Peripheral CD8+CD25+ T lymphocytes from MHC class II-deficient mice exhibit regulatory activity

We characterized CD8(+) T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4(+)CD25(+) T cells, CD8(+)CD25(+) T cells appear late in the periphery during ontogeny. Peripheral CD8(+)CD25(+) T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4(+)CD25(+) T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25(-) T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4(+)CD25(+) T cells. However, unlike CD4(+)CD25(+) T cells CD8(+)CD25(+) T cells from MHC class II-deficient mice strongly proliferate and produce IFN-gamma in vitro in response to stimulation in the absence of exogenous IL-2.     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.     

Latency-associated peptide identifies a novel CD4+CD25+ regulatory T cell subset with TGFbeta-mediated function and enhanced suppression of experimental autoimmune encephalomyelitis

CD4(+)CD25(+) regulatory T cells (Tregs) are essential for maintaining self-tolerance and immune homeostasis. Here we characterize a novel subset of CD4(+)CD25(+) Tregs that express latency-associated peptide (LAP) on their cell surface (CD4(+)CD25(+)LAP(+) cells). CD4(+)CD25(+)LAP(+) cells express elevated levels of Foxp3 and Treg-associated molecules (CTLA4, glucocorticoid-induced TNFR-related gene), secrete TGFbeta, and express both cell surface TGFbeta and surface receptors for TGFbeta. In vitro, the suppressive function of CD4(+)CD25(+)LAP(+) cells is both cell contact and soluble factor dependent; this contrasts with CD4(+)CD25(+)LAP(-) cells, which are mainly cell contact dependent. In a model of experimental autoimmune encephalomyelitis, CD4(+)CD25(+)LAP(+) cells exhibit more potent suppressive activity than CD4(+)CD25(+)LAP(-) cells, and the suppression is TGFbeta dependent. We further show that CD4(+)CD25(+)LAP(+) cells suppress myelin oligodendrocyte glycoprotein-specific immune responses by inducing Foxp3 and by inhibiting IL-17 production. Our findings demonstrate that CD4(+)CD25(+) Tregs are a heterogeneous population and that the CD4(+)CD25(+) subset that expresses LAP functions in a TGFbeta-dependent manner and has greater in vivo suppressive properties. Our work helps elucidate the ambiguity concerning the role of TGFbeta in CD4(+)CD25(+) Treg-mediated suppression and indicates that LAP is an authentic marker able to identify a TGFbeta-expressing CD4(+)CD25(+) Treg subset.     

Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10

Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.     

CD83 expression in CD4+ T cells modulates inflammation and autoimmunity

The transmembrane protein CD83 has been initially described as a maturation marker for dendritic cells. Moreover, there is increasing evidence that CD83 also regulates B cell function, thymic T cell maturation, and peripheral T cell activation. Herein, we show that CD83 expression confers immunosuppressive function to CD4(+) T cells. CD83 mRNA is differentially expressed in naturally occurring CD4(+)CD25(+) regulatory T cells, and upon activation these cells rapidly express large amounts of surface CD83. Transduction of naive CD4(+)CD25(-) T cells with CD83 encoding retroviruses induces a regulatory phenotype in vitro, which is accompanied by the induction of Foxp3. Functional analysis of CD83-transduced T cells in vivo demonstrates that these CD83(+)Foxp3(+) T cells are able to interfere with the effector phase of severe contact hypersensitivity reaction of the skin. Moreover, adoptive transfer of these cells prevents the paralysis associated with experimental autoimmune encephalomyelitis, suppresses proinflammatory cytokines IFN-gamma and IL-17, and increases antiinflammatory IL-10 in recipient mice. Taken together, our data provide the first evidence that CD83 expression can contribute to the immunosuppressive function of CD4(+) T cells in vivo.     

