Function of the IL-2R for thymic and peripheral CD4+CD25+ Foxp3+ T regulatory cells

IL-2 contributes to the production, function, and homeostasis of CD4+CD25+ T(reg) cells. However, it remains uncertain whether IL-2 is essential for the development of T(reg) cells in the thymus, their homeostasis in the periphery, or both. The present study was undertaken to investigate the contribution of IL-2 during thymic T(reg) cell development and its maintenance in peripheral immune tissue. Relying on genetic mouse models where IL-2R signaling was either completely blocked or selectively inhibited in peripheral CD4+CD25+ T(reg) cells, we show that the IL-2/IL-2R interaction is active in the thymus at the earliest stage of the development of T(reg) cells to promote their expansion and to up-regulate Foxp3 and CD25 to normal levels. Furthermore, CD4+CD25+Foxp3+ T(reg) cells with impaired IL-2-induced signaling persist in the periphery and control autoimmunity without constant thymic output. These peripheral T(reg) cells with poor responsiveness to IL-2 exhibited slower growth and extended survival in vivo, somewhat lower suppressive activity, and poor IL-2-dependent survival in vitro. Mixed thymic and bone marrow chimeric mice showed that wild-type-derived T(reg) cells were substantially more effective in populating peripheral immune tissue than T(reg) cells with impaired IL-2 signaling. Collectively, these data support the notion that normally IL-2 is a dominant mechanism controlling the number of thymic and peripheral T(reg) cells.     

CD4+CD25+Foxp3+ regulatory T cells are dispensable for controlling CD8+ T cell-mediated lung inflammation

Every person harbors a population of potentially self-reactive lymphocytes controlled by tightly balanced tolerance mechanisms. Failures in this balance evoke immune activation and autoimmunity. In this study, we investigated the contribution of self-reactive CD8(+) T lymphocytes to chronic pulmonary inflammation and a possible role for naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) in counterbalancing this process. Using a transgenic murine model for autoimmune-mediated lung disease, we demonstrated that despite pulmonary inflammation, lung-specific CD8(+) T cells can reside quiescently in close proximity to self-antigen. Whereas self-reactive CD8(+) T cells in the inflamed lung and lung-draining lymph nodes downregulated the expression of effector molecules, those located in the spleen appeared to be partly Ag-experienced and displayed a memory-like phenotype. Because ex vivo-reisolated self-reactive CD8(+) T cells were very well capable of responding to the Ag in vitro, we investigated a possible contribution of nTregs to the immune control over autoaggressive CD8(+) T cells in the lung. Notably, CD8(+) T cell tolerance established in the lung depends only partially on the function of nTregs, because self-reactive CD8(+) T cells underwent only biased activation and did not acquire effector function after nTreg depletion. However, although transient ablation of nTregs did not expand the population of self-reactive CD8(+) T cells or exacerbate the disease, it provoked rapid accumulation of activated CD103(+)CD62L(lo) Tregs in bronchial lymph nodes, a finding suggesting an adaptive phenotypic switch in the nTreg population that acts in concert with other yet-undefined mechanisms to prevent the detrimental activation of self-reactive CD8(+) T cells.     

Cutting edge: self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T cells

CD4(+)CD25(+) regulatory T cell selection is initiated by high-specificity interactions with self-peptides in the thymus, although how these cells respond to cytokine-derived signals and to re-exposure to self-peptide:MHC complexes in the periphery is not well understood. We have used a transgenic mouse system, in which the peptide that induces thymic selection of a clonal population of CD4(+)CD25(+) regulatory T cells is known, to show that CD4(+)CD25(+) T cells proliferate in response to their selecting self-peptide in vivo. Moreover, they do not proliferate in response to lymphopenia in the absence of the selecting self-peptide, reflecting a low level of expression of the high affinity receptor for IL-7 (CD127) relative to conventional CD4(+) T cells. That their selecting self-peptide is both required for and promotes the peripheral expansion of CD4(+)CD25(+) regulatory T cells may direct their accumulation in sites where the self-peptide is expressed.     

