Glucocorticoid-induced TNFR-related Protein Research Articles

EPA-0919 – Imbalance between glucocorticoid-induced tnfr-related protein (gitr) and il-2 in depressed copd patients modulate immunological self-tolerance mediated by treg cells and may explain amplified systemic inflammation

Mounting evidence underscores the important role of immunological and immunopathological processes in depression. Alterations in immune function secondary to increased psychological stress including enhanced secretion of proinflammatory cytokines affect the brain and elicits various symptoms of depression also in patients suffering from chronic inflammatory conditions including chronic obstructive pulmonary disease (COPD). However, mechanisms underlying increased immune response/decreased immune tolerance in people developing depression including COPD patients are largely unknown. The aim of this study was to explore the relationship between markers of systemic inflammation and depression. We hypothesized that the plausible factor contributing to amplified systemic inflammation in depressed patients is glucocorticoid-induced TNF receptor (GITR). Blood was collected from patients diagnosed with chronic obstructive pulmonary disease and comorbid depressive symptoms (COPD+DS, (N=13), individuals with either COPD (N=16) or recurrent depressive disorder (rDD) alone (N=15) and from healthy controls (N=19). Levels of IL-2, IL-6, IL-8, IFN-g, TNF-a, IL-17 and GITR were detected with ELISA. Surface phenotype expression of T regulatory and T effector cells was analysed with a flow cytometery. We observedthat COPD, depression and COPD with comorbid depression are associated with increased IL-6 levels when compared with healthy controls 42.2±1.87 pg/mL, 41.9±1.51 pg/mL, 41.7 ±1.31 and 34.7±1.36 pg/mL, respectively (p<0.05). Concentrations of IFN-g were significantly increased in COPD+DS patients when compared with controls (24.3±1.49 pg/mL and 17.8±0.70 pg/mL, respectively, p<0.05). IL-2 levels were highest in COPD+DS (3.20±0.389 pg/mL) and differed significantly when this group was compared with controls (2.30±0.176 pg/mL), p£0.05. GITR was significantly higher in COPD patients (0.332±0.04849) when compared with depressed (0.183±0.0492), when compared with COPD +depressive symptoms (0.194± 0.0357) and also when compared with controls (0.1601±0.0191), all p<0.05. We observed strong significant positive correlation between GITR and IL-2 and also between GITR and IFN-g in the study groups (p<0.001). Further, we checked the ratios of IL- 2/GITR and IFN-g /GITR. We observed significantly increased IL-2/GITR ratio in depressed groups (p<0.01). We also observed strong positive correlation between IL-2/GITR and Treg/Teffector (p<0.001). In this study we identified GITR and IL-2 as putative inflammatory agents associated with depressive symptoms in COPD patients. Furthermore our study revealed that alterations of IL-2/GITR balance modulate immunological self-tolerance mediated by Treg cells. Our observations suggest that modulation of IL-2/GITR and its effects on T cell population(s)are putative immune pathways contributing to systemic inflammation in depression.

Reduced Effectiveness of CD4+Foxp3+ Regulatory T Cells in CD28-Deficient NOD.H-2h4 Mice Leads to Increased Severity of Spontaneous Autoimmune Thyroiditis

NOD.H-2h4 mice given NaI in their drinking water develop iodine-accelerated spontaneous autoimmune thyroiditis (ISAT) with chronic inflammation of the thyroid by T and B cells and production of anti-mouse thyroglobulin (MTg) autoantibody. CD28(-/-) NOD.H-2h4 mice, which have reduced numbers of CD4(+)Foxp3(+) regulatory T cells (Tregs), were developed to examine the role of Tregs in ISAT development. CD28(-/-) NOD.H2-h4 mice develop more severe ISAT than do wild-type (WT) mice, with collagen deposition (fibrosis) and low serum T4. CD28(-/-) mice have increased expression of proinflammatory cytokines IFN-γ and IL-6, consistent with increased mononuclear cell infiltration and tissue destruction in thyroids. Importantly, transferring purified CD4(+)Foxp3(+) Tregs from WT mice reduces ISAT severity in CD28(-/-) mice without increasing the total number of Tregs, suggesting that endogenous Tregs in CD28(-/-) mice are functionally ineffective. Endogenous CD28(-/-) Tregs have reduced surface expression of CD27, TNFR2 p75, and glucocorticoid-induced TNFR-related protein compared with transferred CD28(+/+) Tregs. Although anti-MTg autoantibody levels generally correlate with ISAT severity scores in WT mice, CD28(-/-) mice have lower anti-MTg autoantibody responses than do WT mice. The percentages of follicular B cells are decreased and those of marginal zone B cells are increased in spleens of CD28(-/-) mice, and they have fewer thyroid-infiltrating B cells than do WT mice. This suggests that CD28 deficiency has direct and indirect effects on the B cell compartment. B cell-deficient (B(-/-)) NOD.H-2h4 mice are resistant to ISAT, but CD28(-/-)B(-/-) mice develop ISAT comparable to WT mice and have reduced numbers of Tregs compared with WT B(-/-) mice.

