocorticoid induced TNF receptor family related protein (GITR) on peripheral T cells from normal human donors and patients with non-infectious uveitis.
abstract: The expression of the glucocorticoid induced TNF receptor family related gene (GITR) in subsets of T lymphocytes from human peripheral blood was studied. In normal human peripheral blood mononuclear cells, GITR expression on the resting CD4+ T cells was low but markedly increased after activation. The percentage of GITR+ T cells in the CD4+CD25+ T cell subpopulation (15.1%) was significantly higher than that in the CD4+CD25- T cell subpopulation (5.2%, P<0.01), suggesting a preferential co-expression of GITR with CD25. In a group of patients with non-infectious uveitis, a proposed T helper cell mediated autoimmune ocular disease, the GITR expression on the CD4+ T cells in both the active patients (34.5%) and the inactive patients (19.6%) was significantly higher as compared to that in the normal donors (10.7%; P<0.01 vs. active, P<0.05 vs. inactive). This increased GITR expression in T cells was only seen in the CD4 positive T helper cell subpopulation but not in the CD4 negative T cell subpopulation. GITR expression on the CD4+ T cells decreased when the patients became clinically quiescent. Therefore, GITR is an activation marker for the CD4+ T cells and preferentially co-expressed with CD25 on the CD4+ T cells in human peripheral blood. Its expression correlates with the clinical course of non-infectious uveitis.

title: DNA microarray analysis of the gene expression profiles of naïve versus activated tumor-specific T cells.
abstract: T cells are a key element in effective cancer immunity, recognizing MHC-antigen peptide complexes on the surface of antigen presenting cells and translating these signals into cytotoxic effector T cell responses. In this study, we systematically investigated by DNA array analysis the expression profiles of 514 immunologically relevant genes in naïve and SP2/0 tumor-specific activated mouse T cell populations. Our data shows that naïve T cells expressed 37 (i.e., 7.6% of the 514) transcripts with expression level (EL) values of > or =2.0, while the activated T cells expressed 101 such transcripts. The expression levels of 9 (1.75% of 514) of the shared transcripts were equivalent in the two populations of T cells. Ninety-six genes were differently expressed upon T cell activation, with 71 (13.81%) being up-regulated and 25 (4.86%) down-regulated. The list of significantly affected genes includes numerous cytokines and their receptors (e.g., IL-2Ralpha, IL-6Ralpha, IL-7Ralpha, IL-16, IL-17R, TGF-beta), chemokines and chemokine receptors (e.g., RANTES, CCR7, CXCR4), alternate surface proteins (e.g., 4-1BB, GITR, integrins-alphaL and -beta7, L-selectin, CD6, CD45 and EMMPRIN), cytoplasmic signaling intermediates (e,g., GATA-3, 14-3-3-eta, CIS1, SMAD4 and JAK1) and an array of other molecules (e.g., NFkappa-B inducing kinase, LTBP3 and persephin), several of which are associated with Th1 responses, and T cell self-regulation or migration. Taken together, our data contribute to our understanding of the generalized processes that accompany T cell activation and, more specifically, to our understanding of the processes associated with T cell activation during antitumor responses.

title: CD4+CD25+ T regulatory cells induced by LPS-activated bone marrow dendritic cells suppress experimental autoimmune uveoretinitis in vivo
abstract: BackgroundTolerance-inducing DC are considered to be less mature than immunogenic DC, but the conditions promoting a less mature DC phenotype are not clear. We have previously shown that lipopolysaccharide (LPS) can have differential effects on DC function depending on the timing of DC exposure to LPS. Here, we show that early LPS-activated bone marrow derived DC (early DC, eDC), when administered subcutaneously to mice in vivo, promote tolerance to EAU induced via immunisation with interphotoreceptor retinol binding protein (IRBP) peptide 161–180. The effect correlates with the failure of eDC to secrete IL-12, and appears to be mediated in part via expansion of naturally occurring CD4+CD25+ T regulatory cells (Tregs), which also mediate suppression of EAU on adoptive transfer to naive mice followed by immunization with autoantigen.MethodsImmature DC were prepared from BMDC cultures. Early DC (eDC) and late DC (lDC) for tolerance experiments were obtained by differential timing of LPS addition and their cytokine secretion profile was analyzed. eDC and lDC were subcutaneously injected into mice. From the dLN CD4+ CD25+ GITR+ T regulatory cells found to express FoxP3 were isolated and transferred into mice prior to immunisation with IRBP. The immune response was scored by histopathology. Tregs were characterized in vitro by intracellular staining, cytokine secretion assay and transwell experiments.ResultseDC secrete IL-10 but no IL-12 or IFNγ. When injected subcutaneously into naive mice, they expand the population of CD4+CD25+high GITR+ T cells expressing FoxP3 in the dLN, thus increasing the total number of IL-10 producing cells. eDC induced Tregs inhibit CD4+CD25− T effector cell proliferation by a contact dependent process, and both eDC and Tregs suppress retinal damage when adoptively transferred.ConclusionsWe suggest that DC maturation may be necessary for both tolerance and immunity, but differential levels of activation and/or cytokine production direct the outcome of DC-T cell interaction and this is determined by IL-12 production. T regulatory cells induced in vivo by contact with eDC are able to suppress disease in the EAU model by adoptive transfer.

title: Regulatory T Cells Overexpress a Subset of Th2 Gene Transcripts1
abstract: There is now compelling evidence for subpopulations of CD4+ T cells whose role is to prevent immune pathology in both autoimmunity and transplantation. We have cloned CD4+ T cells against a male transplantation Ag that, unlike Th1 or Th2 clones, suppresses the rejection of male skin grafts and are therefore considered examples of regulatory T cells. We have identified, using serial analysis of gene expression, transcripts that are overexpressed in regulatory T cells compared with Th1 and Th2 clones. Some of these transcripts are increased in tolerated rather than rejecting skin grafts and in addition are expressed by the natural regulatory CD4+CD25+ subpopulation of naive mice. These genes include prepro-enkephalin, GM2 ganglioside activator protein, glucocorticoid-induced TNFR superfamily member 18, and integrin αEβ7. They seem to represent a subset of transcripts shared with Th2 cells, suggesting that transplantation tolerance and normal immunoregulation may represent a unique form of Th2-like differentiation.

title: CD4+CD25+ T Regulatory Cells Dependent on ICOS Promote Regulation of Effector Cells in the Prediabetic Lesion
abstract: CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69− cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69− cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory–specific profile. Thus, CD4+CD25+69− Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.

title: Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes.
abstract: CD8+CD25+ cells, which expressed high levels of Foxp3, glucocorticoid-induced tumor necrosis factor receptor (GITR), CCR8, tumor necrosis factor receptor 2 (TNFR2), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) mRNAs, were identified in the fibrous septa and medullary areas of human thymus. Activated CD8+CD25+ thymocytes did not produce cytokines, but most of them expressed surface CTLA-4 and transforming growth factor beta1 (TGF-beta1). Like CD4+CD25+, CD8+CD25+ thymocytes suppressed the proliferation of autologous CD25-T cells via a contact-dependent mechanism. The suppressive activity of CD8+CD25+ thymocytes was abrogated by a mixture of anti-CTLA-4 and anti-TGF-beta1 antibodies and it was mediated by their ability to inhibit the expression of the interleukin 2 receptor alpha chain on target T cells. These results demonstrate the existence of a subset of human CD8+CD25+ thymocytes sharing phenotype, functional features, and mechanism of action with CD4+CD25+ T regulatory cells.

title: Human CD25+CD4+ T Suppressor Cell Clones Produce Transforming Growth Factor β, but not Interleukin 10, and Are Distinct from Type 1 T Regulatory Cells
abstract: T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4+ T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β. The relationship between CD25+CD4+ T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25+CD4+ T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25−CD4+ T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25+CD4+ T cells. Cloned human CD25+CD4+ T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25+CD4+ T cell clones with suppressive function produce IL-10, but all produce TGF-β. Suppression mediated by CD25+CD4+ T cell clones is partially dependent on TGF-β, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25+CD4+ T cells are distinct from IL-10–producing Tr1 cells.

title: Phenotypic characterization of regulatory T cells in the human decidua
abstract: Pregnancy is a unique situation for the maternal immune system. We have studied and identified a CD4+CD25+ regulatory T (Treg) cell population isolated from the human decidua. This mucosal surface in the uterus is in direct contact with semiallogenic fetal cells. We observed that about 14% of the decidual CD4+ T cells have the CD4+CD25+ phenotype. The decidual CD4+CD25+ T cells expressed high frequency of intracellular CTLA‐4 (CTLA‐4i). The majority of CD4+CD25+CTLA‐4i+ cells were also positive for GITR and OX40, typical markers for human Treg cells. The frequency of CD4+CD25+ T cells in the peripheral blood from pregnant women was found to be increased during the first and second trimester of gestation when compared to nonpregnant controls. Being an important molecule for Treg cells, the role of CTLA‐4 in the regulation of indoleamine 2,3‐dioxygenase (IDO) expression was also examined. The stimulation with CTLA‐4Ig did not increase IDO mRNA expression in CD14+ cells from pregnant women, while IFN‐γ was observed to up‐regulate IDO expression. The presence of Treg cells in the human decidua suggests that these cells are important in protecting the fetus from alloreactive immune responses at the maternal–fetal interface.

title: CD4+CD25bright Regulatory T Cells Actively Regulate Inflammation in the Joints of Patients with the Remitting Form of Juvenile Idiopathic Arthritis
abstract: This study investigates the role of CD4+CD25+ regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4+CD25bright T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4+CD25bright T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4+CD25int T cells and therefore of CD4+CD25total in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4+CD25bright T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4+CD25int T cell population. The CD4+CD25bright cells of both patient groups and the CD4+CD25int cells of pers-OA JIA patients were able to suppress responses of CD25neg cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4+CD25bright T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4+CD25+ Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4+CD25bright T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4+CD25bright T cells. These data suggest that CD4+CD25bright Treg cells play a role in determining the patient’s fate toward either a favorable or unfavorable clinical course of disease.

title: CD4+CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells1
abstract: It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4+CD25bright T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4+ T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4+CD25− T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4+CD25+ T cells, however, cells expressing CD25 at high levels (CD4+CD25bright) suppressed the proliferation and various cytokine productions of CD4+CD25− T cells. LP CD4+CD25bright T cells by themselves produced fewer amounts of IL-2, IFN-γ, and IL-10. Interestingly, LP CD4+CD25bright T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4+CD25bright T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.

title: Regulation of Murine Inflammatory Bowel Disease by CD25+ and CD25− CD4+ Glucocorticoid-Induced TNF Receptor Family-Related Gene+ Regulatory T Cells1
abstract: CD4+CD25+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4+CD25− T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4+CD25+ regulatory T cells. In this study, we show evidence that CD4+GITR+ T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4+GITR− T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4+GITR+ population prevented the development of CD4+CD45RBhigh T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RBhigh and CD45RBlow CD4+ T cells. Interestingly, both CD4+CD25+ and CD4+CD25− GITR+ T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4+CD25−GITR+ T cells as well as CD4+CD25+GITR+ T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-β. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.

title: Foxp3 programs the development and function of CD4+CD25+ regulatory T cells
abstract: CD4+CD25+ regulatory T cells are essential for the active suppression of autoimmunity. Here we report that the forkhead transcription factor Foxp3 is specifically expressed in CD4+CD25+ regulatory T cells and is required for their development. The lethal autoimmune syndrome observed in Foxp3-mutant scurfy mice and Foxp3-null mice results from a CD4+CD25+ regulatory T cell deficiency and not from a cell-intrinsic defect of CD4+CD25− T cells. CD4+CD25+ regulatory T cells rescue disease development and preferentially expand when transferred into neonatal Foxp3-deficient mice. Furthermore, ectopic expression of Foxp3 confers suppressor function on peripheral CD4+CD25− T cells. Thus, Foxp3 is a critical regulator of CD4+CD25+ regulatory T cell development and function.

title: Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation*
abstract: Regulatory T cells (Treg) express Foxp3, a forkhead family member that is necessary and sufficient for Treg lineage choice and function. Ectopic expression of Foxp3 in non-Treg leads to repression of the interleukin 2 (IL-2) and interferon γ (IFNγ) genes, gain of suppressor function, and induction of genes such as CD25, GITR, and CTLA-4, but the mode by which Foxp3 enforces this program is unclear. Using chromatin immunoprecipitation, we have demonstrated that Foxp3 binds to the endogenous IL-2 and IFNγ loci in T cells, but only after T cell receptor stimulation. This activation-induced Foxp3 binding was abrogated by cyclosporin A, suggesting a role for the phosphatase calcineurin in Foxp3 function. We have also shown that binding of Foxp3 to the IL-2 and IFNγ genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling and opposes gene transcription. Conversely, binding of Foxp3 to the GITR, CD25, and CTLA-4 genes results in increased histone acetylation. These data indicate that Foxp3 may regulate transcription through direct chromatin remodeling and show that Foxp3 function is influenced by signals from the TCR.

