The role of Fas in the immune system biology of IL-2R alpha knockout mice: interplay among regulatory T cells, inflammation, hemopoiesis, and apoptosis

Introducing lpr mutation prevents early mortality associated with IL-2Ralpha knockout (KO) mice, prompting us to determine the role of Fas in the immune system biology of IL-2Ralpha KO mice. Consistent with a defect in CD4+CD25+ regulatory T (Treg) cell expression, spontaneous lymphocyte activation in lymphoid organs was observed in 6-wk-old mice. In 16- to 22-wk-old mice, infiltration of leukocytes was observed in bone marrow, colon, lung, pancreas, lacrimal gland, and salivary gland, but not in heart, thyroid, liver, stomach, small intestine, ovary, and kidney. In the lymphocytes-infiltrated bone marrow, B cell lymphopoiesis was blocked at pro-B to pre-B/immature B stage, culminating in an age-dependent B cell loss in the periphery. These phenotypes were also observed in IL-2Ralpha KO mice bearing the lpr mutation (DM mice), indicating Treg cell function and the phenotypes attributed directly to Treg cell abnormality are largely Fas-independent. However, anemia and body weight loss were partially prevented, tissue cell apoptosis was inhibited, and lifespan was improved in the DM mice, demonstrating Fas-dependent elements in these processes. Our age-dependent, lifelong analysis of IL-2Ralpha KO and DM mice supports a CD4+CD25+ Treg cell-based mechanism for the abnormal immune system biology observed in IL-2Ralpha KO mice and provides a global view of the interplays among Treg cells, multiorgan inflammation, hemopoiesis, and apoptosis.     

IL-12R beta 2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit beta2 (IL-12Rbeta2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rbeta2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rbeta2(-/-) mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rbeta2-deficient mice to autoimmune diseases. T cells from IL-12Rbeta2(-/-) mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) in the thymus and spleen of IL-12Rbeta2(-/-) mice were comparable to those of WT mice. However, IL-12Rbeta2(-/-) mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-beta, as shown by significantly lower numbers of CD25(+)CD4(+) T cells that expressed Foxp3. Functionally, CD25(+)CD4(+) Tregs derived from IL-12Rbeta2(-/-) mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rbeta2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rbeta2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.     

Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells

IL-2 controls the survival of regulatory T cells (Tregs), but it is unclear whether IL-2 also directly affects Treg suppressive capacity in vivo. We have found that eliminating Bim-dependent apoptosis in IL-2- and CD25-deficient mice restored Treg numbers but failed to cure their lethal autoimmune disease, demonstrating that IL-2-dependent survival and suppressive activity can be uncoupled in Tregs. Treatment with IL-2-anti-IL-2-Ab complexes enhanced the numbers and suppressive capacity of IL-2-deprived Tregs with striking increases in CD25, CTLA-4, and CD39/CD73 expression. Although cytokine treatment induced these suppressive mechanisms in both IL-2(-/-) and IL-2(-/-)Bim(-/-) mice, it only reversed autoimmune disease in the latter. Our results suggest that successful IL-2 therapy of established autoimmune diseases will require a threshold quantity of Tregs present at the start of treatment and show that the suppressive capacity of Tregs critically depends on IL-2 even when Treg survival is independent of this cytokine.     

IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development

Common gamma chain (gammac)-receptor dependent cytokines are required for regulatory T cell (Treg) development as gammac(-/-) mice lack Tregs. However, it is unclear which gammac-dependent cytokines are involved in this process. Furthermore, thymic stromal lymphopoietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing CD4(+)Foxp3(+) Tregs in the thymus express the IL-2Rbeta, IL-4Ralpha, IL-7Ralpha, IL-15Ralpha, and IL-21Ralpha chains, but not the IL9Ralpha or TSLPRalpha chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in CD4(+)Foxp3(+) Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were capable of inducing the conversion of CD4(+)CD25(+)Foxp3(-) thymic Treg progenitors into CD4(+)Foxp3(+) mature Tregs in vitro. To examine this issue in more detail, we generated IL-2Rbeta(-/-) x IL-7Ralpha(-/-) and IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice. We found that IL-2Rbeta(-/-) x IL-7Ralpha(-/-) mice were devoid of Tregs thereby recapitulating the phenotype observed in gammac(-/-) mice; in contrast, the phenotype observed in IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice was comparable to that seen in IL-2Rbeta(-/-) mice. Finally, we observed that Tregs from both IL-2(-/-) and IL-2Rbeta(-/-) mice show elevated expression of IL-7Ralpha and IL-15Ralpha chains. Addition of IL-2 to Tregs from IL-2(-/-) mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the predominant role in Treg development, but that in its absence the IL-7Ralpha and IL-15Ralpha chains are up-regulated and allow for IL-7 and IL-15 to partially compensate for loss of IL-2.     

Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R

Complex interactions between effector T cells and Foxp3⁺ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4⁺Foxp3⁻ T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.     

