Giuseppe Nocentini

GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations

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.

Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-κB–dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4+ T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.

GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily.

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.

GITR/GITRL: more than an effector T cell co-stimulatory system.

Glucocorticoid‐induced TNFR‐related protein (GITR) is a member of the TNFR superfamily, expressed in several cells and tissues including T lymphocytes, NK cells and antigen‐presenting cells (APC). GITR activation, upon interaction with its ligand (GITRL), functions as a co‐activating signal. GITRL is mainly expressed on APC and GITR/GITRL interaction is important for the development of immune response. This review summarizes recent results about the GITR/GITRL system, focusing on the interplay between APC, effector and regulatory T cells.

Balance between Regulatory T and Th17 Cells in Systemic Lupus Erythematosus: The Old and the New

Pathogenic mechanisms underlying the development of systemic lupus erythematosus (SLE) are very complex and not yet entirely clarified. However, the pivotal role of T lymphocytes in the induction and perpetuation of aberrant immune response is well established. Among T cells, IL-17 producing T helper (Th17) cells and regulatory T (Treg) cells represent an intriguing issue to be addressed in SLE pathogenesis, since an imbalance between the two subsets has been observed in the course of the disease. Treg cells appear to be impaired and therefore unable to counteract autoreactive T lymphocytes. Conversely, Th17 cells accumulate in target organs contributing to local IL-17 production and eventually tissue damage. In this setting, targeting Treg/Th17 balance for therapeutic purposes may represent an intriguing and useful tool for SLE treatment in the next future. In this paper, the current knowledge about Treg and Th17 cells interplay in SLE will be discussed.

GITR interacts with the pro-apoptotic protein Siva and induces apoptosis.

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) (Figure 1A). 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. Results in 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 (see Figure 1A). 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 Nature Publishing Group All rights reserved 1350-9047/02

Mechanisms of the anti-inflammatory effects of glucocorticoids: genomic and nongenomic interference with MAPK signaling pathways

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GITR: a modulator of immune response and inflammation.

Glucocorticoids (GCs) are steroid hormones produced by the adrenal gland and regulated by the hypothalamus‐pituitary‐adrenal axis. GCs mediate effects that mostly result in transcriptional regulation of glucocorticoid receptor target genes. Mitogen‐activated protein kinases (MAPKs) comprise a family of signaling proteins that convert extracellular stimuli into the activation of intracellular transduction pathways via phosphorylation of a cascade of substrates. They modulate a variety of physiological cell processes, such as proliferation, apoptosis, and development. However, when MAPKs are improperly activated by proinflammatory and/or extracellular stress stimuli, they contribute to the regulation of proinflammatory transcription factors, thus perpetuating activation of the inflammatory cascade. One of the mechanisms by which GCs exert their anti‐inflammatory effects is negative interference with MAPK signaling pathways. Several functional interactions between GCs and MAPK signaling have been discovered and studied. Some of these interactions involve the GC‐mediated up‐regulation of proteins that in turn interfere with the activation of MAPK, such as glucocorticoid‐induced‐leucine zipper, MAPK phosphatase‐1, and annexin‐1. Other mechanisms include activated GR directly interacting with components of the MAPK pathway and negatively regulating their activation. The multiple interactions between GCs and MAPK pathways and their potential biological relevance in mediating the anti‐inflammatory effects of GCs are reviewed.—Ayroldi, E., Cannarile, L., Migliorati, G., Nocentini, G., Delfino, D. V., Riccardi, C. Mechanisms of the anti‐inflammatory effects of glucocorticoids: genomic and nongenomic interference with MAPK signaling pathways. FASEB J. 26, 4805–4820 (2012). www.fasebj.org

Glucocorticoid-Induced TNFR-Related Protein Lowers the Threshold of CD28 Costimulation in CD8+ T Cells

Glucocorticoid-Induced TNFR-Related (GITR) protein belongs to Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and stimulates both the acquired and innate immunity. It is expressed in several cells and tissues, including T and Natural Killer (NK) cells and is activated by its ligand, GITRL, mainly expressed on Antigen Presenting Cells (APCs) and endothelial cells. GITR/GITRL system participates in the development of autoimmune/inflammatory responses and graft vs. host disease and potentiates response to infection and tumors. These effects are due to several concurrent mechanisms including: co-activation of effector T-cells, inhibition of regulatory T (Treg) cells, NK-cell co-activation, activation of macrophages, modulation of DC function and regulation of the extravasation process. In this chapter we describe: 1) the main structural features of GITR and GITRL, 2) the transduction pathways activated by GITR triggering, 3) the effects derived from GITR/GITRL system interaction, considering the interplay between the different cells of the immune system. Moreover, the potential use of GITR/GITRL modulators in disease treatment is discussed.

Dietary alpha-linolenic acid reduces COX-2 expression and induces apoptosis of hepatoma cells.

CD28 is well characterized as a costimulatory molecule in T cell activation. Recent evidences indicate that TNFR superfamily members, including glucocorticoid-induced TNFR-related protein (GITR), act as costimulatory molecules. In this study, the relationship between GITR and CD28 has been investigated in murine CD8+ T cells. When suboptimal doses of anti-CD3 Ab were used, the absence of GITR lowered CD28-induced activation in these cells whereas the lack of CD28 did not affect the response of CD8+ T cells to GITR costimulus. In fact, costimulation of CD28 in anti-CD3-activated GITR−/− CD8+ T cells resulted in an impaired increase of proliferation, impaired protection from apoptosis, and an impaired rise of activation molecules such as IL-2R, IL-2, and IFN-γ. Most notably, CD28-costimulated GITR−/− CD8+ T cells revealed lower NF-κB activation. As a consequence, up-regulation of Bcl-xL, one of the major target proteins of CD28-dependent NF-κB activation, was defective in costimulated GITR−/− CD8+ T cells. What contributed to the response to CD28 ligation in CD8+ T cells was the early up-regulation of GITR ligand on the same cells, the effect of which was blocked by the addition of a recombinant GITR-Fc protein. Our results indicate that GITR influences CD8+ T cell response to CD28 costimulation, lowering the threshold of CD8+ T cell activation.