title: An essential role for Scurfin in CD4+CD25+ T regulatory cells
abstract: The molecular properties that characterize CD4+CD25+ regulatory T cells (TR cells) remain elusive. Absence of the transcription factor Scurfin (also known as forkhead box P3 and encoded by Foxp3) causes a rapidly fatal lymphoproliferative disease, similar to that seen in mice lacking cytolytic T lymphocyte–associated antigen 4 (CTLA-4). Here we show that Foxp3 is highly expressed by TR cells and is associated with TR cell activity and phenotype. Scurfin-deficient mice lack TR cells, whereas mice that overexpress Foxp3 possess more TR cells. In Foxp3-overexpressing mice, both CD4+CD25− and CD4−CD8+ T cells show suppressive activity and CD4+CD25− cells express glucocorticoid-induced tumor-necrosis factor receptor–related (GITR) protein. The forced expression of Foxp3 also delays disease in CTLA-4−/− mice, indicating that the Scurfin and CTLA-4 pathways may intersect and providing further insight into the TR cell lineage.

title: Inhibition of the Transcription Factor Foxp3 Converts Desmoglein 3-Specific Type 1 Regulatory T Cells into Th2-Like Cells1
abstract: Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disorder and is associated with autoantibodies against desmoglein (Dsg)3 that are regulated by Th2 cells. Recently, Dsg3-specific type 1 regulatory T cells (Tr1) were identified that are presumably critical for the maintenance of tolerance against Dsg3 because there is a much lower Dsg3-specific Tr1:Th2 ratio in the PV patients than in healthy individuals. The aim of this study was to down-regulate the transcription factor Foxp3 in Dsg3-specific Tr1 using antisense oligonucleotides because Foxp3 is constitutively expressed by the Dsg3-specific Tr1. Antisense-treated Dsg3-specific Tr1 clones lost expression of Foxp3, glucocorticoid-induced TNFR family-related receptor, and CTLA-4, and started to secrete IL-2, whereas the secretion of IL-5, TGF-β, and IL-10 remained unchanged. Moreover, antisense treatment induced a proliferative response to Dsg3 of the formerly anergic Tr1 and abrogated their suppressor activity on Dsg3-specific Th2 cell clones. Thus, inhibition of Foxp3 mRNA expression in the Tr1 induced a Th2-like phenotype. In conclusion, Foxp3 expression is inherent to Tr1 function, and modulation of Foxp3 expression in autoaggressive Th2 cells may provide a novel therapeutic approach aimed at restoring tolerance against Dsg3 in PV.

title: Murine vascular endothelium activates and induces the generation of allogeneic CD4+25+Foxp3+ regulatory T cells.
abstract: Unlike graft-resident donor-derived hemopoietic APCs, which decrease in number over time after transplantation, vascular endothelial cells are lifelong residents of a vascularized allograft. Endothelial cells are potent APCs for allogeneic CD8+ T lymphocytes but are unable to induce proliferation of allogeneic CD4+ T lymphocytes. Although the reason for this differential response has been poorly understood, here we report that alloantigen presentation by vascular endothelium to CD4+ T lymphocytes activates and induces CD4+25+Foxp3+ regulatory T cells, which can inhibit proliferation of alloreactive T cells both in vitro and in vivo. This process occurs independently of B7.1 costimulation but is dependent on programmed death ligand 1 (B7-H1). This finding may have important implications for tolerance induction in transplantation.

title: Thrombospondin/CD47 Interaction: A Pathway to Generate Regulatory T Cells from Human CD4+CD25− T Cells in Response to Inflammation1
abstract: Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study, we show that the anti-inflammatory extracellular matrix protein, thrombospondin-1, promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor, CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+CD25− T cells to become suppressive. The latter expressed increased amounts of CTLA-4, OX40, GITR, and Foxp3 and inhibited autologous Th0, Th1, and Th2 cells. Their regulatory activity was contact dependent, TGF-β independent, and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation.

title: Control of immune homeostasis by naturally arising regulatory CD4+ T cells.
abstract: CD4(+)CD25(+) regulatory T (T(R)) cells are essential negative regulators of multiple immune functions. Their development and function are critically dependent on the forkhead transcription factor Foxp3. IL-2R-derived signals are also required for their maturation and/or proliferation. Expression of the TNF receptor family member GITR appears to define this naturally arising, thymically derived lineage more accurately than CD25. T(R) cells suppress virtually all forms of immune responsiveness investigated to date, including both adaptive and innate immunity. T(R) cells are capable of robust antigen-driven proliferation in vivo, and may participate in clonal expansion in response to infection, similar to all other adaptive immune lineages.

title: Intranasal Vaccination with Proinsulin DNA Induces Regulatory CD4+ T Cells That Prevent Experimental Autoimmune Diabetes1
abstract: Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (Treg) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ Treg that suppressed diabetes development, both after adoptive cotransfer with “diabetogenic” spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+CD25+ Treg, CD4+ Treg induced by proinsulin DNA were both CD25+ and CD25− and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of Treg and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective Treg, but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.

title: Differential response of murine CD4+CD25+ and CD4+CD25– T cells to dexamethasone‐induced cell death
abstract: To evaluate the in vivo effect of immunosuppressive glucocorticoids on CD4+CD25+ T regulatory cells, we injected dexamethasone (Dex) into BALB/c mice. Administrationof Dex enhanced the proportion of CD4+CD25+ cells and the ratio of CD4+CD25+ cells to CD4+CD25– cells in the lymphoid organs, especially in thethymus. This correlates with our in vitro observation that CD4+CD25+ T cells express higher levels of glucocorticoid receptor and Bcl‐2, and are therefore more resistant to Dex‐mediated cell death than CD4+CD25– T cells. Furthermore, IL‐2 selectively protected CD4+CD25+ T cells from Dex‐induced cell death, while IL‐7 and IL‐15 didnot exert preferential protective effects. Dex‐treated CD4+CD25+ T cells expressed higher levels of intracellular CTLA‐4 and surface glucocorticoid‐induced TNF receptor than fresh CD4+CD25+ T cells, but still failed to respond to TCR stimulation and inhibited proliferation of CD4+CD25– T cells. These results suggest that, in addition to suppressing cytokine transcription, Dex treatment is permissive for the survival of functional CD4+CD25+ T regulatory cells, and this property may contribute to the anti‐inflammatory and immunosuppressive efficacy of glucocorticoids. Our data also suggest that selective protection of CD4+CD25+ T cell from apoptosis may constitute a role in immune tolerance for IL‐2.

title: TCR-mediated activation promotes GITR upregulation in T cells and resistance to glucocorticoid-induced death.
abstract: T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR-/-). We also compared the action of GC on GITR+ and GITR- T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR-/- mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR-/- cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10(-8) to 10(-6) M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.

title: Osteostat/tumor necrosis factor superfamily 18 inhibits osteoclastogenesis and is selectively expressed by vascular endothelial cells.
abstract: Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN-alpha and IFN-beta. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.

title: Expression of GITR ligand abrogates immunosuppressive function of ocular tissue and differentially modulates inflammatory cytokines and chemokines
abstract: The glucocorticoid‐induced TNF‐related receptor ligand (GITRL) was previously shown to be constitutively expressed at low levels in human eye, including retinal pigment epithelial (RPE) cells. By expressing enhanced yellow fluorescent protein‐tagged human GITRL in human RPE cells, we investigated the significance of expression of GITRL on human ocular tissue. Confocal immunofluorescence microscopy and flow cytometry confirmed the surface expression of GITRL on RPE cells. However, a soluble form of GITRL was also detected. Remarkably, expression of GITRL on the RPE cells abrogated RPE‐mediated immunosuppression of CD3+ T cells, implicated as a possible mechanism for ocular immune privilege. This abrogation of immunosuppression by GITRL‐RPE was dependent on GITR‐GITRL interaction and could not be mimicked by anti‐CD28 antibody. Analysis of cytokine profiles revealed high level of TGF‐beta during the immunosuppression by RPE cells while expression of GITRL abrogated the RPE cell‐induced TGF‐beta secretion. Expression of GITRL also stimulates secretion of an array of proinflammatory cytokines/chemokines from T cells. GITR‐GITRL interaction provides a unique proinflammatory costimulation that may signal through a different pathway than that of CD28‐B7 costimulation. This study implicated that GITRL could be a potential candidate for regulation of the ocular immune privilege and the balance between immune privilege and inflammation.

title: Engagement of Glucocorticoid-Induced TNFR Family-Related Receptor on Effector T Cells by its Ligand Mediates Resistance to Suppression by CD4+CD25+ T Cells
abstract: Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR−/− mice, we report that GITR engagement on CD25−, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR−/−CD25− T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25− T cells, rendering them resistant to CD25+-mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.

title: Staphylococcal enterotoxin B inhibits regulatory T cells by inducing glucocorticoid-induced TNF receptor-related protein ligand on monocytes.
abstract: Superantigens inhibit naturally occurring CD4+CD25+ regulatory T-cell (nTreg) activity, yet the mechanism for this is unknown.



We tested the hypothesis that staphylococcal enterotoxin B (SEB), a prototypic superantigen, inhibits the ability of nTregs to suppress T-effector cell (Teff) proliferation by an antigen-presenting cell-dependent cell contact mechanism and the induction of glucocorticoid-induced TNF receptor-related protein ligand (GITR-L) on monocytes.



Cell proliferation assays were performed on immunomagnetic-bead separated CD4+CD25+, CD4+CD25- T cells and CD14+ monocytes using anti-CD3, SEB, or a neutralizing anti-GITR-L antibody for stimulation. A cytokine permeable membrane insert separating cells in culture was used to evaluate the role of cell contact. Proliferation was measured by [3H]-thymidine incorporation or a carboxyfluorescein diacetate succinimidyl ester assay. GITR-L expression was determined by RT-PCR and immunostaining.



Antigen-presenting cell contact was required for proliferation of nTregs and loss of their ability to suppress Teff proliferation as confirmed by carboxyfluorescein diacetate succinimidyl ester. At baseline, monocytes expressed no GITR-L, but after SEB stimulation, GITR-L gene and protein expression increased significantly over a period of 24 hours (P<.05). When GITR-L activity was neutralized with blocking antibody, as opposed to isotype control, nTregs suppressed Teff proliferation despite the presence of SEB (P<.05). Likewise, loss of GITR-L at 48 hours was associated with restoration of nTreg activity (P<or=.05).



Staphylococcal enterotoxin B upregulates GITR-L on monocytes and inhibits nTreg ability to suppress Teff proliferation via a cell contact interaction.



