Experience-driven development: effector/memory-like alphaE+Foxp3+ regulatory T cells originate from both naive T cells and naturally occurring naive-like regulatory T cells

Naturally occurring Foxp3+CD25+CD4+ regulatory T cells (Treg) have initially been described as anergic cells; however, more recent in vivo studies suggest that Tregs vigorously proliferate under both homeostatic as well as inflammatory conditions. We have previously identified a subset of murine CD4+ Tregs, which is characterized by expression of the integrin alphaEbeta7 and which displays an effector/memory-like phenotype indicative of Ag-specific expansion and differentiation. In the present study, the alphaE+ Treg subset was found to contain a large fraction of cycling cells under homeostatic conditions in healthy mice. Using an adoptive transfer system of Ag-specific T cells, we could demonstrate that the vast majority of transferred natural, naive-like CD25+CD4+ Tregs acquired expression of the integrin alphaEbeta7 upon tolerogenic application of Ag via the oral route. In addition, using the same system, Foxp3+ Tregs could be de novo induced from conventional naive CD25-CD4+ T cells, and this conversion was associated with concomitant expression of alphaE. These findings suggest that Tregs expressing the integrin alphaE are effector/memory Tregs with a high turnover rate that can develop in the periphery upon Ag contact under tolerogenic conditions, both from thymic-derived CD25+CD4+ Tregs with a naive-like phenotype as well as from conventional naive T cells.     

Isolation and characterization of naturally occurring subclasses of human peripheral blood T cells with regulatory functions.

By utilizing naturally occurring autoimmune antibodies from patients with juvenile rheumatoid arthritis, we have isolated and functionally characterized two unique subpopulations of T cells. JRA+ T cells, i.e., those identified by sera from these patients, react poorly in response to allogeneic cells, respond to Con A but not PHA, and do not help in the synthesis and secretion of Ig by B cells. In contrast, JRA- T cells, i.e., those not identified by sera from these patients, respond very well to allogeneic cells, proliferate well in response to PHA but not Con A, and more interestingly, can greatly enhance the secretion of Ig by B cells.     

Control of intestinal inflammation by regulatory T cells

Regulatory T(Treg)-cell populations have been identified in a number of disease models. In this review we focus on the role of naturally occurring Treg cells in the control of intestinal inflammation. Specifically, we discuss their mechanism of action with particular emphasis on the role of anti-inflammatory cytokines and cell surface molecules.     

CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells

CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.     

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice

Background

Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice.

Principal Findings

Both effector (CD25⁻, FoxP3⁻) and suppressor (CD25⁺, FoxP3⁺) CD4⁺ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP tranegenes. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4⁺ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4⁺CD25⁻ T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice.

Conclusion

These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.     

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.     

Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring regulatory T cells but to the induction of regulatory functions in conventional CD4+ T cells

Rapamycin is an immunosuppressive drug currently used in different clinical settings. Although the capacity of rapamycin to inhibit the mammalian target of rapamycin serine/threonine protein kinase and therefore T cell cycle progression is well known, its effects are complex and not completely understood. It has been reported recently that TCR-mediated stimulation of murine CD4+ T cells in the presence of rapamycin results in increased proportions of CD4+ T cells with suppressive functions, suggesting that the drug may also exert its immunosuppressive activity by promoting the selective expansion of naturally occurring CD4+ regulatory T cells (Treg). In this study, we show that stimulation of human circulating CD4+ T cells in the presence of rapamycin results indeed in highly increased suppressor activity. By assessing the effect of rapamycin on the growth of nonregulatory and Treg populations of defined differentiation stages purified ex vivo from circulating CD4+ T cells, we could demonstrate that this phenomenon is not due to a selective expansion of naturally occurring Tregs, but to the capacity of rapamycin to induce, upon TCR-mediated stimulation, suppressor functions in conventional CD4+ T cells. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.     

Peptidoglycan recognition protein Pglyrp2 protects mice from psoriasis-like skin inflammation by promoting regulatory T cells and limiting Th17 responses

Skin protects the body from the environment and is an important component of the innate and adaptive immune systems. Psoriasis is a frequent inflammatory skin disease of unknown cause determined by multigenic predisposition, environmental factors, and aberrant immune response. Peptidoglycan recognition proteins (Pglyrps) are expressed in the skin, and we report in this article that they modulate sensitivity in an experimentally induced mouse model of psoriasis. We demonstrate that Pglyrp2(-/-) mice (but not Pglyrp3(-/-) and Pglyrp4(-/-) mice) are more sensitive to the development of 12-O-tetradecanoylphorbol 13-acetate-induced psoriasis-like inflammation, whereas Pglyrp1(-/-) mice are less sensitive. The mechanism underlying this increased sensitivity of Pglyrp2(-/-) mice to 12-O-tetradecanoylphorbol 13-acetate-induced psoriasis-like inflammation is reduced recruitment of regulatory T cells to the skin and enhanced production and activation of Th17 cells in the skin in Pglyrp2(-/-) mice, which results in more severe inflammation and keratinocyte proliferation. Thus, in wild type mice, Pglyrp2 limits overactivation of Th17 cells by promoting accumulation of regulatory T cells at the site of inflammation, which protects the skin from the exaggerated inflammatory response.     

Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10

Mucosal tissues require constant immune surveillance to clear harmful pathogens while maintaining tolerance to self Ags. Regulatory T cells (Tregs) play a central role in this process and expression of alpha(E)beta(7) has been reported to define a subset of Tregs with tropism for inflamed tissues. However, the signals responsible for recruiting Tregs to epithelial surfaces are poorly understood. We have isolated a subset of CCR10-expressing CD25+CD4+Foxp3+ Tregs with potent anti-inflammatory properties from chronically inflamed human liver. The CCR10+ Tregs were detected around bile ducts that expressed increased levels of the CCR10 ligand CCL28. CCL28 was secreted by primary human cholangiocytes in vitro in response to LPS, IL-1beta, or bile acids. Exposure of CCR10+ Tregs to CCL28 in vitro stimulated migration and adhesion to mucosal addressin cell adhesion molecule-1 and VCAM-1. Liver-derived CCR10+ Tregs expressed low levels of CCR7 but high levels of CXCR3, a chemokine receptor associated with infiltration into inflamed tissue and contained a subset of alpha(E)beta7(+) cells. We propose that CXCR3 promotes the recruitment of Tregs to inflamed tissues and CCR10 allows them to respond to CCL28 secreted by epithelial cells resulting in the accumulation of CCR10+ Tregs at mucosal surfaces.     

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma

Recent data suggest a decreased prevalence of IFN‐γ‐producing T lymphocytes (Type 1 T cells) in tumor‐bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti‐tumor responses.     

Simian T cell leukemia virus type-1-specific killer T cells in naturally infected African green monkey carriers

Studies were made on the cellular immunity of 13 African green monkeys (Cercopithecus aethiops) naturally infected with Simian T cell leukemia virus type 1 (STLV-1), closely related to human T cell leukemia virus type 1. They were classified into 3 groups: 1) progressed carrier, 2) carrier, and 3) normal control. This grouping was made according to their hematologic features, i.e., number of peripheral white blood cells, existence of blastoid cells, presence of STLV-1 Ag on PBL and anti-STLV-1 antibody titers. None of the STLV-1 carriers showed any clinical signs, but STLV-1-specific killer T cells was detected in these PBL without in vitro stimulation. In vitro studies on Ag stimulation showed that the STLV-1-specific killer T cells had been fully activated in vivo, and that no augmentation of in vitro stimulation with STLV-1 Ag was necessary, and that primary in vitro stimulation of normal control PBL was not sufficient to induce specific killer T cells. In addition NK cell activity in PBL of infected monkeys were significantly higher than those in the uninfected.     

Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ-competent subset of human CD4(+)CD127(lo/-)CD25(+) Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ(+) subset by flow cytometry. Purified IFN-γ(+) Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3(+)IFN-γ(+) fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ(+) Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.     

Schistosomes induce regulatory features in human and mouse CD1d(hi) B cells: inhibition of allergic inflammation by IL-10 and regulatory T cells

Chronic helminth infections, such as schistosomes, are negatively associated with allergic disorders. Here, using B cell IL-10-deficient mice, Schistosoma mansoni-mediated protection against experimental ovalbumin-induced allergic airway inflammation (AAI) was shown to be specifically dependent on IL-10-producing B cells. To study the organs involved, we transferred B cells from lungs, mesenteric lymph nodes or spleen of OVA-infected mice to recipient OVA-sensitized mice, and showed that both lung and splenic B cells reduced AAI, but only splenic B cells in an IL-10-dependent manner. Although splenic B cell protection was accompanied by elevated levels of pulmonary FoxP3(+) regulatory T cells, in vivo ablation of FoxP3(+) T cells only moderately restored AAI, indicating an important role for the direct suppressory effect of regulatory B cells. Splenic marginal zone CD1d(+) B cells proved to be the responsible splenic B cell subset as they produced high levels of IL-10 and induced FoxP3(+) T cells in vitro. Indeed, transfer of CD1d(+) MZ-depleted splenic B cells from infected mice restored AAI. Markedly, we found a similarly elevated population of CD1d(hi) B cells in peripheral blood of Schistosoma haematobium-infected Gabonese children compared to uninfected children and these cells produced elevated levels of IL-10. Importantly, the number of IL-10-producing CD1d(hi) B cells was reduced after anti-schistosome treatment. This study points out that in both mice and men schistosomes have the capacity to drive the development of IL-10-producing regulatory CD1d(hi) B cells and furthermore, these are instrumental in reducing experimental allergic inflammation in mice.     

Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.     

Interaction of mannose-binding protein with associated serine proteases: effects of naturally occurring mutations

Mannose-binding protein (MBP; mannose-binding lectin) forms part of the innate immune system. By binding directly to carbohydrates on the surfaces of potential microbial pathogens, MBP and MBP-associated serine proteases (MASPs) can replace antibodies and complement components C1q, C1r, and C1s of the classical complement pathway. In order to investigate the mechanisms of MASP activation by MBP, the cDNAs of rat MASP-1 and -2 have been isolated, and portions encompassing the N-terminal CUB and epidermal growth factor-like domains have been expressed and purified. Biophysical characterization of the purified proteins indicates that each truncated MASP is a Ca(2+)-independent homodimer in solution, in which the interacting modules include the N-terminal two domains. Binding studies reveal that both MASPs associate independently with rat MBP in a Ca(2+)-dependent manner through interactions involving the N-terminal three domains. The biophysical properties of the truncated MASPs indicate that the interactions with MBP leading to complement activation differ significantly from those between components C1q, C1r, and C1s of the classical pathway. Analysis of MASP binding by rat MBP containing naturally occurring mutations equivalent to those associated with human immunodeficiency indicates that binding to both truncated MASP-1 and MASP-2 proteins is defective in such mutants.     

Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy

Mutations in the gene encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) have been reported in patients with congenital generalized lipodystrophy (CGL). AGPAT2, a 278 amino acid protein, belongs to the acyltransferase enzyme family, and has two conserved motifs, NHX(4)D and EGTR, involved in the enzymatic activity. The AGPATs catalyze acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) during the biosynthesis of glycerophospholipids and triglycerides from glycerol-3-phosphate. The present studies were designed to determine the enzymatic activity of AGPAT2 mutants found in CGL patients to provide a molecular explanation for the phenotype and to obtain additional information about the structure-function relationship of AGPAT2 protein. The enzymatic activities of the wild type AGPAT2 and mutants were determined in cell lysates of overexpressing Chinese hamster ovary cells by measuring the conversion of [(3)H]LPA to [(3)H]PA in the presence of oleoyl-coenzyme A. Whereas, the R68X, 221delGT, 252delMRT, D180fsX251, and V167fsX183 mutants had markedly reduced enzymatic activity (median <15% of the wild type), the mutants, 140delF, G136R, and L228P, retained median activity ranging from 15% to 40% of the wild type enzyme. However, the missense mutant, A239V, had 90% of the wild type activity. We suggest that reduction in AGPAT2 enzymatic activity underlies the loss of adipose tissue in CGL. Our observations reveal an important role of various carboxy-terminal residues in determining the enzymatic activity of AGPAT2.     

Alternatively activated myeloid cells limit pathogenicity associated with African trypanosomiasis through the IL-10 inducible gene selenoprotein P

Uncontrolled inflammation is a major cause of tissue injury/pathogenicity often resulting in death of a host infected with African trypanosomes. Thus, comparing the immune response in hosts that develop different degrees of disease severity represents a promising approach to discover processes contributing to trypanosomiasis control. It is known that limitation of pathogenicity requires a transition in the course of infection, from an IFN-gamma-dependent response resulting in the development of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alternatively activated myeloid cells (M2). Herein, mechanisms and downstream effectors by which M2 contribute to lower the pathogenicity and the associated susceptibility to African trypanosomiasis have been explored. Gene expression analysis in IL-10 knockout and wild-type mice, that are susceptible and relatively resistant to Trypanosoma congolense infection, respectively, revealed a number of IL-10-inducible genes expressed by M2, including Sepp1 coding for selenoprotein P. Functional analyses confirm that selenoprotein P contributes to limit disease severity through anti-oxidant activity. Indeed, Sepp1 knockout mice, but not Sepp1(Delta)(240-361) mice retaining the anti-oxidant motif but lacking the selenium transporter domain of selenoprotein P, exhibited increased tissue injury that associated with increased production of reactive oxygen species and increased apoptosis in the liver immune cells, reduced parasite clearance capacity of myeloid cells, and decreased survival. These data validate M2-associated molecules as functioning in reducing the impact of parasite infection on the host.     

TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells

TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-gamma, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-beta, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.