A function for IL-7R for CD4+CD25+Foxp3+ T regulatory cells

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25-Foxp3low T cells. Interestingly, common gamma chain (gammac) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25-Foxp3low subset. Therefore, other gammac-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the gammac-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rbeta and IL-7Ralpha contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the gammac knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rbeta in mice double deficient in IL-2Rbeta and IL-7Ralpha established that IL-2Rbeta is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rbeta-/- mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.     

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.     

Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+ and Foxp3-CD25- CD4+ regulatory T cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (T(reg)). We found that total T(reg), KJ1-26+ T(reg) and CTLA-4+ T(reg) were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by co-administering cholera toxin or by treatment with anti-TGF-beta mAb. CD25+ T(reg), but also CD25-CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ T(reg) from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ T(reg). Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3-CD25-CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ T(reg) cells, which expressed Foxp3 more strongly than naturally developing T(reg) and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25- T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ T(reg) together with the generation of both Foxp3+ and Foxp3-CD25- CD4+ T(reg).     

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.     

Keratinocyte growth factor induces expansion of murine peripheral CD4+Foxp3+ regulatory T cells and increases their thymic output

Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4(+)Foxp3(+) Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4(+)Foxp3(+) Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4(+)Foxp3(-) T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4(+) T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4(+)Foxp3(+) Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.     

Agonist-driven development of CD4+CD25+Foxp3+ regulatory T cells requires a second signal mediated by Stat6

The factors that induce Foxp3 expression and regulatory T (Treg) cell development remain unknown. In this study, we investigated the role of STAT4 and STAT6 in agonist-driven generation of Ag-specific Foxp3-expressing Treg cells. Our findings indicate that fully efficient induction of Foxp3 expression and development of Ag-specific Treg cells requires the synergistic action of two signals: a TCR-mediated signal and a second signal mediated by STAT6. Indeed, by comparing the development of wild-type and STAT4- and STAT6-deficient hemagglutinin-specific T cells in the presence of hemagglutinin Ag, we found that the absence of STAT6 impaired the generation of Ag-specific CD4+CD25+Foxp3+ cells. Moreover, in transgenic mice expressing a constitutively active form of STAT6, we found that the fraction of CD4+Foxp3+ cells exceeds that of control wild-type littermates. Overall these findings support a role for the STAT6 pathway in Treg cell development and maintenance.     

CD25+Foxp3+ regulatory T cells facilitate CD4+ T cell clonal anergy induction during the recovery from lymphopenia

T cell clonal anergy induction in lymphopenic nu/nu mice was found to be ineffective. Exposure to a tolerizing peptide Ag regimen instead induced aggressive CD4(+) cell cycle progression and increased Ag responsiveness (priming). Reconstitution of T cell-deficient mice by an adoptive transfer of mature peripheral lymphocytes was accompanied by the development of a CD25(+)Foxp3(+)CTLA-4(+)CD4(+) regulatory T cell population that acted to dampen Ag-driven cell cycle progression and facilitate the induction of clonal anergy in nearby responder CD25(-)CD4(+) T cells. Thus, an early recovery of CD25(+) regulatory T cells following a lymphopenic event can prevent exuberant Ag-stimulated CD4(+) cell cycle progression and promote the development of clonal anergy.     

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.     

Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin

Naturally occurring CD4(+)CD25(+) regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4(+)CD25(+) non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4(+)CD25(high) T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4(+)CD25(low) or CD4(+)CD25(-) T cells did not. The expanding pure populations of CD4(+)CD25(high) T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4(+)CD25(-) T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4(+)CD25(high) T cell populations retained a broad TCR repertoire and, like CD4(+) CD25(+) T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4(+) and CD8(+) T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.     

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.     

G protein-coupled receptor 83 overexpression in naive CD4+CD25- T cells leads to the induction of Foxp3+ regulatory T cells in vivo

Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3-CD4+CD25- T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25- or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25- T cells. Transduction of naive CD4+CD25- T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.