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.     

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

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

Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis

Regulatory CD4(+)CD25(+) T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-gamma production by CD4(+)CD25(-) and CD8(+) T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-beta, suggesting a mechanism of linked immunosuppression. Tregs/CD4(+)CD25(-) T cell cocultures lead to similar changes in IFN-gamma and IL-10 secretion in patients and controls, whereas increased TGF-beta secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8(+) T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4(+)CD25(-) T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4(+)CD25(-) T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4(+)CD25(-) T cell spontaneous apoptosis may contribute to the development of autoimmunity.     

Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein (residues 178-191) for SJL mice.

We previously described a synthetic peptide of myelin proteolipid protein (PLP), peptide 139-151, which induces experimental allergic encephalomyelitis in SJL/J (H-2s) mice. We have now identified an additional determinant, PLP residues 178-191, that is also a potent encephalitogen in this strain. When PLP peptide 178-191 was compared with peptide 139-151 on an equimolar basis, the day of onset of disease induced by PLP 178-191 was earlier, but the incidence, severity, and histologic features were indistinguishable. Lymph node cells from animals immunized with the whole PLP molecule responded to both PLP 178-191 and 139-151, suggesting immunologic codominance of the two epitopes. PLP 178-191 elicited stronger proliferative responses and this may relate to the earlier onset of disease induced with this peptide. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice. The presence of multiple encephalitogenic codominant PLP epitopes within a single mouse strain emphasizes the complexity of the immune response to PLP and its potential as a target Ag in autoimmune demyelinating diseases.     

CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis

We identified CD8(+)CD122(+) regulatory T cells (Tregs) and demonstrated their importance in the maintenance of immune homeostasis and in the recovery from experimental autoimmune encephalomyelitis. In this paper, we show that CD8(+)CD122(+) Tregs effectively prevent and cure colitis in a mouse model. In our experiments, colitis was induced in lymphocyte-deficient RAG-2(-/-) mice by transferring CD4(+)CD45RB(high) cells that were excluded with CD4(+) Tregs. Cotransfer of CD8(+)CD122(+) cells clearly suppressed the development of colitis, and this suppressive effect was similar to that of CD4(+)CD45RB(low) cells that were mostly CD4(+) Tregs. CD8(+)CD122(+) cells obtained from IL-10(-/-) mice were unable to suppress colitis, indicating that IL-10 is an important effect-transmitting factor in the suppression of colitis. CD8(+)CD122(+) cells showed a suppressive effect when they were transferred 4 wk after CD4(+)CD45RB(high) cells, indicating the therapeutic potential of CD8(+)CD122(+) cells. A mixture of CD8(+)CD122(+) cells and CD4(+)CD45RB(low) cells was far more effective than single Tregs, indicating the synergistic effect of these Tregs. These overall findings demonstrate the potential role of CD8(+) Tregs, and possibly together with CD4(+) Tregs, in the medical care of inflammatory bowel disease patients.     

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.     

Peptide specificity of thymic selection of CD4+CD25+ T cells.

The CD4(+)CD25(+) regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4(+)CD25(+) cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4(+)CD25(+) cells in the thymus. By comparing the development of CD4(+)CD25(-) and CD4(+)CD25(+) cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4(+)CD25(+) cells appear during natural selection of CD4(+) T cells. The proportion of CD4(+)CD25(+) cells in the population of CD4(+) thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4(+)CD25(+) cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4(+) T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4(+)CD25(+) thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.     

Superantigen-induced proliferation of human CD4+CD25- T cells is followed by a switch to a functional regulatory phenotype

Bacterial superantigens are potent T cell activators. In humans they cause toxic shock and scarlet fever, and they are implicated in Kawasaki's disease, autoimmunity, atopy, and sepsis. Their function remains unknown, but it may be to impair host immune responses increasing bacterial carriage and transmission. Regulatory (CD25(+)FOXP3(+)) T cells (Tregs) play a role in controlling inflammatory responses to infection. Approximately 2% of circulating T cells are naturally occurring Tregs (nTregs). Conventional Ag stimulation of naive FOXP3(-) T cells induces Ag-specific Tregs. Polyclonal T cell activation has been shown to produce non-Ag-specific Tregs. Because superantigens are unique among microbial virulence factors in their ability to trigger polyclonal T cell activation, we wanted to determine whether superantigen stimulation of T cells could induce non-Ag-specific Tregs. We assessed the effect of superantigen stimulation of human T cells on activation, regulatory markers, and cytokine production by flow cytometry and T cell suppression assays. Stimulation of PBMCs with staphylococcal exotoxin A and streptococcal pyrogenic exotoxins A and K/L resulted in dose-dependent FOXP3 expression. Characterization of this response for streptococcal pyrogenic exotoxin K/L confirmed its Vβ specificity, that CD25(+)FOXP3(+) cells arose from CD25(-) T cells and required APCs. These cells had increased CTLA-4 and CD127 expression, typical of the recently described activated converted Treg-like cells, and exhibited functional suppressor activity comparable to nTregs. Superantigen-stimulated CD25(+)FOXP3(+) T cells expressed IL-10 at lower superantigen concentrations than was required to trigger IFN-γ production. This study provides a mechanism for bacterial evasion of the immune response through the superantigen induction of Tregs.     