Prevention of GITR-L induction on monocytes by SEB may be a novel target for control of inflammation in superantigen-driven diseases such as atopic dermatitis.

title: In Vivo Kinetics of GITR and GITR Ligand Expression and Their Functional Significance in Regulating Viral Immunopathology
abstract: This report evaluates the role of interaction between glucocorticoid-induced tumor necrosis factor receptor (GITR) and GITR ligand (GITR-L) in the immunoinflammatory response to infection with herpes simplex virus (HSV). Both GITR and GITR-L were transiently upregulated after ocular HSV infection, on antigen-specific T cells and antigen-presenting cells, respectively, in the draining lymph node (DLN). In addition, virus-specific T-cell responses in the DLN and spleen were enhanced by anti-GITR antibody treatment, an outcome expected to result in more severe inflammatory lesions. Intriguingly, the treatment resulted in significantly diminished T-cell-mediated ocular lesions. The explanation for these findings was that anti-GITR antibody treatment caused a reduced production of ocular MMP-9, a molecule involved in ocular angiogenesis, an essential step in the pathogenesis of herpetic keratitis. Our results are the first observations to determine in vivo kinetics of GITR and GITR-L expression after virus infection, and they emphasize the role of GITR-GITR-L interaction to regulate virus-induced immunoinflammatory lesions.

title: Glucocorticoid‐induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock
abstract: In the present study, we used glucocorticoid‐induced tumor necrosis factor (TNF) receptor family gene knockout (GITR‐KO) mice to evaluate a possible role of GITR on the pathogenesis of splanchnic artery occlusion (SAO) shock, which was induced in mice by clamping the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the lipid peroxidation in the ileum of the SAO‐shocked, GITR wild‐type (WT) mice after reperfusion. The absence of GITR significantly reduced the lipid peroxidation in the intestine. SAO‐shocked WT mice developed a significant increase of ileum tissue, TNF‐α, and myeloperoxidase activity and marked histological injury. SAO shock was also associated with a significant mortality (5% survival at 24 h after reperfusion). Reperfused ileum tissue sections from SAO‐shocked WT mice showed positive staining for P‐selectin, intercellular adhesion molecule 1 (ICAM‐1), and E‐selectin. The intensity and degree of P‐selectin, E‐selectin, and ICAM‐1 were markedly reduced in tissue section from SAO‐shocked, GITR‐KO mice. SAO‐shocked, GITR‐KO mice also showed a significant reduction of the TNF‐α production and neutrophil infiltration into the reperfused intestine, an improved histological status of the reperfused tissues, and an improved survival. Taken together, our results clearly demonstrate that GITR plays an important role in the ischemia and reperfusion injury and put forward the hypothesis that modulation of GITR expression may represent a novel and possible strategy.

title: Soluble glucocorticoid‐induced tumor necrosis factor receptor (sGITR) increased MMP‐9 activity in murine macrophage
abstract: Glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, increased production of matrix matalloproteinase (MMP‐9) in murine macrophages. Murine macrophages produced a band of gelatinolytic activity at 100 kDa when stimulated for 18 h with soluble GITR. MMP‐9 was identified by gelatin zymography and Western blot. Previous results demonstrated that murine macrophages express GITR and GITR ligand constitutively. Induction of MMP‐9 was synergistic with co‐treatment of INF‐γ. MMPs could play a critical role in progression and promotion of tissue injury after inflammation stimulated by GITR/ligand system.  2003.

title: Genetic and pharmacological inhibition of GITR‐GITRL interaction reduces chronic lung injury induced by bleomycin instillation
abstract: We have recently identified a gene named GITR (glucocorticoid‐induced TNF receptor related gene). GITR is expressed in different cells and tissues such as T lymphocytes from thymus and spleen and lymph nodes, and also in the lung. GITR ligand (GITRL) is expressed in several cells including macrophages, B cells, denditric cells, and endothelial cells. In the present study, by comparing the responses in wild‐type (WT) mice (GITR+/+) and GITR‐deficient mice (GITR‐/‐), we investigated the role played by GITR‐GITRL interaction in the development of chronic lung injury caused by bleomycin instillation. When compared with bleomycin‐treated GITR+/+ mice, bleomycintreated GITR‐/‐mice exhibited a reduced degree of i) lung infiltration with polymorphonuclear neutrophils (MPO activity); ii) edema formation; iii) histological evidence of lung injury; iv) TNF‐a and interleukin (IL)‐1f production; v) nitrotyrosine formation; and vi) NF‐κB activation. The cotreatment of GITR+/+ mice with Fc‐GITR fusion protein (6.25 µg/mouse) also significantly attenuated all of the above indicators of lung damage and inflammation. Our results clearly demonstrate that GITR‐GITRL interaction plays an important role in the chronic lung injury induced by bleomycin in the mice. Cuzzocrea, S., Ronchetti, S., Genovese, T., Mazzon, E., Agostini, M., Di Paola, R., Esposito, E., Muià, C., Nocentini, G., Riccardi, C. Genetic and pharmacological inhibition of GITR‐GITRL interaction reduces chronic lung injury induced by bleomycin instillation. FASEB J. 21, 117–129 (2007)

title: GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice
abstract: Uncontrolled T cell activation and abnormal function of the innate immune system against normal enteric bacterial flora play a critical part in the pathogenesis of inflammatory bowel disease (IBD). Therefore, pharmacological strategies directed to restore the normal responsiveness of the immune system could be efficacious in the treatment of these pathological conditions. Glucocorticoid-induced tumour necrosis factor receptor (GITR)-related gene is a member of the tumour necrosis factor receptor superfamily that is constitutively expressed at high levels on regulatory T cells and at low levels on unstimulated T cells, B cells and macrophages. GITR triggering leads to activation of T effectors and reversal of suppressive function of regulatory T cells.  To investigate the role of GITR in the development of experimental colitis in mice.  Using GITR−/− mice, GITR deletion protected against 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis by reducing innate immune responses and effector T cell activity. Effector T cells isolated from GITR−/− mice were less effective than T cells isolated from GITR+/+ mice to transfer colitis in immunodeficient mice. Blocking the GITR/ligand for GITR (GITRL) signal by giving soluble GITR prevented TNBS-induced colitis in normal GITR+/+ and also in lymphocyte-deficient SCID mice.  Collectively, these data suggest that GITR plays a critical part in regulating both acquired and innate mucosal immune responses during the development of experimental colitis in mice. Therefore, targeting the GITR/GITRL system signalling may represent a potential pharmacological tool for the treatment of IBD.

title: IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin.
abstract: Regulatory T cells (Tregs) are crucial for the induction and maintenance of self-tolerance and are present in peripheral tissues such as skin and gut under normal, noninflamed conditions. We report isolation and expansion of the Treg population resident in normal human skin. Cutaneous Tregs expressed high levels of CD25, L-selectin, GITR, FOXP3, and intracellular CTLA-4, low levels of CD69, and high levels of the skin-homing addressins CLA, CCR4, and CCR6. Skin Tregs suppressed the proliferation of CD25(lo) T cells from the same skin sample in response to CD3 and CD28 antibodies. Suppression was dependent on cell contact and not affected by neutralizing antibodies to interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Surprisingly, cutaneous Tregs proliferated in an antigen-independent manner when cultured in contact with dermal fibroblasts and IL-15, conditions similar to those found in chronically inflamed skin. We hypothesize that local proliferation of Tregs may occur within inflamed skin and could serve as a brake for cutaneous inflammation as well as a mechanism for the homeostatic proliferation of natural Tregs that has been observed within intact organisms.

title: Role of glucocorticoid‐induced TNF receptor family gene (GITR) in collagen‐induced arthritis
abstract: In rheumatoid arthritis (RA), a widespread autoimmune/inflammatory joint disease, early activation of effector CD4+ T lymphocytes, and cytokine production is followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, and disease. GITR (glucocorticoid‐induced TNFR family‐related gene), a costimulatory molecule for T lymphocytes, increases CD4+CD25− effector T cell activation while inhibiting suppressor activity of CD4+CD25+ T regulatory (Treg) cells. We analyzed the role of GITR in type II collagen (CII) ‐induced arthritis (CIA) using GITR− /− and GITR+ / + mice. Results indicate significantly less CIA induction in GITR−/− mice than in GITR+/+ mice, with marked differences in erythema, edema, neutrophil infiltration, joint injury, and bone erosion. Production of IFNγ, IL‐6, TNFα, MIP‐1α, and MIP‐2, inducible NOS (iNOS), COX‐2, and nitrotyrosine poly‐ADP‐ribose (PAR) were also less in CII‐treated GITR−/− mice. Although CD4+CD25+ Treg cells from GITR+/+ and GITR−/− CII‐challenged mice exerted similar suppressor activity in vitro, GITR triggering abrogated GITR+/+ Treg suppressor activity and costimulated CD4+CD25− GITR+/+ effector cells. Furthermore, Treg cells from GITR−/− protected more than Treg cells from GITR+/+ mice against CIA when cotransferred with Treg‐depleted splenocytes from arthritic GITR+/+ animals into severe combined immunodeficient (SCID) mice. In conclusion, GITR plays a critical role in the immunological response against CII and in the development of CIA. Cuzzocrea, S., Ayroldi, E., Di Paola, R., Agostini, M., Mazzon, E., Bruscoli, S., Genovese, T., Ronchetti, S., Caputi, A. P., Riccardi, C. Role of glucocorticoid‐induced TNF receptor family gene (GITR) in collagen‐induced arthritis. FASEB J. 19, 1253–1265 (2005)

title: Glucocorticoid‐induced TNFR family‐related protein (GITR) activation exacerbates murine asthma and collagen‐induced arthritis
abstract: Glucocorticoid‐induced TNFR family‐related protein (GITR) is expressed at low levels on resting T cells, B cells and macrophages but at high levels on regulatory T cells (Treg). Although GITR expression is up‐regulated on CD4+ effector cells upon activation, the role of GITR in Th1 and Th2 cell development is unclear. We report here that activation of GITR signalling by  antibody markedly enhanced the induction of both Th1 and Th2 cytokine production by naive CD4+CD25– T cells. Consistent with this observation,  antibody significantly enhanced the expression of the key Th1 (T‐bet) and Th2 (GATA3) transcription factors in vitro. Administration of  mAb in a murine model of arthritis significantly exacerbated the severity and onset of joint inflammation with elevated production of TNF‐α, IFN‐γ, IL‐5, and collagen‐specific IgG1. Administration of  mAb also significantly exacerbated murine allergic airways inflammation with elevated production of OVA‐specific IFN‐γ, IL‐2, IL‐4, IL‐5, and IgE. Finally, we demonstrated that adoptive transfer of CD4+GITR+ T cells effectively abolished airway inflammation induced in SCID mice reconstituted with CD4+GITR– T cells. Our results therefore provide direct evidence that GITR can modulate both Th1‐ and Th2‐mediated inflammatory diseases, and may be a potential target for therapeutic intervention.

title: CD4+ CD25+ T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis
abstract: CD4+ CD25+ regulatory T (Treg) cells play a critical role in the maintenance of peripheral tolerance and the prevention of autoimmunity. In the present study, we have explored the characteristics of CD4+ CD25+ Treg cells in patients with rheumatoid arthritis (RA). The frequency and phenotype of CD4+ CD25+ T cells in paired samples of synovial fluid (SF) and peripheral blood (PB) from patients with RA and PB from normal controls were analysed. An increased frequency of CD4+ cells T cells expressing CD25 was detected in SF compared to PB from patients with RA. No significant difference was observed in the numbers of CD4+ CD25+ T cells in PB from patients and controls. SF CD4+ CD25+ T cells expressed high levels of CTLA‐4 (both surface and intracellular), GITR and OX40, as well as Foxp3 transcripts. Functionally, SF CD4+ CD25+ T cells were impaired in their proliferative responses and could suppress the proliferation of their CD4+ CD25– counterparts. In conclusion, these data demonstrate that CD4+ CD25+ Treg cells, with the potential to regulate the function of effector T cells and antigen‐presenting cells, accumulate in the synovium of patients with RA.

title: Interference with CD4+CD25+ T-cell-mediated tolerance to experimental autoimmune thyroiditis by glucocorticoid-induced tumor necrosis factor receptor monoclonal antibody.
abstract: Experimental autoimmune thyroiditis (EAT), a murine model for Hashimoto's thyroiditis, is inducible with mouse thyroglobulin (mTg), and characterized by mononuclear cell infiltration and destruction of the thyroid gland. Pretreatment with mTg leads to CD4+CD25+ T-cell-mediated resistance to subsequent EAT induction. We have recently demonstrated that in vivo administration of a monoclonal antibody (mAb) to CD137, a member of the tumor necrosis factor receptor (TNFR) family, interferes with both the development and mediation of induced EAT tolerance. Here, we examined the influence of another TNFR family member, glucocorticoid-induced TNFR (GITR), which has been reported to modulate the function of CD4+CD25+ T cells in other models. We found that in vivo administration of GITR mAb inhibited EAT tolerance induction and abrogated established tolerance, enabling thyroiditis induction. In in vitro assays, GITR mAb inhibited suppression of mTg-primed T cells by CD4+CD25+ T cells isolated from mTg-pretreated mice. The target of GITR mAb appears to be CD4+CD25- T cells, rather than CD4+CD25+ T cells from tolerized mice, suggesting that GITR signaling likely interferes with EAT tolerance by enabling thyroiditogenic T cells to circumvent suppression by CD4+CD25+ regulatory T cells.

title: The TNF ligand superfamily and its relevance for human diseases.
abstract: The tumor necrosis factor (TNF) receptor superfamily comprises 10 different members of type I integral membrane glycoproteins with characteristic limited sequence homology for the extracellular cysteine-rich repeats. A parallel existing TNF ligand superfamily has been discovered by cloning of ligands for all of the TNF receptor superfamily members. These molecules are type II membrane glycoproteins, with the exception of LT-alpha, which is the only secreted protein of the family. TNF and CD95L also exist in biologically active shed soluble form. The TNF ligand superfamily presently contains nine different proteins. In addition, the NGFR p75 binds to a second family of proteins. These NGF-like dimeric soluble molecules are basic neurotrophic factors, and the five members are not related to the TNF superfamily ligands. The TNF-like ligands share some common biological activities, but other activities appear to be shared only by some ligands or are unique. The diverse biological effects mediated through the interaction of the members of the TNF receptor and ligand superfamilies have provided information on the regulation of cellular activation, including the involvement of T-cell-dependent immune responses as well as associations with human diseases.