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.     

Th3 cells in peripheral tolerance. II. TGF-beta-transgenic Th3 cells rescue IL-2-deficient mice from autoimmunity

We developed a transgenic (Tg) mouse that expresses TGF-beta under control of the IL-2 promoter to investigate Th3 cell differentiation both in vitro and in vivo. We previously found that repetitive in vitro Ag stimulation results in constant expression of Foxp3 in TGF-beta-Tg Th3 cells that acquire regulatory function independent of surface expression of CD25. To examine the differentiation and function of Th3 cells in vivo and to compare them with thymic-derived CD4(+)CD25(+) regulatory T cells (Treg), we introduced the TGF-beta transgene into T cells of IL-2-deficient (IL-2(-/-)) mice. We found that the induction, differentiation, and function of TGF-beta-derived Foxp3(+) Th3 cells were independent of IL-2, which differs from thymic Tregs. In an environment that lacks functional CD25(+) thymic-derived Tregs, expression of the TGF-beta transgene in IL-2(-/-) mice led to the induction of distinct CD25(-) regulatory cells in the periphery. These cells expressed Foxp3 and efficiently controlled hyperproliferation of T cells and rescued the IL-2(-/-) mouse from lethal autoimmunity. Unlike IL-2(-/-) animals, TGF-beta/IL-2(-/-) mice had normal numbers of T cells, B cells, macrophages, and dendritic cells and did not have splenomegaly, lymphadenopathy, or inflammation in multiple organs. Accumulation of Foxp3(+) cells over time, however, was dependent on IL-2. Our results suggest that TGF-beta-derived Foxp3(+)CD25(+/-) Th3 regulatory cells represent a different cell lineage from thymic-derived CD25(+) Tregs in the periphery but may play an important role in maintaining thymic Tregs in the peripheral immune compartment by secretion of TGF-beta.     

Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-gamma plays a major pathogenic effect. The role of the IFN-gamma-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-gamma and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4(+)/CD8(+) self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18R alpha-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18R beta is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18R beta could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-gamma production and development of IFN-gamma-dependent autoimmune pathology.     

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.     

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4⁺ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2^−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2^−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2^−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.     

The influence of excessive IL-6 production in vivo on the development and function of Foxp3+ regulatory T cells

IL-6 is a proinflammatory cytokine and its overproduction is implicated in a variety of inflammatory disorders. Recent in vitro analyses suggest that IL-6 is a key cytokine that determines the balance between Foxp3(+) regulatory T cells (Tregs) and Th17 cells. However, it remains unclear whether excessive IL-6 production in vivo alters the development and function of Foxp3(+) Tregs. In this study, we analyzed IL-6 transgenic (Tg) mice in which serum IL-6 levels are constitutively elevated. Interestingly, in IL-6 Tg mice, whereas peripheral lymphoid organs were enlarged, and T cells exhibited activated phenotype, Tregs were not reduced but rather increased compared with wild-type mice. In addition, Tregs from Tg mice normally suppressed proliferation of naive T cells in vitro. Furthermore, Tregs cotransferred with naive CD4 T cells into SCID-IL-6 Tg mice inhibited colitis as successfully as those transferred into control SCID mice. These results indicate that overproduction of IL-6 does not inhibit development or function of Foxp3(+) Tregs in vivo. However, when naive CD4 T cells alone were transferred, Foxp3(+) Tregs retrieved from SCID-IL-6 Tg mice were reduced compared with SCID mice. Moreover, the Helios(-) subpopulation of Foxp3(+) Tregs, recently defined as extrathymic Tregs, was significantly reduced in IL-6 Tg mice compared with wild-type mice. Collectively, these results suggest that IL-6 overproduced in vivo inhibits inducible Treg generation from naive T cells, but does not affect the development and function of natural Tregs.     

Lipopolysaccharide-activated IL-10-secreting dendritic cells suppress experimental autoimmune uveoretinitis by MHCII-dependent activation of CD62L-expressing regulatory T cells

Dendritic cells (DC) are key regulators of immune responses. Mature DC are traditionally considered to be immunogenic, although there is accumulating evidence that they can also be tolerogenic and induce Ag-specific regulatory T cells (Tregs). However, the mechanism of this Treg induction and the site of Treg action in vivo are yet to be defined. In this study, using the experimental model of interphotoreceptor retinoid-binding protein peptide (1-20)-induced experimental autoimmune uveoretinitis, we show that s.c. inoculation of IRBP-peptide-pulsed IL-10-producing LPS-activated mature DC (IL-10-DC) at one site (the cervical region) suppresses autoimmunity induced at a separate site (the inguinal region). Our data show that s.c. IL-10-DC correlates with an increase in the number of CD4(+)CD25(+)Foxp3(+) Tregs at the DC-draining lymph nodes (DC-dLN). However, although MHCII(-/-) IL-10-DC also induces Treg expansion at this DC-dLN, they failed to suppress experimental autoimmune uveoretinitis. Furthermore, unlike wild-type IL-10-DC, MHCII(-/-) IL-10-DC did not correlate with an increase in the percentage of Tregs expressing CD62L at the DC-dLN, nor did they associate with an increase in Treg number at a distal site. Similar effects were also observed after s.c. hen egg lysozyme-pulsed IL-10-DC, which produced a strong reduction in the number and activation of proliferating Ag-specific CD4(+) 3A9 T effector cells. We therefore propose that IL-10-DC require MHCII-dependent Ag presentation, and hence TCR ligation, to promote CD62L-mediated trafficking of Tregs to the site of T effector cell priming, where they suppress autoimmunity.     