Inducible CD4+LAP+Foxp3- regulatory T cells suppress allergic inflammation

Regulatory T cells (Tregs) play a critical role in the maintenance of airway tolerance. We report that inhaled soluble Ag induces adaptive Foxp3(+) Tregs, as well as a regulatory population of CD4(+) T cells in the lungs and lung-draining lymph nodes that express latency-associated peptide (LAP) on their cell surface but do not express Foxp3. Blocking the cytokine IL-10 or TGF-β prevented the generation of LAP(+) Tregs and Foxp3(+) Tregs in vivo, and the LAP(+) Tregs could also be generated concomitantly with Foxp3(+) Tregs in vitro by culturing naive CD4(+) T cells with Ag and exogenous TGF-β. The LAP(+) Tregs strongly suppressed naive CD4(+) T cell proliferation, and transfer of sorted OVA-specific LAP(+) Tregs in vivo inhibited allergic eosinophilia and Th2 cytokine expression in the lung, either when present at the time of Th2 sensitization or when injected after Th2 cells were formed. Furthermore, inflammatory innate stimuli from house dust mite extract, nucleotide-binding oligomerization domain containing 2 ligand, and LPS, which are sufficient for blocking airway tolerance, strongly decreased the induction of LAP(+) Tregs. Taken together, we concluded that inducible Ag-specific LAP(+) Tregs can suppress asthmatic lung inflammation and constitute a mediator of airway tolerance together with Foxp3(+) Tregs.     

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

Strong impact of CD4+ Foxp3+ regulatory T cells and limited effect of T cell-derived IL-10 on pathogen clearance during Plasmodium yoelii infection

It is well established that CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play a crucial role in the course of different infectious diseases. However, contradictory results have been published regarding to malaria infection. In this study, we report that specific ablation of Foxp3(+) Tregs in Plasmodium yoelii-infected DEREG-BALB/c mice leads to an increase in T cell activation accompanied by a significant decrease in parasitemia. To better understand how Foxp3(+) Tregs orchestrate this phenotype, we used microarrays to analyze CD4(+)CD25(+)Foxp3(+) Tregs and CD4(+)CD25(-)Foxp3(-) T cells in the course of P. yoelii infection. Using this approach we identified genes specifically upregulated in CD4(+)CD25(+)Foxp3(+) Tregs in the course of infection, such as G-protein-coupled receptor 83 and Socs2. This analysis also revealed that both CD4(+)CD25(+)Foxp3(+) Tregs and CD4(+)CD25(-)Foxp3(-) T cells upregulate CTLA-4, granzyme B, and, more strikingly, IL-10 during acute blood infection. Therefore, we aimed to define the function of T cell-derived IL-10 in this context by Cre/loxP-mediated selective conditional inactivation of the IL-10 gene in T cells. Unexpectedly, IL-10 ablation in T cells exerts only a minor effect on parasite clearance, even though CD8(+) T cells are more strongly activated, the production of IFN-γ and TNF-α by CD4(+)CD25(-) T cells is increased, and the suppressive activity of CD4(+)CD25(+) Tregs is reduced upon infection. In summary, these results suggest that CD4(+)Foxp3(+) Tregs modulate the course of P. yoelii infection in BALB/c mice. Moreover, CD4(+) T cell-derived IL-10 affects T effector function and Treg activity, but has only a limited direct effect on parasite clearance in this model.     

Impact of protective IL-2 allelic variants on CD4+ Foxp3+ regulatory T cell function in situ and resistance to autoimmune diabetes in NOD mice