title: Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection.
abstract: The establishment of viral persistence generally requires evasion of the host CD8(+) T cell response. Here we describe a form of evasion wherein the CD8(+) T cells are fully capable of recognizing their cognate antigen but their effector functions are suppressed by regulatory T cells. Virus-specific CD8(+) T cells adoptively transferred into mice persistently infected with Friend virus proliferated and appeared activated, but failed to produce IFNgamma or reduce virus loads. Cotransfer experiments revealed that a subpopulation of CD4(+) T cells from persistently infected mice suppressed IFNgamma production by the CD8(+) T cells. Treatment of persistently infected mice with anti-GITR antibody to ameliorate suppression by regulatory T cells significantly improved IFNgamma production by transferred CD8(+) T cells and allowed a significant reduction in viral loads. The results indicate that CD4(+) regulatory T cells contribute to viral persistence and demonstrate an immunotherapy for treating chronic retroviral infections.

title: The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages.
abstract: In order to clarify the mechanism by which soluble GITR (sGITR) inhibits the survival of murine macrophages we examined its effect on the macrophage cell cycle. Soluble GITR induced G1 phase arrest followed by apoptosis. It also reduced the expression of cyclins D2 and A, and of cdk4, resulting in reduced cdk2 and cdk4 activities. These findings suggest that sGITR arrests division of the macrophages in G1 by lowering the activities of cdk2 and cdk4, and that this leads to apoptosis.

title: ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow.
abstract: In vivo, leukocyte transendothelial migration (TEM) occurs at endothelial cell junctions (paracellular) and nonjunctional (transcellular) locations, whereas in vitro models report that TEM is mostly paracellular. The mechanisms that control the route of leukocyte TEM remain unknown. Here we tested the hypothesis that elevated intercellular adhesion molecule-1 (ICAM-1) expression regulates the location of polymorphonuclear leukocyte (PMN) TEM. We used an in vitro flow model of tumor necrosis factor-alpha (TNF-alpha)-activated human umbilical vein endothelium cells (HUVECs) or an HUVEC cell line transfected with ICAM-1GFP (green fluorescent protein) and live-cell fluorescence microscopy to quantify the location of PMN adhesion and TEM. We observed robust transcellular TEM with TNF-alpha-activated HUVECs and ICAM-1GFP immortalized HUVECS (iHUVECs). In contrast, primary CD3+ T lymphocytes exclusively used a paracellular route. Endothelial ICAM-1 was identified as essential for both paracellular and transcellular PMN transmigration, and interfering with ICAM-1 cytoplasmic tail function preferentially reduced transcellular TEM. We also found that ICAM-1 surface density and distribution as well as endothelial cell shape contributed to transcellular TEM. In summary, ICAM-1 promotes junctional and nonjunctional TEM across inflamed vascular endothelium via distinct cytoplasmic tail associations.

title: Pharmacological action of melatonin in shock, inflammation and ischemia/reperfusion injury.
abstract: A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially, superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation, shock and ischemia/reperfusion injury. The aim of this review is to describe recent developments in the field of oxidative stress research. The first part of the review focuses on the roles of reactive oxygen species in shock, inflammation and ischemia/reperfusion injury. The second part of the review described the pharmacological action of melatonin in shock, ischemia/reperfusion, and inflammation. The role of reactive oxygen species: Immunohistochemical and biochemical evidence demonstrate the production of reactive oxygen species in shock, inflammation and ischemia/reperfusion injury. Reactive oxygen species can initiate a wide range of toxic oxidative reactions. These include the initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3 phosphate dehydrogenase, inhibition of membrane sodium/potassium ATP-ase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation and ischemia and reperfusion. Treatment with melatonin has been shown to prevent in vivo the delayed vascular decompensation and the cellular energetic failure associated with shock, inflammation and ischemia/reperfusion injury. Reactive oxygen species (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single-strand breakage, with subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. Recently, it has been demonstrated that melatonin inhibits the activation of poly (ADP-ribose) synthetase, and prevents the organ injury associated with shock, inflammation and ischemia and reperfusion.

title: New insights into the mode of action of anti-inflammatory drugs
abstract: The discovery of a second cyclooxygenase has provided fresh impetus to the search for new anti-inflammatory drugs. The second enzyme is effectively absent from healthy tissues but its levels rise dramatically during inflammation. It can be induced in migratory cells by bacterial lipopolysaccharide, cytokines and growth factors. The constitutive cyclooxygenase-1 (COX-1) can thus be considered a “housekeeping” enzyme, in contrast to cyclooxygenase-2 (COX-2) which is activated by tissue damage. Both enzymes have a molecular weight of around 70kDa and similar Km and Vmax values for their reaction with arachidonic acid. Several non steroid anti-inflammatory drugs which have more than 1,000 fold selectivity for COX-2 over COX-1 are in the early stages of drug development.

title: Soluble glucocorticoid-induced tumor necrosis factor receptor stimulates osteoclastogenesis by down-regulation of osteoprotegerin in bone marrow stromal cells.
abstract: Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) is a potent stimulator of osteoclastogenesis. The mechanism by which it induces osteoclastogenesis was studied by culturing bone-marrow-derived macrophages (BMM) with conditioned medium from mouse bone marrow stromal cells. GITR and GITR ligand (GITRL) were expressed on the surface of bone marrow stromal cells, and sGITR-induced osteoclastogenesis was inhibited by anti-GITRL Ab, indicating that stimulatory effect of osteoclastogenesis by sGITR involved signaling via GITRL. Bone marrow stromal cells up-regulated cyclooxygenase-2 (COX-2) and produced prostaglandin E(2) (PGE(2)) early in their response to sGITR, and the stimulation of osteoclastogenesis was markedly inhibited by NS398, a COX-2 inhibitor. Later, sGITR markedly reduced the steady-state level of osteoprotegerin (OPG) mRNA and increased receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA. NS398 blocked the sGITR-induced reduction of OPG mRNA but did not significantly affect the sGITR-induced rise in RANKL mRNA. This suggests that down-regulation of OPG by PGE(2) is involved in sGITR-induced osteoclast (OC) formation in the presence of conditioned medium from mouse bone marrow stromal cells.

title: Induction of nitric oxide synthase (NOS) by soluble glucocorticoid induced tumor necrosis factor receptor (sGITR) is modulated by IFN-gamma in murine macrophage.
abstract: Earlier study showed that glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, activated murine macrophages to express inducible nitric oxide synthase (iNOS) and to generate nitric oxide (NO). A possible involvement of pro-inflammatory cytokines on NO production by GITR was investigated in vitro systems and signaling molecules contributing to sGITR-induced iNOS production are determined in Raw 264.7 cells, a murine macrophage cell line. The result showed that the synergy was afforded by the combination of GITR with IFN-g in a dose-dependent manner but IFN-gamma alone was not able to induce NOS. No effects were observed with TNF-alpha, IL-1beta, or IL-6 co-treated with GITR. To determine signaling molecules contributing to sGITR-induced iNOS production, a specific inhibitor for signal pathway proteins tested showed that PDTC (NF-kappaB) and genistein (tyrosine kinase) inhibited NOS induction significantly, while sodium orthovanadate (tyrosine phosphatase) potentiated NOS expression. These results suggest that activations of NF-kappaB were involved in induction of iNOS by GITR and IFN-gamma priming caused earlier and stronger NF-kappaB activation.

title: Recombinant glucocorticoid induced tumor necrosis factor receptor (rGITR) induces NOS in murine macrophage
abstract: Glucocorticoid induced tumor necrosis factor receptor (GITR) is a new member of the tumor necrosis factor–nerve growth factor receptor superfamily of which the function has not been well studied. The extracellular domain of GITR was produced in Escherichia coli and purified as a single band of predicted M r of 18.0 kDa. GITR and GITR ligand were expressed constitutively on the surface of Raw 264.7 macrophage cell line and murine peritoneal macrophages. An extracellular domain of GITR can activate murine macrophages to express inducible nitric oxide synthase and to generate nitric oxide in a dose‐ and time‐dependent manner.

title: Glucocorticoid‐induced tumour necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques
abstract: Glucocorticoid‐induced tumour necrosis factor receptor family related protein (GITR) is the 18th member of the tumour necrosis factor receptor superfamily (TNFRSF18) and is known to interact with its cognate ligand GITRL (TNFSF18). We investigated the potential role of GITR in the pro‐inflammatory activation of macrophages. Immunohistochemistry and in situ hybridization analyses of human atherosclerotic plaques demonstrated that GITR and its ligand are expressed mainly in lipid‐rich macrophages. We then investigated the role of GITR in human and mouse monocyte/macrophage functions. Stimulation of GITR caused nuclear factor (NF)‐κB‐dependent activation of matrix metalloproteinase‐9 (MMP‐9) and pro‐inflammatory cytokine expression in both the human and mouse monocytic/macrophage cell lines, THP‐1 and RAW264.7, respectively. These cellular responses were also observed when the THP‐1 cells were treated with phorbol‐12 myristate‐13 acetate (PMA), which is known to induce macrophage differentiation. To demonstrate that these responses are not restricted to cultured cell lines, we tested primary macrophages. Both peritoneal and bone marrow‐derived macrophages responded to GITR stimulation with induction of MMP‐9 and tumour necrosis factor‐α (TNF‐α). Furthermore, the GITR staining pattern overlapped with those of MMP‐9 and TNF‐α in atherosclerotic plaques. These data indicate that GITR‐mediated macrophage activation may promote atherogenesis via the induction of pro‐atherogenic cytokines/chemokines, and destabilize the atherosclerotic plaques via the induction of the matrix‐degrading enzyme, MMP‐9.

title: Induction of MMP-13 expression by soluble human glucocorticoid-induced tumor necrosis factor receptor in fibroblast-like synovial cells.
abstract: We tested the hypothesis that human glucocorticoid-induced tumor necrosis factor receptor (hGITR/TR11) expressed on the surface of activated CD4(+) T cells is responsible for up-regulating the production of matrix metalloproteinase (MMP)-13 by fibroblast-like synoviocytes (FLSs).



The level of MMP-13 was measured by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR). Expressions of hGITR ligand (hGITRL) on the surface of FLSs and hGITR on the surface of human CD4(+) T cells were analyzed by flow cytometry and RT-PCR. Neutralizing antibodies (Abs) were used to block hGITRL and hGITR on the surface of FLSs and human CD4(+) T cells, respectively. Human CD4(+) T cells were cocultured with FLSs to facilitate interaction between hGITR on CD4(+) T cells and hGITRL on FLSs.