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.

Mechanistic investigation of immunosuppression in patients with condyloma acuminata

Condyloma acuminatum (CA) is a common sexually transmitted disease caused by human papillomavirus (HPV) infection. Previous studies have identified that the occurrence, relapse and cancerization of CA is relevant to immune imbalance caused by immune hypofunction or immunoregulatory dysfunction. However, to date, the specific mechanisms accounting for immune imbalance in CA patients have remained elusive. In the present study, changes in the expression levels of myeloid differentiation factor 88 (MyD88) and toll‑like receptors (TLRs) were determined in lesion tissues and peripheral blood samples obtained from CA patients by fluorescence quantitative PCR and western blot analysis. The results indicated that TLRs and MyD88 expression was upregulated in the lesion tissues only. In addition, the expression of forkhead box P3, a characteristic marker of regulatory T cells (Tregs), transforming growth factor‑β1 and interleukin (IL)‑10, inhibitory factors secreted by Tregs and inhibitory costimulatory molecules, cytotoxic T-lymphocyte antigen 4, glucocorticoid-induced TNFR-related protein and programmed cell death protein 1 was observed to be upregulated, indicating that immunosuppression of Tregs was enhanced significantly. However, the expression levels of NKG2D and NKp46, natural killer (NK) cell activation receptors located on the surface of NK cells, decreased markedly indicating that HPV infection inhibits the activation of NK cells. The secretion levels of various cytokines in the peripheral blood of CA patients were detected by enzyme‑linked immunosorbent assay revealing that IL‑2, IL‑12 and interferon‑γ levels were markedly lower than that of healthy subjects. By contrast, the expression levels of tumor necrosis factor‑α, IL‑4 and IL‑10 were markedly increased in CA samples compared with the control, with the exception of IL‑6. Taken together, these results are consistent with the hypothesis of immunosuppression in CA patients. Increased expression of MyD88 and TLRs is likely to enhance immunosuppression of Tregs, leading to the imbalance of Th1/Th2, cytotoxic T cell type 1 (Tc1)/Tc2 cells and secreted cytokines.

AB0415 High expression of gitr in serum and labial salivary glands from patients with primary sjögren’s syndrome

ObjectivesGlucocorticoid-induced TNFR-related (GITR) is a gene coding for a member of the TNF receptor superfamily. After activated by its ligand GITRL, GITR could influence the activity of effector and regulatory...

A role for the GITR pathway in anaphylaxis (P4326)

Abstract Immune modulation using monoclonal antibodies has a significant impact on the overall survival of patients with metastatic melanoma. Monoclonal antibodies that engage members of the tumor necrosis factor receptor (TNFR) superfamily have shown promising tumor protection in preclinical models. Glucocorticoid-induced TNFR-related protein (GITR) is a particularly potent target for tumor immunotherapy. A single dose of GITR agonist antibody results in 60% protection from established B16 melanoma tumors. Therefore, several doses of anti-GITR may be necessary in order to fully eradicate established tumors. Because GITR is expressed throughout the immune system, we investigated whether multiple doses of anti-GITR result in adverse immune-related events. We observed that three doses of anti-GITR cause anaphylaxis, a side effect that has not been reported using this or other TNFR superfamily agonist antibodies. Anaphylaxis was not observed upon the first or second doses of anti-GITR or in GITR knockout mice. Anti-GITR-induced anaphylaxis is associated with a severe drop in body temperature and elevated serum proinflammatory cytokine levels. We are further investigating the causes of toxicity resulting from repetitive anti-GITR treatment and whether this effect can be reversed or prevented while preserving the antitumor activity of anti-GITR, an important step given the translational potential of this therapy.