Type I diabetes (T1D) susceptibility is inherited through multiple insulin-dependent diabetes (Idd) genes. NOD.B6 Idd3 congenic mice, introgressed with an Idd3 allele from T1D-resistant C57BL/6 mice (Idd3(B6)), show a marked resistance to T1D compared with control NOD mice. The protective function of the Idd3 locus is confined to the Il2 gene, whose expression is critical for naturally occurring CD4(+)Foxp3(+) regulatory T (nT(reg)) cell development and function. In this study, we asked whether Idd3(B6) protective alleles in the NOD mouse model confer T1D resistance by promoting the cellular frequency, function, or homeostasis of nT(reg) cells in vivo. We show that resistance to T1D in NOD.B6 Idd3 congenic mice correlates with increased levels of IL-2 mRNA and protein production in Ag-activated diabetogenic CD4(+) T cells. We also observe that protective IL2 allelic variants (Idd3(B6) resistance allele) also favor the expansion and suppressive functions of CD4(+)Foxp3(+) nT(reg) cells in vitro, as well as restrain the proliferation, IL-17 production, and pathogenicity of diabetogenic CD4(+) T cells in vivo more efficiently than control do nT(reg) cells. Lastly, the resistance to T1D in Idd3 congenic mice does not correlate with an augmented systemic frequency of CD4(+)Foxp3(+) nT(reg) cells but more so with the ability of protective IL2 allelic variants to promote the expansion of CD4(+)Foxp3(+) nT(reg) cells directly in the target organ undergoing autoimmune attack. Thus, protective, IL2 allelic variants impinge the development of organ-specific autoimmunity by bolstering the IL-2 producing capacity of self-reactive CD4(+) T cells and, in turn, favor the function and homeostasis of CD4(+)Foxp3(+) nT(reg) cells in vivo.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection, with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10-producing cells in the lung during acute RSV infection were CD4(+) T cells. The IL-10-producing CD4(+) T cells included Foxp3(+) regulatory T cells, Foxp3(-) CD4(+) T cells that coproduce IFN-γ, and Foxp3(-) CD4(+) T cells that do not coproduce IFN-γ. RSV infection of IL-10-deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared with control mice. We also observed an increase in the magnitude of the RSV-induced CD8(+) and CD4(+) T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4(+) T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4(+) T cells and a concomitant decrease in Foxp3(+) regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.     

Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells

Defects in the Bcl-2-regulated apoptotic pathway inhibit the deletion of self-reactive T cells. What is unresolved, however, is the nature and fate of such self-reactive T cells escaping deletion. In this study, we report that mice with such defects contained increased numbers of CD25(low)Foxp3(+) cells in the thymus and peripheral lymph tissues. The increased CD25(low)Foxp3(+) population contained a large fraction of cells bearing self-reactive TCRs, evident from a prominent increase in self-superantigen-specific Foxp3(+)Vβ5(+)CD4(+) T cells in BALB/c Bim(-/-) mice compared with control animals. The survival rate of the expanded CD25(low)Foxp3(+) cells was similar to that of CD25(high)Foxp3(+) CD4 T cells in vitro and in vivo. IL-2R stimulation, but not TCR ligation, upregulated CD25 on CD25(low)Foxp3(+)CD4(+) T cells in vitro and in vivo. The expanded CD25(low)Foxp3(+)CD4(+) T cells from Bim(-/-) mice were anergic but also had weaker regulatory function than CD25(high)Foxp3(+) CD4(+) T cells from the same mice. Analysis of Bim(-/-) mice that also lacked Fas showed that the peripheral homeostasis of this expanded population was in part regulated by this death receptor. In conclusion, these results show that self-reactive T cell escapes from thymic deletion in mice defective in the Bcl-2-regulated apoptotic pathway upregulate Foxp3 and become unresponsive upon encountering self-Ag without necessarily gaining potent regulatory function. This clonal functional diversion may help to curtail autoaggressiveness of escaped self-reactive CD4(+) T cells and thereby safeguard immunological tolerance.     

Cutting edge: regulatory T cells induce CD4+CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta

Recent studies have shown that TGF-beta together with IL-6 induce the differentiation of IL-17-producing T cells (Th17) T cells. We therefore examined whether CD4(+)CD25(+)Foxp3(+) regulatory T cells, i.e., cells previously shown to produce TGF-beta, serve as Th17 inducers. We found that upon activation purified CD25(+) T cells (or sorted GFP(+) T cells obtained from Foxp3-GFP knockin mice) produce high amounts of soluble TGF-beta and when cultured with CD4(+)CD25(-)Foxp3(-) T cells in the presence of IL-6 induce the latter to differentiate into Th17 cells. Perhaps more importantly, upon activation, CD4(+)CD25(+)Foxp3(+)(GFP(+)) T cells themselves differentiate into Th17 cells in the presence of IL-6 (and in the absence of exogenous TGF-beta). These results indicate that CD4(+)CD25(+)Foxp3(+) regulatory T cells can function as inducers of Th17 cells and can differentiate into Th17 cells. They thus have important implications to our understanding of regulatory T cell function and their possible therapeutic use.     

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3(+) regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3(+) Tregs unleashes the robust expansion and activation of peptide-stimulated CD8(+) T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8(+) T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8(+) T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8(+) T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3(+) Treg suppressive potency. Therefore, Foxp3(+) Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8(+) T cells.