Soluble hGITR (shGITR) stimulated FLSs to produce MMP-13, and blockade of hGITRL reduced this effect. Direct contact between activated CD4(+) T and FLSs also induced the production of MMP-13, and neutralization of hGITR on activated CD4(+) T cells during coculture decreased the amount of MMP-13 produced by FLSs.



shGITR stimulated FLSs to produce MMP-13 via a signal through hGITRL. Direct contact between activated CD4(+) T cells and FLSs facilitated hGITR-hGITRL interaction, and resulted in inducing MMP-13. This effect may increase tissue destruction in chronic inflammation such as rheumatoid arthritis (RA).

title: Neonatal immune responses to TLR2 stimulation: Influence of maternal atopy on Foxp3 and IL-10 expression
abstract: BackgroundMaternal atopic background and stimulation of the adaptive immune system with allergen interact in the development of allergic disease. Stimulation of the innate immune system through microbial exposure, such as activation of the innate Toll-like-receptor 2 (TLR2), may reduce the development of allergy in childhood. However, little is known about the immunological effects of microbial stimulation on early immune responses and in association with maternal atopy.MethodsWe analyzed immune responses of cord blood mononuclear cells (CBMC) from 50 healthy neonates (31 non-atopic and 19 atopic mothers). Cells were stimulated with the TLR2 agonist peptidoglycan (Ppg) or the allergen house dust mite Dermatophagoides farinae (Derf1), and results compared to unstimulated cells. We analyzed lymphocyte proliferation and cytokine secretion of CBMC. In addition, we assessed gene expression associated with T regulatory cells including the transcription factor Foxp3, the glucocorticoid-induced TNF receptor (GITR), and the cytotoxic lymphocyte antigen 4 (CTLA4). Lymphocyte proliferation was measured by 3H-Thymidine uptake, cytokine concentrations determined by ELISA, mRNA expression of T cell markers by real-time RT-PCR.ResultsPpg stimulation induced primarily IL-10 cytokine production, in addition to IFN-γ, IL-13 and TNF-α secretion. GITR was increased following Ppg stimulation (p = 0.07). Ppg-induced IL-10 production and induction of Foxp3 were higher in CBMC without, than with maternal atopy (p = 0.04, p = 0.049). IL-10 production was highly correlated with increased expression of Foxp3 (r = 0.53, p = 0.001), GITR (r = 0.47, p = 0.004) and CTLA4 (r = 0.49, p = 0.003), independent of maternal atopy.ConclusionTLR2 stimulation with Ppg induces IL-10 and genes associated with T regulatory cells, influenced by maternal atopy. Increased IL-10 and Foxp3 induction in CBMC of non-atopic compared to atopic mothers, may indicate an increased capacity to respond to microbial stimuli.

title: Glucocorticoid-Induced TNF Receptor Functions as a Costimulatory Receptor That Promotes Survival in Early Phases of T Cell Activation1
abstract: Glucocorticoid-induced TNFR (GITR) is a member of the TNFR family that can inhibit the suppressive function of regulatory T cells and promote the survival and activation of T cells. However, little is known about the molecular mechanisms regulating T cell survival and activation downstream of GITR. To gain further insight into the cellular events and signaling pathways triggered by GITR, survival, proliferation, and cytokine production as well as activation of MAPKs and NF-κB were monitored after cross-linking of the receptor on naive and activated T cells. GITR cross-linking provided costimulation of naive and activated T cells and resulted in activation of MAPKs and NF-κB. Although GITR-induced signaling pathways augmented the survival of naive T cells, they were not sufficient to inhibit activation-induced cell death triggered by CD3 cross-linking of activated T cells. Differences in the contributions of GITR to cell survival between naive and activated T cells suggest that the receptor triggers specific pathways depending on the activation state of the T cell.

title: Cutting Edge: Ligation of the Glucocorticoid-Induced TNF Receptor Enhances Autoreactive CD4+ T Cell Activation and Experimental Autoimmune Encephalomyelitis1
abstract: The glucocorticoid-induced TNFR (GITR) is expressed at high levels on resting CD4+CD25+ T regulatory (TR) cells and regulates their suppressive phenotype. Accordingly, we show that anti-GITR mAb treatment of SJL mice with proteolipid protein 139–151-induced experimental autoimmune encephalomyelitis significantly exacerbated clinical disease severity and CNS inflammation, and induced elevated levels of Ag-specific T cell proliferation and cytokine production. Interestingly, prior depletion of TR cells failed to result in exacerbated experimental autoimmune encephalomyelitis suggesting alternative targets for the anti-GITR mAb treatment. Importantly, naive CD4+CD25− T cells up-regulated GITR expression in an activation-dependent manner and anti-GITR mAb treatment enhanced the level of CD4+ T cell activation, proliferation, and cytokine production in the absence of TR cells both in vivo and in vitro. Taken together, these findings suggest a dual functional role for GITR as GITR cross-linking both inactivates TR cells and increases CD4+CD25− T cell effector function, thus enhancing T cell immunity.

title: CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms.
abstract: The glucocorticoid-induced TNF-related gene receptor (GITR) is the newest member of the costimulatory molecule family and is expressed on both resting CD4+CD25+ regulatory T (T(R)) cells and activated CD4+ T cells. We investigated the endogenous mechanisms that regulate GITR expression on both T(R) and CD4+ T cells, as well as the functional interaction between GITR and other costimulatory molecules. CD28 stimulation increased GITR expression on both T(R) and CD4+ T cells via IL-2-dependent mechanisms. In addition, ligation of GITR and/or CD28 increased the level of CD4+ T cell proliferation and effector function under both APC-dependent and -independent conditions, suggesting that these costimulatory molecules cooperate to regulate CD4+ T cell activation and function by directly signaling to the CD4+ T cell. Thus, GITR may serve opposing functional roles on CD4+ T(R) and effector cells and alterations in GITR expression and/or function may tip the balance between immune tolerance and effector function.

title: Conversion of Alloantigen-Specific CD8+ T Cell Anergy to CD8+ T Cell Priming through In Vivo Ligation of Glucocorticoid-Induced TNF Receptor1
abstract: In this study, we investigated the effect of an agonistic mAb (DTA-1) against glucocorticoid-induced TNF receptor (GITR) in a murine model of systemic lupus erythematosus-like chronic graft-vs-host disease (cGVHD). A single dose of DTA-1 inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. DTA-1-treated mice showed clinical and pathological signs of acute GVHD (aGVHD), such as lymphopenia, loss of body weight, increase of donor cell engraftment, and intestinal damage, indicating that DTA-1 shifted cGVHD toward aGVHD. The conversion of cGVHD to aGVHD occurred because DTA-1 prevented donor CD8+ T cell anergy. Functionally active donor CD8+ T cells produced high levels of IFN-γ and had an elevated CTL activity against host Ags. In in vitro MLR, anergic responder CD8+ T cells were generated, and DTA-1 stimulated the activation of these anergic CD8+ T cells. We further confirmed in vivo that donor CD8+ T cells, but not donor CD4+ T cells, were responsible for the DTA-1-mediated conversion of cGVHD to aGVHD. These results indicate that donor CD8+ T cell anergy is a restriction factor in the development of aGVHD and that in vivo ligation of GITR prevents CD8+ T cell anergy by activating donor CD8+ T cells that otherwise become anergic. In sum, our data suggest GITR as an important costimulatory molecule regulating cGVHD vs aGVHD and as a target for therapeutic intervention in a variety of related diseases.

title: GITR Activation Induces an Opposite Effect on Alloreactive CD4+ and CD8+ T Cells in Graft-Versus-Host Disease
abstract: Glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) is a member of the tumor necrosis factor receptor (TNFR) family that is expressed at low levels on unstimulated T cells, B cells, and macrophages. Upon activation, CD4+ and CD8+ T cells up-regulate GITR expression, whereas immunoregulatory T cells constitutively express high levels of GITR. Here, we show that GITR may regulate alloreactive responses during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Using a BMT model with major histocompatibility complex class I and class II disparity, we demonstrate that GITR stimulation in vitro and in vivo enhances alloreactive CD8+CD25− T cell proliferation, whereas it decreases alloreactive CD4+CD25− proliferation. Allo-stimulated CD4+CD25− cells show increased apoptosis upon GITR stimulation that is dependent on the Fas–FasL pathway. Recipients of an allograft containing CD8+CD25− donor T cells had increased GVHD morbidity and mortality in the presence of GITR-activating antibody (Ab). Conversely, recipients of an allograft with CD4+CD25− T cells showed a significant decrease in GVHD when treated with a GITR-activating Ab. Our findings indicate that GITR has opposite effects on the regulation of alloreactive CD4+ and CD8+ T cells.

title: CD4+CD25− T Cells in Aged Mice Are Hyporesponsive and Exhibit Suppressive Activity 1
abstract: Studies on humans and rodents have established that functional deterioration of CD4 T cells occurs with aging. We report in this study that ∼70% of CD4+CD25− T cell preparations from individual 24-mo-old mice are hyporesponsive to in vitro stimulation with anti-CD3 Ab. The remaining 30% of CD4+CD25− T cell preparations showing the intermediate or normal responsiveness in the primary stimulation also exhibit the hyporesponsive properties after primary stimulation. Both of these hyporesponsive aged CD4+CD25− T cells could inhibit the proliferation of cocultured CD4+CD25− T cells from young mice, like CD4+CD25+ T cells, which have recently been demonstrated as an immune regulator in young mice. Another experiment revealed that hyporesponsive aged CD4+CD25− T cells arrest the cell division of cocultured young CD4+CD25− T cells. The suppressive activity observed in aged CD4+CD25− T cells is aging-dependent, not mediated by humoral factors, cell-contact dependent, and broken by the addition of IL-2 or anti-GITR Ab, but not by anti-CTLA-4 Ab. These studies show that aging not only leads to a decline in the ability to mount CD4+CD25− T cell responses, but at the same time, also renders these aged CD4+CD25− T cells suppressive.

title: Functional assay for human CD4+CD25+ Treg cells reveals an age‐dependent loss of suppressive activity
abstract: CD4+CD25+ regulatory T cells (Treg cells) prevent T cell‐mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS‐ or bead‐sorted CD4+CD25+ T cells from healthy donors to inhibit anti‐CD3/CD28 activation of CD4+CD25− indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25− indicator cells by both CD4+CD25+high and CD4+CD25+low T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO− (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell–cell contact, and was reversible by interleukin‐2 (IL‐2). Of general interest is that a wider sampling of 27 healthy donors revealed an age‐ but not gender‐dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA‐4 and GITR than CD4+CD25− T cells from the same donors. Moreover, antibody neutralization of CTLA‐4, GITR, IL‐10, or IL‐17 completely reversed Treg‐induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.

title: Control of Autoimmune Myocarditis and Multiorgan Inflammation by Glucocorticoid-Induced TNF Receptor Family-Related Proteinhigh, Foxp3-Expressing CD25+ and CD25− Regulatory T Cells1
abstract: Anomalies of naturally occurring CD4+ regulatory T cells (Treg) cause severe autoimmune/inflammatory diseases in humans and rodents. The transcription factor Foxp3 is currently the most specific marker for natural CD4+ Treg, but it would be useful if other Treg markers, particularly cell surface molecules, could be elucidated. We demonstrate in this study that the vast majority of Foxp3-expressing CD4+ T cells (whether CD25+ or CD25−) show constitutive high-level expression of glucocorticoid-induced TNFR family-related gene/protein (GITR). Transfer of T cell or thymocyte suspensions depleted of GITRhigh cells produces in BALB/c nude mice a wider spectrum and more severe forms of autoimmune diseases than does transfer of similar cell suspensions depleted of CD25+CD4+ T cells only. Notably, mice that receive cells depleted of GITRhigh populations develop severe multiorgan inflammation that includes fatal autoimmune myocarditis resembling giant cell myocarditis in humans, accompanying high-titer anti-myosin autoantibodies. Similar transfer of GITRhigh-depleted cells from prediabetic NOD mice to NOD-SCID mice accelerates the development of diabetes and induces skeletal muscle myositis and other autoimmune/inflammatory diseases. We conclude that GITRhigh, Foxp3-expressing natural Treg, containing both CD25+ and CD25− cell populations, contribute to preventing a variety of autoimmune/inflammatory diseases, and depletion of these cells allows the activation of even weak or rare autoreactive T cells yielding widespread severe autoimmune disease. Diseases induced in this way include many which have been suspected of an autoimmune etiology in humans without much evidence. GITRhigh, Foxp3-expressing natural Treg represent a potential target for the treatment and prevention of these diseases.

title: Cutting Edge: Contact-Mediated Suppression by CD4+CD25+ Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism1
abstract: CD4+CD25+ regulatory T cells (Treg) are potent immunosuppressive cells that are pivotal in the regulation of peripheral tolerance. In this report, we identify granzyme B (GZ-B) as one of the key components of Treg-mediated suppression. Induction of regulatory activity is correlated with the up-regulation of GZ-B expression. Proof of a functional involvement of GZ-B in contact-mediated suppression by Treg is shown by the reduced ability of Treg from GZ-B−/− mice to suppress as efficiently as Treg from WT mice. GZ-B-mediated suppression is perforin independent, because suppression by Treg from perforin−/− and WT is indistinguishable. Additionally, suppression mediated by Treg appears to be mediated, in part, by the induction of apoptosis in the CD4+CD25− effector cell. In summary, GZ-B is one of the key mechanisms through which CD4+CD25+ Treg induce cell contact-mediated suppression.

title: Glucocorticoid-Induced TNF Receptor Family Related Gene Activation Overcomes Tolerance/Ignorance to Melanoma Differentiation Antigens and Enhances Antitumor Immunity1
abstract: Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-γ and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.

