Characterization of IL-10-secreting T cells derived from regulatory CD4+CD25+ cells by the TIRC7 surface marker

Natural CD25(+)CD4(+) regulatory T cells (Treg) are essential for self-tolerance and for the control of T cell-mediated immune pathologies. However, the identification of Tregs in an ongoing immune response or in inflamed tissues remains elusive. Our experiments indicate that TIRC7, T cell immune response cDNA 7, a novel membrane molecule involved in the regulation of T lymphocyte activation, identifies two Treg subsets (CD25(low)TIRC7(+) and CD25(high)TIRC7(-)) that are characterized by the expression of Foxp3 and a suppressive activity in vitro and in vivo. We also showed that the CD25(low)TIRC7(+) subset represents IL-10-secreting Tregs in steady state, which is accumulated intratumorally in a tumor-bearing mice model. Blockade of the effect of IL-10 reversed the suppression imposed by the CD25(low)TIRC7(+) subset. Interestingly, these IL-10-secreting cells derived from the CD25(high)TIRC7(-) subset, both in vitro and in vivo, in response to tumoral Ags. Our present results strongly support the notion that, in the pool of natural Tregs, some cells can recognize foreign Ags and that this recognition is an essential step in their expansion and suppressive activity in vivo.     

CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4

Regulatory T cells are critical in regulating the immune response, and therefore play an important role in the defense against infection and control of autoimmune diseases. However, a therapeutic role of regulatory T cells in an established disease has not been fully established. In this study, we provide direct evidence that CD4(+)CD25(+) regulatory T cells can cure an established, severe, and progressive colitis. SCID mice developed severe colitis when adoptively transferred with naive CD4(+)CD25(-) T cells and infected with the protozoan parasite Leishmania major. The disease development can be completely halted and symptoms reversed, with a healthy outcome, by transferring freshly isolated or activated CD4(+)CD25(+) T cells from syngeneic donors. The therapeutic effect of the regulatory T cells was completely blocked by treatment of the recipients with anti-IL-10R, anti-CTLA4, or anti-TGF-beta Ab. However, the resurgence of colitis under these treatments was not accompanied by the reactivation of Th1 or Th2 response nor was it correlated to the parasite load. These results therefore demonstrate that CD4(+)CD25(+) T cells are therapeutic and that the effect is mediated by both IL-10/TGF-beta-dependent and independent mechanisms. Furthermore, colitis can manifest independent of Th1 and Th2 responses.     

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.     

Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells

CD4(+)CD25(+) regulatory T cells have been shown to prevent T cell-mediated immune pathology; however, their ability to ameliorate established inflammation has not been tested. Using the CD4(+)CD45RB(high) T cell transfer model of inflammatory bowel disease, we show that CD4(+)CD25(+) but not CD4(+)CD25(-)CD45RB(low) T cells are able to cure intestinal inflammation. Transfer of CD4(+)CD25(+) T cells into mice with colitis led to resolution of the lamina propria infiltrate in the intestine and reappearance of normal intestinal architecture. CD4(+)CD25(+) T cells were found to proliferate in the mesenteric lymph nodes and inflamed colon. They were located between clusters of CD11c(+) cells and pathogenic T cells and found to be in contact with both cell types. These studies suggest that manipulation of CD4(+)CD25(+) T cells may be beneficial in the treatment of chronic inflammatory diseases.     

IL-10 from CD4CD25Foxp3CD127 adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection

The outcome of malaria infection is determined, in part, by the balance of pro-inflammatory and regulatory immune responses. Failure to develop an effective pro-inflammatory response can lead to unrestricted parasite replication, whilst failure to regulate this response leads to the development of severe immunopathology. IL-10 and TGF-beta are known to be important components of the regulatory response, but the cellular source of these cytokines is still unknown. Here we have examined the role of natural and adaptive regulatory T cells in the control of malaria infection and find that classical CD4+CD25(hi) (and Foxp3+) regulatory T cells do not significantly influence the outcome of infections with the lethal (17XL) strain of Plasmodium yoelii (PyL). In contrast, we find that adaptive IL-10-producing, CD4+ T cells (which are CD25-, Foxp3-, and CD127- and do not produce Th1, Th2, or Th17 associated cytokines) that are generated during both PyL and non-lethal P. yoelii 17X (PyNL) infections are able to down-regulate pro-inflammatory responses and impede parasite clearance. In summary, we have identified a population of induced Foxp3- regulatory (Tr1) T cells, characterised by production of IL-10 and down regulation of IL-7Ralpha, that modulates the inflammatory response to malaria.     

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.     