GITR-Dependent Regulation of 4-1BB Expression: Implications for T Cell Memory and Anti–4-1BB–Induced Pathology

The TNFR family member 4-1BB plays a key role in the survival of activated and memory CD8 T cells. However, the mechanisms that regulate 4-1BB re-expression on memory CD8 T cells after Ag clearance are unknown. In unimmunized mice, ∼10% of CD8 CD44(hi) memory T cells in the bone marrow (BM) and liver express 4-1BB, with minimal 4-1BB expression in spleen and lymph node. IL-2, IL-15, and IL-7 are collectively dispensable for 4-1BB expression on the memory CD8 T cells. Rather, T cell-intrinsic glucocorticoid-induced TNFR-related protein (GITR) contributes to 4-1BB expression on CD8 T cells upon their entry into the BM or liver. Consistent with its role in regulation of 4-1BB, GITR is required on memory CD8 T cells for their persistence in vivo. These findings reveal site-specific effects of the BM and liver microenvironment on CD8 memory T cells. Previous work has demonstrated that 4-1BB agonists given to unimmunized mice induce splenomegaly, hepatitis, and other immune system anomalies. Moreover, severe liver pathology has been observed in a subset of anti-4-1BB-treated melanoma patients. Remarkably, the absence of GITR in mice almost completely abrogates cellular expansions, splenomegaly, and liver inflammation associated with anti-4-1BB agonist treatment of unimmunized mice. In contrast, lack of CD8 T cells selectively improves liver pathology, but not splenomegaly in the mice. Thus, the regulation of 4-1BB expression by GITR on CD8 T cells, as well as on other cells, contributes to the pathological effects of anti-4-1BB in unimmunized mice.

Glucocorticoid-induced TNFR-related protein stimulation reverses cardiac allograft acceptance induced by CD40-CD40L blockade.

CD40-CD40L blockade has potent immunosuppressive effects in cardiac allograft rejection but is less effective in the presence of inflammatory signals. To better understand the factors that mediate CD40-CD40L blockade-resistant rejection, we studied the effects of stimulation through glucocorticoid-induced TNFR-related protein (GITR), a costimulatory protein expressed by regulatory and effector T cells. Stimulation of CD40−/− or wild-type recipient mice treated with anti-CD40L mAb (WT+anti-CD40L) and with agonistic anti-GITR mAb resulted in cardiac allograft rejection. GITR stimulation did not induce rejection once long-term graft acceptance was established. In vitro, GITR stimulation increased proliferation of effector T cells and decreased regulatory T cell (Treg) differentiation in both treatment groups. GITR-stimulated CD40−/− recipients rejected their allografts more rapidly compared to GITR-stimulated WT+anti-CD40L recipients, and this rejection, characterized by a robust Th2 response and significant eosinophilic infiltrate, could be mediated by CD4+ T cells alone. In contrast, both CD4+ and CD8+ T cells were required to induce rejection in GITR-stimulated WT+anti-CD40L-treated recipients, and the pathology of rejection was less severe. Hence, early GITR stimulation could initiate graft rejection despite CD40 deficiency or anti-CD40L mAb treatment, though the recipient response was dependent on the mechanism of CD40-CD40L disruption.

