Simona Ronchetti

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 Tu2004lymphocytes, 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.

Role of glucocorticoid-induced TNF receptor family gene (GITR) in collagen-induced arthritis

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)

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

Pharmacological modulation of GITRL/GITR system: therapeutic perspectives

Glucocorticoid‐induced TNFR‐related (gitr) is a gene coding for a member of the TNF receptor superfamily. GITR activation by its ligand (GITRL) influences the activity of effector and regulatory T cells, thus participating in the development of immune response against tumours and infectious agents, as well as in autoimmune and inflammatory diseases. Notably, treating animals with GITR‐Fc fusion protein ameliorates autoimmune/inflammatory diseases while GITR triggering, by treatment with anti‐GITR mAb, is effective in treating viral, bacterial and parasitic infections, as well in boosting immune response against tumours. GITR modulation has been indicated as one of the top 25 most promising research areas by the American National Cancer Institute, and a clinical trial testing the efficacy of an anti‐GITR mAb in melanoma patients has been started. In this review, we summarize results regarding: (i) the mechanisms by which GITRL/GITR system modulates immune response; (ii) the structural and functional studies clearly demonstrating differences between GITRL/GITR systems of mice and humans; (iii) the molecules with pharmacological activities including anti‐GITR mAbs, GITR‐Fc and GITRL‐Fc fusion proteins, GITRL in monomer or multimer conformation; and (iv) the possible risks deriving from GITRL/GITR system pharmacological modulation. In conclusion, GITR triggering and inhibition could be useful in treating tumours, infectious diseases, as well as autoimmune and inflammatory diseases. However, differences between mouse and human GITRL/GITR systems suggest that further preclinical studies are needed to better understand how safe therapeutic results can be obtained and to design appropriate clinical trials.

Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation

Glucocorticoid-induced TNFR-related gene (GITR) participates in the immune/inflammatory response. Because GITR expression has been described in cells other than T lymphocytes, we investigated whether it also modulates acute inflammatory response. Using GITR-deficient (GITR−/−) mice, we analyzed the role of GITR in the development of carrageenan-induced lung inflammation (pleurisy) by studying several proinflammatory markers 2–8 h after carrageenan injection. When compared with GITR+/+, GITR−/− mice exhibited decreased production of turbid exudate containing a lower number of leukocytes. This was correlated with the reduction of inflammatory markers (including TNF-α, IL-1β, myeloperoxidase, inducible NO synthase, and cyclooxygenase 2) in the pleural exudate and/or in the lung. Moreover, endothelial cells expressed lower levels of adhesion molecules. In lungs of GITR+/+ mice, GITR ligand expression was not modulated during pleurisy, while that of GITR increased, as a consequence of increased infiltration by GITR-expressing cells and of GITR up-regulation in macrophages and endothelial cells. Finally, cotreatment of GITR+/+ mice with carrageenan and Fc-GITR fusion protein decreased the number of inflammatory cells (pleural macrophages and lung neutrophils) as compared with carrageenan treatment alone, confirming that GITR plays a role in the modulation of pleurisy.

CD4+CD25lowGITR+ cells: A novel human CD4+ T-cell population with regulatory activity

Treg subsets play a role in sustaining peripheral tolerance, are characterized by markers such as forkhead winged‐helix transcription factor (FOXP3) and CD25, and produce suppressive cytokines, such as IL‐10 and TGF‐β. Glucocorticoid‐induced TNF receptor family‐related (GITR) protein has been suggested to regulate Treg activity in mice. The aim of our study was to investigate GITR expression in human CD4+ T lymphocytes and its possible role in Treg function. Results indicate that a subset of CD4+ T cells in the peripheral blood expresses GITR and low levels of CD25 (CD4+CD25lowGITR+). These cells show Treg features as they express FOXP3, IL‐10, TGF‐β and are anergic but, as opposed to natural Tregs, express low levels of CTLA‐4 and are CD127high. CD4+CD25lowGITR+ cells represent a low percentage of the CD4+ T‐cell population (0.32–1.74%) and are mostly memory cells. Functional experiments demonstrated that CD4+CD25lowGITR+ cells have relevant suppressive activity that depends on TGF‐β. Moreover, an anti‐GITR Ab inhibited their suppressive activity, as observed in CD4+CD25+ murine Tregs. Taken together, these data indicate that human CD4+CD25lowGITR+ cells represent a distinct Treg subpopulation.

Glucocorticoid‐induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock

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.

Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation

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)

Glucocorticoid-Induced Tumour Necrosis Factor Receptor-Related Protein: A Key Marker of Functional Regulatory T Cells

Glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR, TNFRSF18, and CD357) is expressed at high levels in activated T cells and regulatory T cells (Tregs). In this review, we present data from mouse and human studies suggesting that GITR is a crucial player in the differentiation of thymic Tregs (tTregs), and expansion of both tTregs and peripheral Tregs (pTregs). The role of GITR in Treg expansion is confirmed by the association of GITR expression with markers of memory T cells. In this context, it is not surprising that GITR appears to be a marker of active Tregs, as suggested by the association of GITR expression with other markers of Treg activation or cytokines with suppressive activity (e.g., IL-10 and TGF-β), the presence of GITR+ cells in tissues where Tregs are active (e.g., solid tumours), or functional studies on Tregs. Furthermore, some Treg subsets including Tr1 cells express either low or no classical Treg markers (e.g., FoxP3 and CD25) and do express GITR. Therefore, when evaluating changes in the number of Tregs in human diseases, GITR expression must be evaluated. Moreover, GITR should be considered as a marker for isolating Tregs.

GITR+ regulatory T cells in the treatment of autoimmune diseases

Autoimmune diseases decrease life expectancy and quality of life for millions of women and men. Although treatments can slow disease progression and improve quality of life, all currently available drugs have adverse effects and none of them are curative; therefore, requiring patients to take immunosuppressive drugs for the remainder of their lives. A curative therapy that is safe and effective is urgently needed. We believe that therapies promoting the in vivo expansion of regulatory T cells (Tregs) or injection of in vitro expanded autologous/heterologous Tregs (cellular therapy) can alter the natural history of autoimmune diseases. In this review, we present data from murine and human studies suggesting that 1) glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) plays a crucial role in thymic Treg (tTreg) differentiation and expansion; 2) GITR plays a crucial role in peripheral Treg (pTreg) expansion; 3) in patients with Sjögren syndrome and systemic lupus erythematosus, CD4(+)GITR(+) pTregs are expanded in patients with milder forms of the disease; and 4) GITR is superior to other cell surface markers to differentiate Tregs from other CD4(+) T cells. In this context, we consider two potential new approaches for treating autoimmune diseases consisting of the in vivo expansion of GITR(+) Tregs by GITR-triggering drugs and in vitro expansion of autologous or heterologous GITR(+) Tregs to be infused in patients. Advantages of such an approach, technical problems, and safety issues are discussed.