title: Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells
abstract: T cell stimulation via glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) can evoke effective tumor immunity. A single administration of agonistic anti-GITR monoclonal antibody (mAb) to tumor-bearing mice intravenously or directly into tumors provoked potent tumor-specific immunity and eradicated established tumors without eliciting overt autoimmune disease. A large number of CD4+ and CD8+ T cells, including interferon (IFN)-gamma-secreting cells, infiltrated regressing tumors. Tumor-specific IFN-gamma-secreting CD4+ and CD8+ T cells also increased in the spleen. The treatment led to tumor rejection in IFN-gamma-intact mice but not IFN-gamma-deficient mice. Furthermore, coadministration of anti-GITR and anti-CTLA-4 mAbs had a synergistic effect, leading to eradication of more advanced tumors. In contrast, coadministration of anti-CD25 and anti-GITR mAbs was less effective than anti-GITR treatment alone, because anti-CD25 depleted both CD25+-activated effector T cells and CD25+CD4+ naturally occurring regulatory T (T reg) cells. Importantly, CD4+ T cells expressing the T reg-specific transcription factor Foxp3 predominantly infiltrated growing tumors in control mice, indicating that tumor-infiltrating natural Foxp3+CD25+CD4+ T reg cells may hamper the development of effective tumor immunity. Taken together, T cell stimulation through GITR attenuates T reg-mediated suppression or enhances tumor-killing by CD4+ and CD8+ effector T cells, including those secreting IFN-gamma, or both. Agonistic anti-GITR mAb is therefore instrumental in treating advanced cancers.

title: Reduction of Retrovirus-Induced Immunosuppression by In Vivo Modulation of T Cells during Acute Infection
abstract: Chronic infection with Friend retrovirus is associated with suppressed antitumor immune responses. In the present study we investigated whether modulation of T-cell responses during acute infection would restore antitumor immunity in persistently infected mice. T-cell modulation was done by treatments with DTA-1 anti- glucocorticoid-induced tumor necrosis factor receptor monoclonal antibodies. The DTA-1 monoclonal antibody is nondepleting and delivers costimulatory signals that both enhance the activation of effector T cells and inhibit suppression by regulatory T cells. DTA-1 therapy produced faster Th1 immune responses, significant reductions in both acute virus loads and pathology and, most importantly, long-term improvement of CD8+ T-cell-mediated antitumor responses.

title: The Glucocorticoid-Induced Tumor Necrosis Factor Receptor-Related Gene Modulates the Response to Candida albicans Infection
abstract: The glucocorticoid-induced tumor necrosis factor (TNF) receptor-related gene (GITR; TNFRSF18) modulates immune response activating coaccessory signals in T cells and is expressed at high levels in CD4+CD25+ cells. Its ligand (GITRL) is expressed in antigen-presenting cells, where it is capable of promoting signaling. We investigated the role of GITR/GITRL interaction during disseminated candidiasis in GITR knockout (GITR−/−) mice. GITR−/− mice survived longer and had a significantly decreased yeast load in kidneys and brain compared to GITR+/+ mice. Since protective immunity to the fungus is mediated by antigen-specific T helper (Th) 1 cells, we studied in vitro cytokine production following infection. CD4+ T cells of GITR−/− mice demonstrated a more efficient Th1 polarization as suggested by a two- to threefold decreased production of interleukin- (IL-)4 and IL-10 and a four- to fivefold increased production of gamma interferon compared to GITR+/+ mice. This effect was not due to differences in lymphocyte and dendritic cell (DC) subpopulations in infected mice as demonstrated by flow cytometric studies. To verify whether DC activity was differently modulated, DCs were cocultured with CD4+ T cells in the presence of heat-inactivated Candida albicans. DCs, cocultured with GITR+/+ CD4+CD25+ cells produced a lower amount of IL-12 than DCs cocultured with GITR−/− CD4+CD25+ T cells. These results suggest that GITR regulates susceptibility to systemic candidiasis by negatively modulating IL-12 production and promoting polarization of CD4+ T cells towards Th2 by analogy with OX40, another TNF receptor superfamily member.

title: Gene structure and chromosomal assignment of mouse GITR, a member of the tumor necrosis factor/nerve growth factor receptor family.
abstract: GITR is a type I transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor (TNF/NGFR) family. This receptor is preferentially expressed in activated T lymphocytes and may function as signaling molecule during T-cell development. In the present study, we examined the genomic organization of the entire mouse GITR (mGITR) gene. The gene spans a 2543-bp region and consists of five exons (with a length ranging from 88 bp to 395 bp) and four introns (67 bp to 778 bp). In agreement with GITR expression in activated T cells, consensus elements for transcription factors involved in T-cell development and activation were identified in the 5' flanking region, including a consensus element for NF-kappaB. Two highly significant binding sites for MyoD and one binding site for myogenin were also found, suggesting involvement of GITR in muscle development. The mGITR gene contains 17 transcription initiation sites distributed over a 76-bp region, all used with the same frequency. We localized mGITR to the murine chromosome 4 (E region), where other 4 TNF/NGFR members localize, including m4-1BB and mOX40. These results further indicate that GITR shares several features with OX40, 4-1BB, and CD27, suggesting the existence of a new subfamily of the TNFR family, as also confirmed by the similarity of their cytoplasmic domains.

title: Determination of tumor necrosis factor binding protein disulfide structure: deviation of the fourth domain structure from the TNFR/NGFR family cysteine-rich region signature.
abstract: Tumor necrosis factor binding protein is a soluble molecule derived from the extracellular domain of the 55 kDa human tumor necrosis factor receptor, which can block the biological function of tumor necrosis factor by binding to the growth factor. This cysteine-rich molecule is subdivided into four domains, each containing six conserved cysteines that form three intrachain disulfide linkages known as the tumor necrosis factor receptor/nerve growth factor receptor family cysteine-rich region signature structure. In an effort to elucidate the molecular integrity of the molecule, we performed detailed analysis and searched for strategies to elucidate the complete disulfide structure of the E. coli-derived tumor necrosis factor binding protein and to determine the disulfide arrangement in the fourth domain of Chinese hamster ovary cell-derived molecule. The methods employed included various proteolytic digestions, peptide mapping, partial reduction, and assignment of disulfides by N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry with post-source decay. The first three domains of the molecule were confirmed to have disulfide structures identical to the cysteine-rich region signature structure found in the above-mentioned receptor superfamily. The fourth domain has a different structure from the first three domains where the last four cysteines form two disulfide bonds in opposite positions.

title: Requirement of Cysteine-rich Repeats of the Fas Receptor for Binding by the Fas Ligand*
abstract: The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr andlpr cg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.

title: Nerve growth factor binding domain of the nerve growth factor receptor.
abstract: A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptor to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cysteine-rich sequence of the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.

title: GITR interacts with the pro-apoptotic protein Siva and induces apoptosis
abstract: We have previously identified and characterised a murine member of the tumour necrosis factor receptor superfamily (TNFRSF), named GITR (TNFR18). ± 4 Most TNFRSF members modulate cell death and induce or counter apoptosis depending on activation of a specific transduction pathway and we showed GITR counters T cell apoptosis. This effect correlates in part with GITR/TRAF-2 binding and consequent NF-kB activation. The GITR cytoplasmic region shows a striking homology with the cytoplasmic region of other TNFRSF members such as CD40 (TNFRSF5), OX40 (TNFRSF4), 4-1BB (TNFRSF9) and CD27 (TNFRSF7) . CD27 stimulation is reported to either protect from, or induce apoptosis, and, in fact, CD27 binding to Siva, a death-domain containing cytoplasmic molecule that is involved in the apoptotic response to virus infection and oxidative stress, ± 9 induces apoptosis activation. On the basis of CD27 and GITR homology at the cytoplasmic domain (Figure 1A) we hypothesised that murine GITR (mGITR) could bind murine Siva (mSiva). To verify whether mGITR and mOX40 bind mSiva, we in vitro translated their cytoplasmic domains together with the mCD27 domain as positive control. The [S]translated products were challenged with the fusion protein GSTmSiva (glutathione S-transferase-GST-fused to mSiva and bound to a sepharose resin) and extensively washed. A GST protein was used as negative control. Figure 1B shows that mGITR, mOX40 and mCD27 bound mSiva. A symmetric experiment using GST-mGITR (cytoplasmic region) and in vitro translated mSiva, confirmed mGITR/ mSiva binding (Figure 1C). To test whether mGITR binds mSiva in vivo, we performed co-immunoprecipitation studies with tagged proteins (mGITR-myc and mSiva-Xpress), overexpressed in Cos7 cells. mSiva-Xpress or mGITR-myc were coimmunoprecipitated with the anti-myc or anti-Xpress antibody (Ab) respectively and revealed by Western blot analysis.  Figure 1D indicate that mGITR binds mSiva in vivo. To check any aspecificities due to immunoprecipitating antibodies, lysates from GITR-myc and Siva-Xpress single transfection, immunoprecipitated with anti-Xpress or anti-myc Ab respectively, were used as negative controls. We also evaluated the level of mGITRmyc expression in comparison to that of mGITR in DEXtreated hybridoma T-cells or activated T-lymphocytes by semiquantitative PCR or cytofluorimetric experiments, respectively. Results indicate that similar levels of expression are detected in transfected Cos7 cells and either activated or DEX treated T-cells (data not shown). To identify the mGITR motif responsible for mSiva binding, we assessed the binding efficacy of 10 in vitro translated mutants of mGITR cytoplasmic domain (Figure 1E). The percentage of the product bound to the fusion protein GST-mSiva compared to the input was quantitated upon SDS ± PAGE separation. Figure 1F and G show that mGITR deleted in its C-terminal domain (GITRD30, mutant 1) did not bind mSiva, suggesting the 30 C-terminal amino acidic residues are necessary for mSiva binding. We then investigated the binding ability of several deleted mutants lacking 7 ± 10 amino acidic residues in the C-terminal region (Figure 1E) and observed that GITRDc (mutant 4) did not bind mSiva; GITRDa (mutant 2), GITRDb (mutant 3) and GITRDd (mutant 5) bound mSiva less than GITR; and GITRDe (mutant 6) bound mSiva to a similar extent as GITR (Figure 1F and G). The lack of GITRDc-mSiva binding suggests that the amino acidic residues that are not present in GITRDc (SFQFPEEE) are required for mGITR/mSiva binding. The low binding degree of GITRDa-mSiva suggests that the amino acidic residues that are absent in GITRDa, and N-flanking those missing in GITRDc (QLSAEDAC) play some role. Consequently, we prepared and studied specific mGITR protein mutants (Figure 1E) and found that GITR(S180A) (mutant 7), GITR(P190A) (mutant 9) and GITR(E191R,E192V,E193V) (mutant 10) did not bind mSiva, while GITR(E182R,C185G) (mutant 8) bound mSiva to a similar extent as mGITR (Figure 1F and G). The lack of binding of GITRDc, mutant 7, mutant 9 and mutant 10 suggests that the GITR-Siva binding requires specific amino acidic domains. In particular, results indicate that the SFQFPEEE domain (position 186 ± 193) is crucial for mSiva binding, with the PEEE sequence playing an important role. In human GITR (hGITR), the QFPEEE sequence is conserved, suggesting this sequence plays a functional role. Prasad et al and the present results demonstrate that mCD27, hCD27 as well as mOX40 bind Siva. Within these TNFRSF members a PIQE sequence is found in the region corresponding to the PEEE sequence of mGITR and hGITR . Thus, the core motif responsible for Siva binding might be P(I/E)(Q/E)E. The same motif is also present in human 4-1BB and a similar one (PQEE) is found in murine 4-1BB. In human and murine CD40 the PIQE motif is also present, though in different positions of the cytoplasmic domain. Therefore Cell Death and Differentiation (2002) 9, 1382 ± 1384 ã 2002  1350-9047/02 $25.00

title: The nerve growth factor/tumor necrosis factor receptor family
abstract: Receptors in the nerve growth factor/tumor necrosis factor receptor family are characterized by the presence of cysteine‐rich motifs of ~40 amino acids in the extracellular domain. The ligands are type II transmembrane proteins with β‐strands that form a jelly‐roll β‐sandwich. The receptors recognize soluble or cell‐surface‐bound ligands and mediate diverse cellular responses. Activation of intracellular signals is mediated at least in part by the association of proteins with a RING finger motif or a death domain to the cytoplasmic domains of the receptors. In addition to cell‐membrane‐bound receptors soluble forms have been described for most of the receptors. Activation of intracellular signals not only occurs through ligand binding to the receptors but cross‐linking of at least some members of the ligand family can regulate cell functions.

title: LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing.
abstract: Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the "death domain." We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.

title: Identification of three novel mRNA splice variants of GITR
abstract: GITR is a gene belonging to the nerve growth factor-tumor necrosis factor receptor (NGF/TNFR) family that is preferentially expressed in lymphoid tissues, is overexpressed following dexamethasone treatment or T cell receptor (TCR) stimulation and is able to modulate T cell apoptosis. Like other members of the NGF/TNFR family, GITR is composed of an intracellular domain, a transmembrane domain and an extracellular domain that is crucial for the binding with the specific ligand. In particular, the GITR extracellular domain, like other family members, comprises three pseudorepeats of approximately 40 amino acids containing 4 ± 6 cysteine residues. Moreover, murine GITR gene comprises five exons. The first three exons code for the extracellular domain, exon 4 codes for a small part of the extracellular domain, the transmembrane domain and part of the cytoplasmic domain while exon 5 codes for the cytoplasmic domain (see Figure 1A). Several members of NGF/TNFR family are characterized by splicing variants. We performed experiments to analyze whether different splicing products of GITR could be expressed in T cells. We first analyzed a number of clones isolated by a library screening of a hybridoma T cell line, and found clones different from GITR mRNA. In particular, one of the isolated clones (GITR-B) showed 11 bases added between exon 4 and exon 5, belonging to the 3' end of intron 4 (Figure 1A). Thus, in GITR-B exon 5 is 11 bp longer than the exon 5 found in GITR. Moreover, the 3' intron/5' exon boundary is flanked by the consensus splice site sequence CAG/N. Of note, the protein putatively coded by GITR-B is different in the cytoplasmic domain (Figure 1B), due to the reading frame shift. In particular, a region of this cytoplasmic domain  has a significant homology with a cytoplasmic region of CD4 and CD8 receptors known to bind p56 kinase. By performing RT ± PCR using a DNAse-treated RNA from normal thymocytes, another GITR splicing was observed (GITR-C, Figure 1A). In GITR-C, the intron between exon 4 and exon 5 is not spliced out. To ensure that the cloned PCR product derived from mRNA and not from DNA still present after DNAse treatment, we also performed RT ± PCR experiments with a forward primer located in the exon 3 and demonstrated that the GITR-C is indeed a GITR splicing (not shown). In fact, intron 3 resulted spliced out while intron 4 was not. The protein putatively coded by GITR-C is different from GITR and GITR-B in the cytoplasmic domain, due to the addition of 67 bp (intron 4) and a reading frame shift (Figure 1B). This cytoplasmic domain does not show any significant homology with other known proteins. Soluble forms have been described for almost all members of the NGF/TNFR family, as the result of proteolitic shedding or mRNA splicing. ± 9 To investigate whether any alternative splicing variant of GITR gene could produce a soluble form, some RT ± PCRs were performed with primers located in the exons 3 and 5, using RNA derived from thymocytes as template. We cloned a new GITR splicing (GITR-D, Figure 1A), that lacks exon 4. The protein putatively coded by GITR-D (Figure 1C) is identical to GITR in the NH2terminal residues, comprising the signal peptide, the first and the second pseudorepeat and part of the third pseudorepeat motif truncated after the second cysteine, of the extracellular domain, and lacks the transmembrane and intracellular domains. Thus, GITR-D is a secreted protein which could bind the GITR ligand and function as a decoy receptor. We also performed experiments to analyze the expression level of GITR splicings in normal cells. Results indicate that in thymocytes the amount of GITR-D mRNA is comparable to the amount of GITR mRNA, while the levels of GITR-C and GITR-B are lower . Therefore, we can hypothesize that GITR and GITR-D proteins are expressed at similar levels in resting T cells so that GITR-D could efficiently compete with GITR for the ligand binding. GITR-C and GITR-B, although expressed at lower level, might contribute to regulate T-cell activation and death by transduction pathways different from the ones activated by GITR. As an example, GITR may bind TRAF2 through the PxEE motif and GITR-B may bind p56 kinase through the CXC motif (Nocentini et al. manuscript in preparation) thus cooperating in lymphocyte activation. We have previously demonstrated that the expression of GITR gene increases about fourfold in ConA activated thymocytes. However, the amount of the PCR product deriving from GITR increases in the activated thymocytes, while the PCR product deriving from GITR-D decreases as a result of the increased competition by GITR in the PCR assay (Figure 1D, lane 2). This change of the GITR/GITR-D ratio might have a physiological role and suggests that in activated cells GITR-D may be less able to compete with GITR so that the GITR binding with its ligand might be favoured. In conclusion, the four different splicing products of murine GITR gene originate a complex receptor system. The balance among the expression levels of the alternative splicings change with the T-cell activation stage and might result in the activation of different intracellular pathways, upon ligand binding, and modulate the triggering of GITR receptors through the modulation of the amount of freeligand by means of the decoy target. Future studies will be devoted to further analyze the possible role of the alternative splicings of GITR in the regulation of T cell activation and death.

title: TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs.
abstract: TNF family members and their receptors contribute to increased gene expression for inflammatory processes and intracellular cascades leading to programmed cell death, both via activation of NF-kappaB. TNF receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins binding to various receptors of the TNFR family. In an attempt to delineate the role of individual TRAFs, we compared NF-kappaB activation by CD40(wt) and CD40 mutants with different TRAF recruitment patterns. Recognized only recently, NF-kappaB signaling occurs at least via two different pathways. Each pathway results in nuclear translocation of two different Reldimers, the canonical p50/RelA and the noncanonical p52/RelB. Here, we show that via TRAF6, CD40 mediates only the activation of the canonical NF-kappaB pathway. Via TRAF2/5, CD40 activates both the canonical and the noncanonical NF-kappaB pathways. We observed that TRAF3 specifically blocked the NF-kappaB activation via TRAF2/5. This inhibitory effect of TRAF3 depends on the presence of an intact zinc finger domain. Paradoxically, suppression of TRAF2/5-mediated NF-kappaB activation by TRAF3 resulted in enhanced transcriptional activity of TRAF6-mediated canonical NF-kappaB emanating from CD40. We also observed that 12 TNFR family members (p75TNFR, LTbetaR, RANK, HVEM, CD40, CD30, CD27, 4-1BB, GITR, BCMA, OX40, and TACI) are each capable of activating the alternative NF-kappaB pathway and conclude that TRAF3 serves as a negative regulator of this pathway for all tested receptors.

title: Structural basis for self-association and receptor recognition of human TRAF2
abstract: Tumour necrosis factor (TNF)-receptor-associated factors (TRAFs) form a family of cytoplasmic adapter proteins that mediate signal transduction from many members of the TNF-receptor superfamily and the interleukin-1 receptor. They are important in the regulation of cell survival and cell death. The carboxy-terminal region of TRAFs (the TRAF domain) is required for self-association and interaction with receptors. The domain contains a predicted coiled-coil region that is followed by a highly conserved TRAF-C domain. Here we report the crystal structure of the TRAF domain of human TRAF2, both alone and in complex with a peptide from TNF receptor-2 (TNF-R2). The structures reveal a trimeric self-association of the TRAF domain, which we confirm by studies in solution. The TRAF-C domain forms a new, eight-stranded antiparallel β-sandwich structure. The TNF-R2 peptide binds to a conserved shallow surface depression on one TRAF-C domain and does not contact the other protomers of the trimer. The nature of the interaction indicates that an SXXE motif may be a TRAF2-binding consensus sequence. The trimeric structure of the TRAF domain provides an avidity-based explanation for the dependence of TRAF recruitment on the oligomerization of the receptors by their trimeric extracellular ligands.

title: Distinct molecular mechanism for initiating TRAF6 signalling
abstract: Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40° difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide–TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.

title: Glucocorticoid-Induced TNF Receptor, a Costimulatory Receptor on Naive and Activated T Cells, Uses TNF Receptor-Associated Factor 2 in a Novel Fashion as an Inhibitor of NF-κB Activation1
abstract: Glucocorticoid-induced TNFR (GITR) has been implicated as an essential regulator of immune responses to self tissues and pathogens. We have recently shown that GITR-induced cellular events promote survival of naive T cells, but are insufficient to protect against activation-induced cell death. However, the molecular mechanisms of GITR-induced signal transduction that influence physiologic and pathologic immune responses are not well understood. TNFR-associated factors (TRAFs) are pivotal adapter proteins involved in signal transduction pathways of TNFR-related proteins. Yeast two-hybrid assays and studies in HEK293 cells and primary lymphocytes indicated interactions between TRAF2 and GITR mediated by acidic residues in the cytoplasmic domain of the receptor. GITR-induced activation of NF-κB is blocked by A20, an NF-κB-inducible protein that interacts with TRAFs and functions in a negative feedback mechanism downstream of other TNFRs. Interestingly, in contrast with its effects on signaling triggered by other TNFRs, our functional studies revealed that TRAF2 plays a novel inhibitory role in GITR-triggered NF-κB activation.

title: Tumor Necrosis Factor Receptor (TNFR)-associated Factor 5 Is a Critical Intermediate of Costimulatory Signaling Pathways Triggered by Glucocorticoid-induced TNFR in T Cells*
abstract: Tumor necrosis factor receptor (TNFR) family members such as glucocorticoid-induced TNFR (GITR) control T cell activation, differentiation, and effector functions. Importantly, GITR functions as a pivotal regulator of physiologic and pathologic immune responses by abrogating the suppressive effects of T regulatory cells and costimulating T effector cells. However, the molecular mechanisms underlying GITR-triggered signal transduction pathways remain unclear. Interestingly, GITR-induced stimulation of TNFR-associated factor (TRAF) 5-deficient T cells resulted in decreased activation of nuclear factor κB as well as the mitogen-activated protein kinases p38 and extracellular signal-regulated protein kinase, whereas activation of c-Jun N-terminal kinase was less affected. Consistent with impaired signaling, costimulatory effects of GITR were diminished in TRAF5-/- T cells. In sum, our studies indicate that TRAF5 plays a crucial role in GITR-induced signaling pathways that augment T cell activation.

title: CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein.
abstract: Members of the tumor necrosis factor receptor (TNFR) superfamily are important for cell growth and survival. In addition to providing costimulatory signals for cell proliferation, ligation of both TNFR1 and Fas can result in programmed cell death or apoptosis. The underlying mechanism requires an intact 80-aa stretch present in the cytoplasmic tails of both TNFR1 and Fas, termed the death domain (DD). Here we show that CD27, a member of the TNFR family, expressed on discrete subpopulations of T and B cells and known to provide costimulatory signals for T and B cell proliferation and B cell Ig production, can also induce apoptosis. Co-crosslinking of surface Ig receptors along with ligation of CD27 augments CD27-mediated apoptosis.  TNFR1 and Fas, the cytoplasmic tail of CD27 is relatively short and lacks the DD. Using the yeast two-hybrid system, we have cloned a novel protein (Siva) that binds to the CD27 cytoplasmic tail. It has a DD homology region, a box-B-like ring finger, and a zinc finger-like domain. Overexpression of Siva in various cell lines induces apoptosis, suggesting an important role for Siva in the CD27-transduced apoptotic pathway.

title: Siva-1 and an Alternative Splice Form Lacking the Death Domain, Siva-2, Similarly Induce Apoptosis in T Lymphocytes via a Caspase-Dependent Mitochondrial Pathway1
abstract: Siva-1 is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27 and of the glucocorticoid-induced TNFR family-related gene, which are two members of the TNFR family expressed on lymphoid cells. Although Siva-1 expression is up-regulated in multiple pathological processes, little is known about the signaling pathway underlying the Siva-induced apoptosis. In this study, we investigated the mechanism of the proapoptotic activity of Siva-1 and an alternative splice form lacking the death domain of Siva-1, Siva-2, in T lymphocytes in which Siva proteins, CD27, and glucocorticoid-induced TNFR family-related gene are primarily expressed. Overexpression of Siva proteins triggers a typical apoptotic process manifested by cell shrinkage and surface exposure of phosphatidylserine, and confirmed by ultrastructural features. Siva-induced apoptosis is related to the CD27-mediated apoptotic pathway and results in activation of both initiator and effector caspases. This pathway involves a mitochondrial step evidenced by activation of Bid and cytochrome c release, and is modulated by overexpression of Bcl-2 or Bcl-xL. The determinants for Siva-induced apoptosis are not contained within the death domain found in the central part of Siva-1, but rather in both the N-terminal and C-terminal regions shared by both Siva proteins. The N-terminal region also participates in the translocation of both Siva proteins into the nuclear compartment. These results indicate that Siva-1 and Siva-2 mediate apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway that likely involves both cytoplasmic and nuclear events.

title: NFκB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation1
abstract: CD4+CD25+ regulatory T cells (Treg) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on Treg has been shown to be a key player of regulating Treg-mediated suppression. A recent study reports that NF-κB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of Treg but not for its suppression capacity. In this report, we have shown that Treg from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive Treg proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type Treg. Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62Llow Treg both in thymus and periphery than their wild-type littermates. This CD62low subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.