CD25- T cells generate CD25+Foxp3+ regulatory T cells by peripheral expansion

Naturally occurring CD4(+) regulatory T cells are generally identified through their expression of CD25. However, in several experimental systems considerable T(reg) activity has been observed in the CD4(+)CD25(-) fraction. Upon adoptive transfer, the expression of CD25 in donor-derived cells is not stable, with CD4(+)CD25(+) cells appearing in CD4(+)CD25(-) T cell-injected animals and vice versa. We show in this study that CD25(+) cells arising from donor CD25(-) cells upon homeostatic proliferation in recipient mice express markers of freshly isolated T(reg) cells, display an anergic state, and suppress the proliferation of other cells in vitro. The maintenance of CD25 expression by CD4(+)CD25(+) cells depends on IL-2 secreted by cotransferred CD4(+)CD25(-) or by Ag-stimulated T cells in peripheral lymphoid organs.     

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.     

CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25(+) subpopulation of naturally activated (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4(+) cells, we conclude that expansion control and disease prevention are largely independent processes. CD25(+) CD45RB(low) CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.     

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.     

Nasal anti-CD3 antibody ameliorates lupus by inducing an IL-10-secreting CD4+ CD25- LAP+ regulatory T cell and is associated with down-regulation of IL-17+ CD4+ ICOS+ CXCR5+ follicular helper T cells

Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4(+)CD25(+) regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4(+)CD25(-)latency-associated peptide (LAP)(+) regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-beta-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4(+)CD25(-)LAP(+) regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4(+)ICOS(+)CXCR5(+) follicular helper T cells that are required for autoantibody production. CD4(+)ICOS(+)CXCR5(+) follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4(+)CD25(-)LAP(+) regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.     

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.     

Selective availability of IL-2 is a major determinant controlling the production of CD4+CD25+Foxp3+ T regulatory cells

The development and maintenance of T regulatory (Treg) cells critically depend on IL-2. This requirement for IL-2 might be due to specificity associated with IL-2R signal transduction or because IL-2 was uniquely present in the niche in which Treg cells reside. To address this issue, we examined the capacity of IL-7R-dependent signaling to support Treg cell production and prevent autoimmunity in IL-2Rbeta(-/-) mice. Expression of transgenic wild-type IL-7R or a chimeric receptor that consisted of the extracytoplasmic domain of the IL-7R alpha-chain and the cytoplasmic domain of IL-2R beta-chain in IL-2Rbeta(-/-) mice did not prevent autoimmunity. Importantly, expression of a chimeric receptor that consisted of the extracytoplasmic domain of the IL-2R beta-chain and the cytoplasmic domain of IL-7R alpha-chain in IL-2Rbeta(-/-) mice led to Treg cells production in the thymus and periphery and prevented autoimmunity. Signaling through the IL-2R or chimeric IL-2Rbeta/IL-7Ralpha in vivo or the culture of thymocytes from IL-2Rbeta(-/-) mice with IL-7 led to up-regulation of Foxp3 and CD25 on Treg cells. These findings indicate that IL-7R signal transduction is competent to promote Treg cell production, but this signaling requires triggering through IL-2 by binding to the extracytoplasmic portion of the IL-2R via this chimeric receptor. Thus, a major factor controlling the nonredundant activity of the IL-2R is selective compartmentalization of IL-2-producing cells with Treg cells in vivo.     

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.     

CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127^lowPD-1^highTIM-3^high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.     

Regulatory CD4+ CD25+ Foxp3+ T cells expand during experimental Plasmodium infection but do not prevent cerebral malaria

Pathogenic CD8+ T cells are implicated in the physiopathological mechanisms leading to experimental cerebral malaria (CM) in Plasmodium berghei ANKA (PbA) infected mice. Therefore, we hypothesised that in CM susceptible mice the neuropathology could be, at least in part, the result of an inefficient control of pathogenic effector T cells by CD4+ CD25+ Treg cells. Remarkably, the number of CD4+ CD25high T cells expressing Foxp3 increased in the spleen during the course of infection. These cells displayed an activated phenotype and consistent with that, CD4+ CD25high Treg cells isolated from PbA-infected mice showed an enhanced regulatory activity in vitro. Surprisingly, these cells do not migrate to the brain at the time of neurological symptoms as the conventional CD4+ T cells do. CM was not exacerbated in anti-CD25 treated mice when infected with PbA one month after treatment, even if splenic CD8+ T cells expressing CD69 increased in these mice. Taken together, these results show that P. berghei infection leads to an increase of the number of splenic CD4+ CD25high Treg cells exhibiting in vitro suppressive function, but they do not seem to be involved in vivo in the protection against CM.