Alloreactivity-Based Medical Conditions

Alloreactivity is a response of the immune system to individual antigenic differences within species. These responses in general occur following exposure to alloantigens as a consequence of medical intervention (such as transfusion or tissue transplantation) or during pregnancy. Responses to alloantigens form a basis of a broad spectrum of medical conditions, such as graft rejection, graft versus host disease, reaction to blood (products), or biopharmaceuticals and fetal and neonatal diseases (thrombocytopenia, hemolytic anemia, hemochromatosis, biliary atresia, and glomerulopathy). On the other hand, normal pregnancies are often accompanied by strongly tolerogenic responses to fetal alloantigens. Diverse immunologic components, including T cells, antibodies (B cells), and NK cells, promote alloreactivity, and this complexity makes the pathogenesis and tolerogenesis of these conditions distinct and unique. This special issue contains seven manuscripts touching on various aspects of alloreactivity in medicine. Blocking CD40-CD40L interaction can induce acceptance of cardiac allografts. However, the blockade is less efficient under inflammatory conditions. In “Glucocorticoid-induced TNFR-related protein reverses cardiac allograft acceptance induced by CD40-CD40L blockade,” Krill et al. demonstrate that stimulation of GITR (a cell surface molecule displayed by effector and regulatory T cells) can initiate cardiac graft rejection irrespective of the presence or absence of CD40-CD40L blockade, defining thus at least one possible molecular mechanism of resistance to CD40-CD40L suppression. The balance between the effector and regulatory T cells is critical for development of graft-specific immune responses in general. Franzese et al. review the usefulness of this balance in predicting or establishing the diagnosis of graft rejection in “Regulatory T cells in the immunodiagnosis and outcome of kidney allograft rejection.” Allogeneic hematopoietic stem cell transplantation is used for treatment of autoimmune diseases, multiple organ transplantation, consequences of supralethal irradiation and advanced malignancies and other serious conditions refractory to conventional methods. In “New allogeneic hematopoietic cell transplantation method: hematopoietic cell transplantation plus thymus transplantation for intractable diseases,” Hosaka demonstrates that allogeneic hematopoietic stem cell transplantation combined with the same donor thymus transplantation has a superior effect producing more efficient T cell function and reduced graft versus host disease. Redzovic et al., in “Mucins help to avoid alloreactivity at the maternal fetal interface,” review roles of mucin-1 and tumor associated glycoprotein-72 in regulating immune responses. Parallels are drawn between the ability of these molecules to promote tumor growth, invasion, and metastasis and to prevent trophoblast invasion. Removing glycoproteins TAG-72 and Muc 1 from the eutopic implantation site likely contributes to better control of trophoblast invasion by T cells and NK cells. This enables tolerance to paternal antigens of the fetus and normal course and outcome of the pregnancy. Three manuscripts explore distinct aspects of alloantibody formation. Preformed donor HLA class I-specific antibodies are a risk factor for rejection of kidney, heart, and lung grafts, but the role of these antibodies is less clear in liver transplantation, where rejection rates are lower. Yoshizawa et al., in “Significance of semiquantitative assessment of preformed donor-specific antibody using luminex single bead assay in living related liver transplantation,” describe a quantitative assay for donor specific antibodies that has a predictive value for living donor liver transplantation. In “Nuclear antigens and auto/alloantibody responses: friend or foe in transplant immunology,” Nakano et al. review current thinking about the role of nuclear antigens and antibodies specific for nuclear antigens in initiation and regulation of immune responses and graft rejection. Finally, transfusion of allogeneic red blood cells may in some recipients induce alloantibodies. Tatari-Calderone et al. in “The association of CD81 polymorphisms with alloimmunization in sickle cell disease” suggest that genetic elements in the gene encoding B-cell molecule CD81 may be predictive markers of alloimmunization. The huge breadth of the field of alloreactivity-based medical conditions allows the special issue only to scratch under the surface of any specific topic. Our hope, however, is that this issue will help raise awareness of the immunological basis of these conditions and will foster further efforts to better understand their pathogenesis. Ultimately, this should help us design better treatments or prevent the development of these medical conditions altogether.

Serum Autoantibodies: From Identification to Clinical Relevance

Serum Autoantibodies: From Identification to Clinical Relevance

Expansion of CD4+CD25-GITR+ regulatory T-cell subset in the peripheral blood of patients with primary Sjögren's syndrome: correlation with disease activity

CD4+CD25high regulatory T cells (TREG) represent a suppressive T cell subset deeply involved in the modulation of immune responses and eventually in the prevention of autoimmunity. Growing evidence demonstrated that patients with autoimmune and inflammatory chronic diseases display an impairment of TREG cells or activated effector T cells unresponsive to TREG. Glucocorticoid-induced TNFR-related protein (GITR) is a widely accepted marker of murine TREG cells, but little is known in humans. Aim of the present study was to investigate the characteristics of different subsets of TREG cells in Sjögren's syndrome and the potential role of GITR as marker of human TREG cells. Fifteen patients with primary Sjogren's syndrome (SS) and 10 sex- and age-matched normal controls (NC) were enrolled. CD4+ T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies. Disease activity was assessed using the EULAR Sjögren's syndrome disease activity index (ESSDAI). Although the proportion of circulating CD25highGITRhigh subset was similar in SS patients and NC, an expansion of the CD25-GITRhigh cell population was observed in the peripheral blood of SS patients. Interestingly, this expansion was more relevant in patients with inactive rather than active disease. The number of CD4+CD25-GITRhigh cells is increased in SS as compared to NC. Moreover, the fact that the expansion of this cell subset is prevalently observed in patients with inactive disease suggests that these cells may play a role in counteracting inflammatory response.

IL-15–Dependent Upregulation of GITR on CD8 Memory Phenotype T Cells in the Bone Marrow Relative to Spleen and Lymph Node Suggests the Bone Marrow as a Site of Superior Bioavailability of IL-15

CD8 memory T cells are enriched in the bone marrow, a site where these cells are thought to receive homeostatic signals. However, the primary site where CD8 memory T cells receive their cytokine-induced homeostatic signals has recently come under debate. In this study, we demonstrate that the bone marrow contains a fraction of CD8 memory phenotype T cells with elevated expression of glucocorticoid-induced TNFR-related protein (GITR). In contrast, splenic and lymph node memory phenotype T cells have GITR levels similar to those on naive T cells. The bone marrow GITR(hi) memory T cells have a phenotype indicative of cytokine activation, with higher CD122 and lower CD127 than do the GITR(basal) memory T cells. Remarkably, these bone marrow-specific GITR(hi) cells are almost completely ablated in the absence of IL-15, whereas TNFR2 and 4-1BB expression on the CD8 memory T cells are IL-15 independent. Furthermore, adoptively transferred splenic CD8 memory phenotype T cells show IL-15-dependent GITR upregulation upon entry into the bone marrow. This result implies that the selective appearance of GITR(hi) memory phenotype T cells in the bone marrow reflects the local microenvironment rather than a different subset of memory T cells. GITR(-/-) mice have a lower frequency of CD8 memory phenotype cells in the bone marrow, yet the GITR(-/-) cells hyperproliferate compared with those in wild-type mice. Taken together, these data suggest that GITR plays a role in the survival of CD8 memory phenotype T cells and that GITR upregulation represents a precise marker of cells that have responded to IL-15.

Enhanced GITR/GITRL interactions augment IL‐27 expression and induce IL‐10‐producing Tr‐1 like cells

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

Correlation of Circulating Glucocorticoid-Induced TNFR-Related Protein Ligand Levels with Disease Activity in Patients with Systemic Lupus Erythematosus

The aim of this paper is to investigate the correlation of glucocorticoid-induced tumor necrosis factor receptor- (TNFR-) related protein ligand (GITRL) with disease activity and organ involvement in patients with systemic lupus erythematosus (SLE). Serum GITRL levels were measured in 58 patients with SLE and 30 healthy controls matched for age and sex. Patients were assessed for clinical and laboratory variables. Correlations of serum GITRL levels with SLEDAI, laboratory values, and clinical manifestations were assessed. Serum GITRL levels were determined by ELISA. Serum GITRL levels were markedly increased in patients with SLE compared with healthy controls (mean 401.3 ng/mL and 36.59 ng/mL, resp.; P < 0.0001). SLE patients with active disease showed higher serum GITRL levels compared to those with inactive disease (mean 403.3 ng/mL and 136.3 ng/mL, resp; P = 0.0043) as well as normal controls (36.59 ng/mL; P < 0.0001). Serum GITRL levels were positively correlated with SLEDAI, titers of anti-dsDNA antibody, erythrocyte sedimentation rate (ESR), and IgM and negatively correlated with complement3 (C3). Serum GITRL levels were higher in SLE patients with renal involvement and vasculitis compared with patients without the above-mentioned manifestations.

Glucocorticoid-induced TNFR-related protein (GITR) ligand modulates cytokine release and NK cell reactivity in chronic lymphocytic leukemia (CLL)

Natural killer (NK) cells play an important role in the immunosurveillance of hematopoietic malignancies. Their reactivity is influenced by activating and inhibitory signals mediated by tumor-expressed ligands for NK receptors. Many members of the tumor necrosis factor (TNF) family modulate differentiation, proliferation, activation and death of both tumor and immune effector cells. The TNF receptor family member glucocorticoid-induced TNFR-related protein (GITR) stimulates anti-tumor immunity in mice, but available data indicate that GITR may mediate different effects in mice and men and impairs the reactivity of human NK cells. Here, we comprehensively studied the expression and function of GITR ligand (GITRL) in leukemia. Among the different leukemia entities, pronounced expression of GITRL on leukemic cells was observed in chronic lymphocytic leukemia (CLL), and the GITR receptor was expressed at significantly higher levels on NK cells of CLL patients compared with healthy controls. Upon GITR-GITRL interaction, signaling via GITRL into the leukemia cells induced the release of interleukin (IL)-6, IL-8 and TNF, which act as growth and survival factors for CLL cells. In addition, GITRL impaired both direct and Rituximab-induced degranulation, cytotoxicity and interferon-γ production of NK cells, which could be restored by GITR blocking antibodies. Thus, GITRL may contribute to disease pathophysiology and resistance to direct and Rituximab-induced NK reactivity in CLL.

Enhancement of anti‐tumor immunity through local modulation of CTLA‐4 and GITR by dendritic cells

Cancer vaccines have now demonstrated clinical efficacy, but immune modulatory mechanisms that prevent autoimmunity limit their effectiveness. Systemic administration of mAbs targeting the immune modulatory receptors CTLA-4 and glucocorticoid-induced TNFR-related protein (GITR) on Treg and effector T cells augments anti-tumor immunity both experimentally and clinically, but can induce life-threatening autoimmunity. We hypothesized that local delivery of anti-CTLA-4 and anti-GITR mAbs to the sites where T cells and tumor antigen-loaded DC vaccines interact would enhance the induction of anti-tumor immunity while avoiding autoimmunity. To achieve this goal, DCs transfected with mRNA encoding the H and L chains of anti-mouse CTLA-4 and GITR mAbs were co-administered with tumor antigen mRNA-transfected DCs. We observed enhanced induction of anti-tumor immunity and significantly improved survival in melanoma-bearing mice, without signs of autoimmunity. Using in vitro assays with human DCs, we demonstrated that DCs transfected with mRNA encoding a humanized anti-CTLA-4 mAb and mRNA encoding a soluble human GITR-L fusion protein enhance the induction of anti-tumor CTLs in response to DCs transfected with mRNAs encoding either melanoma or breast cancer antigens. Based on these results, this approach of using local delivery of immune modulators to enhance vaccine-induced immunity is currently being evaluated in a phase I clinical cancer immunotherapy trial.

Th2 Responses to Helminth Parasites Can Be Therapeutically Enhanced by, but Are Not Dependent upon, GITR–GITR Ligand Costimulation In Vivo

The immune suppression that characterizes human helminth infections can hinder the development of protective immunity or help to reduce pathogenic inflammation. Signaling through the T cell costimulator glucocorticoid-induced TNFR-related protein (GITR) counteracts immune downregulation by augmenting effector T cell responses and abrogating suppression by Foxp3(+) regulatory T cells. Thus, superphysiological Ab-mediated GITR costimulation represents a novel therapy for promoting protective immunity toward parasitic helminths, whereas blocking physiological GITR-GITR ligand (GITRL) interactions may provide a mechanism for dampening pathogenic Th2 inflammation. We investigated the superphysiological and physiological roles of the GITR-GITRL pathway in the development of protective and pathogenic Th2 responses in murine infection models of filariasis (Litomosoides sigmodontis) and schistosomiasis (Schistosoma mansoni). Providing superphysiological GITR costimulation using an agonistic anti-GITR mAb over the first 12 d of L. sigmodontis infection initially increased the quantity of Th2 cells, as well as their ability to produce Th2 cytokines. However, as infection progressed, the Th2 responses reverted to normal infection levels, and parasite killing remained unaffected. Despite the Th2-promoting role of superphysiological GITR costimulation, Ab-mediated blockade of the GITR-GITRL pathway did not affect Th2 cell priming or maintenance during L. sigmodontis infection. Blockade of GITR-GITRL interactions during the acute egg phase of S. mansoni infection resulted in reduced Th2 responses, but this effect was confined to the spleen and did not lead to changes in liver pathology. Thus, although superphysiological GITR costimulation can therapeutically enhance Th2 responses, physiological GITR-GITRL interactions are not required for the development of Th2-mediated resistance or pathology in murine models of filariasis and schistosomiasis.