title: Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase.
abstract: The NF-kappaB-inducing kinase (NIK) induces proteolytic processing of NF-kappaB2/p100 and, hence, the generation of NF-kappaB dimers such as p52:RelB but was suggested not to signal for the processing of IkappaB. Here, we show that although the induction of IkappaB degradation in lymphocytes by TNF is independent of NIK, its induction by CD70, CD40 ligand, and BLyS/BAFF, which all also induce NF-kappaB2/p100 processing, does depend on NIK function. Both CD70 and TNF induce recruitment of the IKK kinase complex to their receptors. In the case of CD70, but not TNF, this process is associated with NIK recruitment and is followed by prolonged receptor association of just IKK1 and NIK. Recruitment of the IKK complex to CD27, but not that of NIK, depends on NIK kinase function. Our findings indicate that NIK participates in a unique set of proximal signaling events initiated by specific inducers, which activate both canonical and noncanonical NF-kappaB dimers.

title: The alternative NF-kappaB pathway from biochemistry to biology: pitfalls and promises for future drug development.
abstract: The past two decades have led to a tremendous work on the transcription factor NF-kappaB and its molecular mechanisms of activation. The nuclear translocation of NF-kappaB is controlled by two main pathways: the classical and the alternative NF-kappaB pathways. The classical NF-kappaB pathway activates the IKK complex that controls the inducible degradation of most IkappaB family members that are IkappaBalpha, IkappaBbeta, IkappaBvarepsilon and p105. The alternative NF-kappaB pathway induces p100 processing and p52 generation through the activation of at least two kinases, which are NIK and IKKalpha. Genetic studies have shown that IKKgamma is dispensable for the alternative pathway, which suggests the existence of an alternative IKKalpha-containing complex. It is noteworthy that activation of particular p52 heterodimers like p52/RelB requires solely the alternative pathway while activation of p52/p65 or p52/c-Rel involves a "hybrid pathway". Among others, LTbetaR, BAFF-R, CD40 and RANK have the ability to induce the alternative pathway. The latter plays some roles in biological functions controlled by these receptors, which are the development of secondary lymphoid organs, the proliferation, survival and maturation of B cell, and the osteoclastogenesis. Exacerbated activation of the alternative pathway is potentially associated to a wide range of disorders like rheumatoid arthritis, ulcerative colitis or B cell lymphomas. Therefore, inhibitors of the alternative pathway could be valuable tools for the treatment of inflammatory disorders and cancers.

title: Siva-1 negatively regulates NF-κB activity: effect on T-cell receptor-mediated activation-induced cell death (AICD)
abstract: Ligation of TCRs on stimulated T cells leads to activation-induced cell death (AICD) resulting in the downregulation of immune responses, a process essential for T-cell homeostasis. In this study, using transformed T-cell lines such as Jurkat and Do11.10 as cellular models of TCR-mediated AICD, we have demonstrated that the proapoptotic protein Siva-1 is required for TCR-induced apoptosis. Knockdown of Siva-1 rendered T cells specifically resistant to anti-CD3 but not Fas-induced apoptosis. Further, we observed that in Siva-1 knockout Jurkat cells, TCR-mediated activation of the canonical and non-canonical limbs of the NF-κB pathway are significantly enhanced as reflected by elevated nuclear levels of p65 and RelB, respectively. In addition, loss of endogenous Siva-1 also resulted in the enhanced expression of NF-κB- responsive anti-apoptotic genes such as Bcl-xL and c-FLIP. Interestingly, the c-FLIPshort was detected only in TCR-ligated Siva-1 knockdown Jurkat cells. These results demonstrate a significant role for endogenous Siva-1, through its inhibitory effect on NF-κB activity, in TCR-mediated AICD with implications in peripheral tolerance, T-cell homeostasis and cancer.

title: The gene PC3(TIS21/BTG2), prototype member of the PC3/BTG/TOB family: regulator in control of cell growth, differentiation, and DNA repair?
abstract: PC3(TIS21/BTG2) is the founding member of a family of genes endowed with antiproliferative properties, namely BTG1, ANA/BTG3, PC3B, TOB, and TOB2. PC3 was originally isolated as a gene induced by nerve growth factor during neuronal differentiation of rat PC12 cells, or by TPA in NIH3T3 cells (named TIS21), and is a marker for neuronal birth in vivo. This and other findings suggested its implication in the process of neurogenesis as mediator of the growth arrest before differentiation. Remarkably, its human homolog, named BTG2, was shown to be p53-inducible, in conditions of genotoxic damage. PC3(TIS21/BTG2) impairs G(1)-S progression, either by a Rb-dependent pathway through inhibition of cyclin D1 transcription, or in a Rb-independent fashion by cyclin E downregulation. PC3(TIS21/BTG2) might also control the G(2) checkpoint. Furthermore, PC3(TIS21/BTG2) interacts with carbon catabolite repressor protein-associated factor 1 (CAF-1), a molecule that associates to the yeast transcriptional complex CCR4 and might influence cell cycle, with the transcription factor Hoxb9, and with the protein-arginine methyltransferase 1, that might control transcription through histone methylation. Current evidence suggests a physiological role of PC3(TIS21/BTG2) in the control of cell cycle arrest following DNA damage and other types of cellular stress, or before differentiation of the neuron and other cell types. The molecular function of PC3(TIS21/BTG2) is still unknown, but its ability to modulate cyclin D1 transcription, or to synergize with the transcription factor Hoxb9, suggests that it behaves as a transcriptional co-regulator.

title: The Mammalian Immediate-early TIS21 Protein and the Leukemia-associated BTG1 Protein Interact with a Protein-arginine N-Methyltransferase*
abstract: The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC) gene “PRMT1,” for rotein-aginine ethylransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation.

title: Ordered Cooperative Functions of PRMT1, p300, and CARM1 in Transcriptional Activation by p53
abstract: Transcriptional coactivators that modify histones represent an increasingly important group of regulatory factors, although their ability to modify other factors as well precludes common assumptions that they necessarily act by histone modification. In an extension of previous studies showing a role for acetyltransferase p300/CBP in p53 function, we have used systems reconstituted with recombinant chromatin templates and (co)activators to demonstrate (1) the additional involvement of protein arginine methyltransferases PRMT1 and CARM1 in p53 function; (2) both independent and ordered cooperative functions of p300, PRMT1, and CARM1; and (3) mechanisms that involve direct interactions with p53 and, most importantly, obligatory modifications of corresponding histone substrates. ChIP analyses have confirmed the ordered accumulation of these (and other) coactivators and cognate histone modifications on the GADD45 gene following ectopic p53 expression and/or UV irradiation. These studies thus define diverse cofactor functions, as well as underlying mechanisms involving distinct histone modifications, in p53-dependent gene activation.

title: Dynamics of Human Protein Arginine Methyltransferase 1(PRMT1) in Vivo*
abstract: Arginine methylation is a posttranslational protein modification catalyzed by a family of protein arginine methyltransferases (PRMT), the predominant member of which is PRMT1. Despite its major role in arginine methylation of nuclear proteins, surprisingly little is known about the subcellular localization and dynamics of PRMT1. We show here that only a fraction of PRMT1 is located in the nucleus, but the protein is predominantly cytoplasmic. Fluorescence recovery after photobleaching experiments reveal that PRMT1 is highly mobile both in the cytoplasm and the nucleus. However, inhibition of methylation leads to a significant nuclear accumulation of PRMT1, concomitant with the appearance of an immobile fraction of the protein in the nucleus, but not the cytoplasm. Both the accumulation and immobility of PRMT1 is reversed when re-methylation is allowed, suggesting a mechanism where PRMT1 is trapped by unmethylated substrates such as core histones and heterogeneous nuclear ribonucleoprotein proteins until it has executed the methylation reaction.

title: Protein methyltransferase 2 inhibits NF-kappaB function and promotes apoptosis.
abstract: The protein arginine methyltransferases (PRMTs) include a family of proteins with related putative methyltransferase domains that modify chromatin and regulate cellular transcription. Although some family members, PRMT1 and PRMT4, have been implicated in transcriptional modulation or intracellular signaling, the roles of other PRMTs in diverse cellular processes have not been fully established. Here, we report that PRMT2 inhibits NF-kappaB-dependent transcription and promotes apoptosis. PRMT2 exerted this effect by blocking nuclear export of IkappaB-alpha through a leptomycin-sensitive pathway, increasing nuclear IkappaB-alpha and decreasing NF-kappaB DNA binding. The highly conserved S-adenosylmethionine-binding domain of PRMT2 mediated this effect. PRMT2 also rendered cells susceptible to apoptosis by cytokines or cytotoxic drugs, likely due to its effects on NF-kappaB. Mouse embryo fibroblasts from PRMT2 genetic knockouts showed elevated NF-kappaB activity and decreased susceptibility to apoptosis compared to wild-type or complemented cells. Taken together, these data suggest that PRMT2 inhibits cell activation and promotes programmed cell death through this NF-kappaB-dependent mechanism.

title: Effect of Shear Stress on Asymmetric Dimethylarginine Release From Vascular Endothelial Cells
abstract: We demonstrated recently that plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are increased by high salt intake concomitantly with a decrease in plasma levels of NO in human hypertension. We investigated the effect of shear stress on ADMA release in 2 types of cells: transformed human umbilical vein endothelial cells (HUVECs; cell line ECV-304) and HUVECs. Exposure of ECV-304 cells and HUVECs to shear stress with the use of a cone-plate viscometer enhanced gene expression of protein arginine methyltransferase (PRMT-1), ADMA synthase. In HUVECs, the ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased by 2-fold by a shear stress of ≥15 dyne/cm2. A dominant-negative mutant of I&kgr;B kinase &agr; and troglitazone at 8 &mgr;mol/L, an activator of peroxisome proliferator–activated receptor &ggr;, abolished the shear stress–induced increase in PRMT-1 gene expression in parallel with the blockade of nuclear factor (NF)-&kgr;B translocation into the nucleus. The activity of dimethylarginine dimethylaminohydrolase, the degradation enzyme of ADMA, was unchanged after shear stress ≤15 dyne/cm2 and was enhanced by 1.48±0.06-fold (P <0.05) by shear stress at 25 dyne/cm2. The release of ADMA was increased by 1.64±0.10-fold (P <0.05) by shear stress at 15 dyne/cm2 but was not affected by shear stress at 25 dyne/cm2. These results indicate that shear stress enhances gene expression of PRMT-1 and ADMA release via activation of the NF-&kgr;B pathway. Shear stress at higher magnitudes facilitates the degradation of ADMA, thus returning ADMA release levels to baseline.

title: Tumor Necrosis Factor Receptor–Associated Factor (Traf)2 Represses the T Helper Cell Type 2 Response through Interaction with Nfat-Interacting Protein (Nip45)
abstract: Recently we have identified a novel protein NIP45 (nuclear factor of activated T cells [NFAT]-interacting protein) which substantially augments interleukin (IL)-4 gene transcription. The provision of NIP45 together with NFAT and the T helper cell type 2 (Th2)-specific transcription factor c-Maf to cells normally refractory to IL-4 production, such as B cells or Th1 clones, results in substantial IL-4 secretion to levels that approximate those produced by primary Th2 cells. In studies designed to further our understanding of NIP45 activity, we have uncovered a novel facet of IL-4 gene regulation. We present evidence that members of the tumor necrosis factor receptor–associated factor (TRAF) family of proteins, generally known to function as adapter proteins that transduce signals from the tumor necrosis factor receptor superfamily, contribute to the repression of IL-4 gene transcription and that this effect is mediated through their interaction with NIP45.

title: TNF Receptor-Associated Factor 5 Limits the Induction of Th2 Immune Responses1
abstract: The TNF receptor-associated factor (TRAF) family of molecules acts as adapter proteins for signaling pathways initiated by several members of the TNF receptor (TNFR) superfamily. TRAF5−/− animals are viable and have normal development of the immune system despite interacting with several TNFR family members. A clear role for TRAF5 has yet to emerge. OX40 (CD134) interacts with TRAF5, suggesting that this pathway could be involved in regulating T cell differentiation into Th1 or Th2 cells. In tissue culture, OX40 stimulation of TRAF5−/− T cells resulted in a pronounced Th2 phenotype with elevated levels of IL-4 and IL-5. Similarly, in vivo immunization with protein in adjuvant in the presence of an agonist anti-OX40 Ab resulted in enhanced Th2 development in TRAF5−/− mice. Additionally, lung inflammation induced by T cells, which is critically controlled by OX40, was more pronounced in TRAF5−/− mice, characterized by higher levels of Th2 cytokines. These results suggest that TRAF5 can limit the induction of Th2 responses, and that TRAF5 can play a role in modulating responses driven by OX40 costimulation.