ICOS-dependent homeostasis and function of Foxp3+ regulatory T cells in islets of nonobese diabetic mice

A progressive waning in Foxp3(+) regulatory T cell (Treg) functions is thought to provoke autoimmunity in the NOD model of type 1 diabetes (T1D). A deficiency in IL-2 is one of the main triggers for the defective function of Tregs in islets. Notably, abrogation of the ICOS pathway in NOD neonates or BDC2.5-NOD (BDC2.5) mice exacerbates T1D, suggesting an important role for this costimulatory pathway in tolerance to islet Ags. Thus, we hypothesize that ICOS selectively promotes Foxp3(+) Treg functions in BDC2.5 mice. We show that ICOS expression discriminates effector Foxp3(-) T cells from Foxp3(+) Tregs and specifically designates a dominant subset of intra-islet Tregs, endowed with an increased potential to expand, secrete IL-10, and mediate suppressive activity in vitro and in vivo. Consistently, Ab-mediated blockade or genetic deficiency of ICOS selectively abrogates Treg-mediated functions and T1D protection and exacerbates disease in BDC2.5 mice. Moreover, T1D progression in BDC2.5 mice is associated with a decline in ICOS expression in and expansion and suppression by intra-islet Foxp3(+) Tregs. We further show that the ICOS(+) Tregs, in contrast to their ICOS(-) counterparts, are more sensitive to IL-2, a critical signal for their survival and functional stability. Lastly, the temporal loss in ICOS(+) Tregs is readily corrected by IL-2 therapy or protective Il2 gene variation. Overall, ICOS is critical for the homeostasis and functional stability of Foxp3(+) Tregs in prediabetic islets and maintenance of T1D protection.     

Transient local depletion of Foxp3+ regulatory T cells during recovery from colitis via Fas/Fas ligand-induced death

Regulatory T cells (Tregs) play a fundamental role in regulating the immune system in health and disease. Considerable evidence exists demonstrating that transfer of Tregs can cure colitis and a variety of other inflammatory disorders. However, little is known about the effects of inflammation on resident Tregs. Mice (BALB/c or C57BL/6) treated with an intrarectal instillation of the haptenizing agent 2,4-dinitrobenzene sulfonic acid (DNBS) develop an acute inflammatory disease, the histopathology of which peaks at 3 days posttreatment and resolves spontaneously thereafter. In this study we demonstrate that DNBS (or oxazolone)-induced colitis causes a depletion of colonic Foxp3+ Tregs 8 days posttreatment, while the proportion of Foxp3+ cells in the ileum, mesenteric lymph nodes, and spleen remains unchanged. Replenishment of the colonic Treg population was associated with the reappearance of mucosal homing (alpha4beta7+) CD4+Foxp3+ Tregs. Assessing the mechanism of local Treg depletion, we found no evidence to implicate cytokine-induced phenotypic switching in the Foxp3+ population or increased SMAD7 expression despite the essential role that TGF-beta has in Foxp3+ Treg biology. Increased Fas ligand (FasL) expression was observed in the colon of colitic mice and in vitro stimulation with a Fas cross-linking Ab resulted in apoptosis of CD4+Foxp3+ but not CD4+Foxp3- cells. Furthermore, DNBS-induced colitis in Fas/FasL-deficient mice did not result in depletion of colonic Tregs. Finally, adoptively transferred synergic Fas-/- but not Fas+/+ Tregs were protected from depletion in the colon 8 days post-DNBS treatment, thus substantiating the hypothesis that inflammation-induced local depletion of Foxp3+ Tregs in the colon of mice occurs via Fas/FasL-mediated death.     

Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells

IL-10-producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10-producing B cells (IL-10(-/-)B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-10(-/-) B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (μMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-10(-/-) mice increased Foxp3(+) Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10(+/+) B cells established longer contact times with arthritogenic CD4(+)CD25(-) T cells compared with IL-10(-/-) B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4(+) T cells. Thus, IL-10-producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells.     

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.     

Suppression of murine allergic airway disease by IL-2:anti-IL-2 monoclonal antibody-induced regulatory T cells

Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10(+)CD4(+)CD25(+) and Foxp3(+)CD4(+)CD25(+)) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10(+/+), but not IL-10(-/-) Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease.