Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

The regulatory CD4+CD25+ T cells have a limited role on pathogenesis of infection with Trypanosoma cruzi

Recent reports have established an important role of CD4+CD25+ T cells in the immune regulation of infectious diseases, autoimmune disorders and cancer. In the present work, we investigated whether these cells had a regulatory role during Trypanosoma cruzi infection, using the Colombian strain. Inactivation of CD4+CD25+ cells in vivo conferred mice slightly more resistant to infection with the Colombian strain of T. cruzi, as evidenced by lower parasitemia and mortality rates. The augmented resistance to infection with Colombian strain did correlate with increased activation of effector CD4 cells. It was antibody-independent, since no difference in levels of IgM, IgG, IgG1 and IgG2a(b) recognizing T. cruzi antigens was observed throughout the infection of CD25-inactivated and control mice. Regarding pathogenesis, inflammatory infiltrate and frequency of CD4 and CD8 T cells or macrophages in the cardiac tissue was similar in both groups. Together, our data indicate that CD4+CD25+ cells have a limited role on host resistance during early T. cruzi infection. Despite exhaustive investigation, we did not observe any role for these regulatory cells in the pathogenesis of experimental chronic Chagas' disease.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

Regulation of murine inflammatory bowel disease by CD25+ and CD25- CD4+ glucocorticoid-induced TNF receptor family-related gene+ regulatory T cells

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(+)CD45RB(high) T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RB(high) and CD45RB(low) 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-beta. 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.     

Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells

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.     

Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis

The glucocorticoid-induced TNFR (GITR) is expressed at high levels on resting CD4(+)CD25(+) T regulatory (T(R)) 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 T(R) 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 T(R) cells both in vivo and in vitro. Taken together, these findings suggest a dual functional role for GITR as GITR cross-linking both inactivates T(R) cells and increases CD4(+)CD25(-) T cell effector function, thus enhancing T cell immunity.     

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-alpha blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-alpha) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-alpha levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-alpha, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-alpha+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-alpha treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-alpha-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-alpha treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.     

In vitro expansion improves in vivo regulation by CD4+CD25+ regulatory T cells

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFbeta while down-regulating IFN-gamma, GM-CSF, IL5, and TNF-alpha production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFbeta to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.     

Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity

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-gamma 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.     

Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from human CD4+ CD25- T cells in response to inflammation

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-beta 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.     

In vivo infection by Trypanosoma cruzi: the conserved FLY domain of the gp85/trans-sialidase family potentiates host infection

Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas' disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 μg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+ T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+ T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas' disease.     

Plasmodium vivax: Induction of CD4+CD25+FoxP3+ Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Circulation CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-β) as well as pro-inflammatory (IFN-γ, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4⁺CD25⁺FoxP3⁺GITR⁺ or CD4⁺CD25⁺FoxP3⁺CTLA-4⁺ and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.     

CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms

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.     

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.     

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.     

CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to filarial parasites in vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.     

Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells

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.     

Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.     

Increased natural CD4+CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis

Regulatory T cells (Tregs), including natural CD4+CD25+ Tregs and inducible IL-10 producing T regulatory type 1 (T(R)1) cells, maintain tolerance and inhibit autoimmunity. Recently, increased percentages of Tregs have been observed in the blood of septic patients, and ex vivo-activated Tregs were shown to prevent polymicrobial sepsis mortality. Whether endogenous Tregs contribute to sepsis outcome remains unclear. Polymicrobial sepsis, induced by cecal ligation and puncture, caused an increased number of splenic Tregs compared with sham-treated mice. Splenic CD4+CD25+ T cells from septic mice expressed higher levels of Foxp3 mRNA and were more efficient suppressors of CD4+CD25- T effector cell proliferation. Isolated CD4+ T cells from septic mice displayed increased intracellular IL-10 staining following stimulation, indicating that T(R)1 cells may also be elevated in sepsis. Surprisingly, Ab depletion of total CD4+ or CD4+CD25+ populations did not affect mortality. Furthermore, no difference in survival outcome was found between CD25 or IL-10 null mice and wild-type littermates, indicating that Treg or T(R)1-generated IL-10 are not required for survival. These results demonstrate that, although sepsis causes a relative increase in Treg number and increases their suppressive function, their presence does not contribute significantly to overall survival in this model.