Glucocorticoid induced TNFR-related protein (GITR) as marker of human regulatory T cells: expansion of the GITR+CD25- cell subset in patients with systemic lupus erythematosus

Regulatory T cells (T(REG)) represent a T cell subset able to modulate immune response by suppressing autoreactive T-lymphocytes. The evidence of a reduced number and an impaired function of this cell population in autoimmune/inflammatory chronic diseases led to the hypothesis of its involvement in the pathogenesis of these disorders. Glucocorticoid-induced TNFR-related protein (GITR) is a well known marker of murine T(REG) cells, but little is known in humans. The aim of this study was to investigate the characteristics of T(REG) cells in systemic lupus erythematosus (SLE) and the potential role of GITR as marker of human T(REG). Nineteen SLE patients and 15 sex- and age-matched normal controls (NC) were enrolled. CD4(+) T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies and real time polymerase-chain reaction (PCR) using TaqMan probes. The CD25(high)GITR(high) subset was significantly decreased in SLE patients with respect to NC (0.37±0.21% vs 0.72±0.19%; p<0.05). On the opposite, the CD25⁻GITR(high) cell population was expanded in the peripheral blood of SLE patients (3.5±2.25 vs 0.70±0.32%, p<0.01). Interestingly, FoxP3 at mRNA level was expressed in both CD25⁻GITR(high) and CD25(high)GITR(high) cells, suggesting that both cell subsets have regulatory activity. CD4(+)CD25⁻GITR(high) cells are increased in SLE as compared to NC. The expression of high level of GITR, but not CD25, on FoxP3+ cells appears to point to a regulatory phenotype of this peculiar T cell subset.

CD8 T Cell-Intrinsic GITR Is Required for T Cell Clonal Expansion and Mouse Survival following Severe Influenza Infection

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-x(L). In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphocyte survival, they clearly have nonredundant functions in protection from severe infection.

Tolerogenic Dendritic Cells Induce CD4+CD25hiFoxp3+ Regulatory T Cell Differentiation from CD4+CD25−/loFoxp3− Effector T Cells

IL-10-differentiated dendritic cells (DC10) induce allergen tolerance in asthmatic mice, during which their lung Th2 effector T cells (Teffs) are displaced by activated CD4(+)CD25(hi)Foxp3(+) T cells. Intestinal DCs promote oral tolerance by inducing Ag-naive T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), but whether DCs can induce Teffs to differentiate into Tregs remains uncertain. In this study, we addressed this question in OVA-asthmatic mice that were treated with DC10. OVA-presenting DC10 treatment maximally activated lung Tregs in these animals at 3 wk posttreatment, as determined by upregulation of activation markers (ICOS, programmed cell death-1, glucocorticoid-induced TNFR-related protein, LAG3, and CTLA-4) and in functional assays. This in vitro regulatory activity was ≥90% reduced by treatment with anti-IL-10 but not anti-TGF-β Abs. In parallel cultures, OVA- but not house dust mite (HDM)-presenting DC10 induced ≈43% of CFSE-labeled CD25(-/lo)Foxp3(-) Teffs from asthmatic OVA-TCR transgenic mice to differentiate into tolerogenic CD25(hi)Foxp3(+) Tregs. We recapitulated this in vivo using OVA-asthmatic mice that were coinjected with OVA- or HDM-presenting DC10 (i.p.) and CFSE-labeled CD4(+)CD25(-/lo)Foxp3(-) Teffs (i.v.) from the lungs of asthmatic DO11.10 mice. From ≈7 to 21% of the activated (i.e., dividing) DO11.10 Teffs that were recovered from the lungs, lung-draining lymph nodes, or spleens of the OVA-DC10 recipients had differentiated into CD4(+)CD25(hi)Foxp3(+) Tregs, whereas no CFSE-positive Tregs were recovered from the HDM-DC10-treated animals. These data indicate that DC10 treatments induce tolerance at least in part by inducing Teffs to differentiate into CD4(+)CD25(hi)Foxp3(+) Tregs.