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+ and CD4+CD2+high regulatory T cells in disseminated and localized forms of allergic contact dermatitis: relation to specific cytokines

The aim of this study was to evaluate regulatory T lymphocytes (Tregs) in the course of allergic contact dermatitis (ACD) and to elucidate the role of IL-10 and TGF-β in Tregs activity. Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) cells were determined by flow cytometry in patients with acute disseminated ACD ('ad', n = 36), acute localized ACD ('al', n = 26), and disseminated ACD during remission ('rd', n = 27) as well as in controls (n = 22). Serum levels of cytokines were measured using ELISA. The mean percentage of CD4(+)CD25(+) and CD4(+)CD25(high) cells in patients with ad ACD was significantly higher than in controls (p < 0.01) and the remaining patients (p < 0.05). Both cell populations were significantly elevated in persons with widespread skin lesions (p < 0.05). In ad patients the CD4(+)CD25(+) increased during three weeks of disease, although the significant increase of CD4(+)CD25(high) was noted only in the third week. Patients with ad ACD showed a significantly decreased serum level of TGF-β1 as compared with controls and the remaining ACD patients. IL-10 level did not differ between all groups. The elevated population of CD4(+)CD25(high) cells in ad ACD patients, and its dependence on the extension of skin lesions, suggest a role of Tregs in regulating the course of ACD. The growing Tregs percentages may indicate their peripheral generation during ACD. The development of lesions despite an increased population of Tregs suggests their functional defect. The role of TGF-β1 in the suppressive activity of Tregs cannot be excluded.     

Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells

TNFR2 is predominantly expressed by a subset of human and mouse CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2(+) Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30-40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2(-) Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4(+)CD25(-)TNFR2(+) cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2(+) Tregs was considerably more potent than that of reportedly highly suppressive CD103(+) Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2(+) Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.     

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.     

Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.     

CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4(+)CD25(+) population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4(+) cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4(+) T cell line derived from BALB/c mice immunized with peptide 21-35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4(+)CD25(+) T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.     

GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4(+) T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4(+) T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4(+) CD25(+) T regulatory cells (mRNA levels 10-fold higher than naive CD25(-) T cells). Further investigation demonstrated that CD25(+) Foxp3(+) antigen-specific T cells were induced after a "tolerizing-administration" of antigen and that GRAIL expression correlated with the CD25(+) Foxp3(+) antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25(+) T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity.     

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.     

Regulatory T cell properties of chicken CD4+CD25+ cells

Chicken CD4(+)CD25(+) cells were characterized for mammalian regulatory T cells' suppressive and cytokine production properties. Anti-chicken CD25 mAb was produced in mice and conjugated with a fluorescent tag. The specificity of the Ab against chicken CD25 was confirmed by evaluating Con A-induced CD25 upregulation in thymocytes and by quantifying the CD25 mRNA content of positive and negative cells identified by anti-chicken CD25 Ab. The percentage of CD4(+)CD25(+) cells, expressed as a percentage of CD4(+) cells, in thymus and blood was ～3-7%, in spleen was 10%, and in cecal tonsil, lung, and bone marrow was ～15%. Bursa had no detectable CD4(+)CD25(+) cells. CD25(+) cells were mostly CD4(+) in the thymus, whereas in every other organ studied, CD25(+) cells were distributed between CD4(+) and CD4(-) cells. Chicken thymic CD4(+)CD25(+) cells did not proliferate in vitro in the absence of recombinant chicken IL-2 (rCIL-2). In the presence of rCIL-2, PMA plus ionomycin or Con A stimulated CD4(+)CD25(+) cell proliferation, whereas anti-CD3 plus CD28 did not stimulate CD4(+)CD25(+) cell proliferation. Naive CD4(+)CD25(+) cells had 29-fold more IL-10 mRNA and 15-fold more TGF-β mRNA than the naive CD4(+)CD25(-) cells. Naive CD4(+)CD25(+) had no detectable IL-2 mRNA. Both naive and PMA plus ionomycin-stimulated thymic CD4(+)CD25(+) cells suppressed naive T cell proliferation. The suppressive properties were partially contact dependent. Supplementing CD4(+)CD25(+) cell coculture with rCIL-2 reversed the suppressive properties of CD4(+)CD25(+) cells. Chicken CD4(+)CD25(+) cells have suppressive properties similar to that of mammalian regulatory T cells.     

Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.     

Human peripheral blood T regulatory cells (Tregs), functionally primed CCR4+ Tregs and unprimed CCR4- Tregs, regulate effector T cells using FasL

Regulatory CD25(+)CD4(+) T cells (Tregs) play an important role in the control of peripheral tolerance. In this study we demonstrate that human peripheral blood Tregs can be divided into two distinct populations based on the expression of CCR4. The majority ( approximately 75%) of freshly isolated Tregs express CCR4 and presumably represent memory-type Tregs. Interestingly, CCR4(-) Tregs require anti-CD3 Ab-mediated activation to acquire a regulatory activity, while CCR4(+) Tregs appear to be already primed to suppress the proliferation of CD8(+) T cells. CCR4 is also expressed on CD25(low)CD4(+) T cells (CCR4(+) non-Tregs) that mostly suppress Th1-type polarization without affecting T cell proliferation, presumably via the production of immunomodulatory cytokines like IL-10. In contrast, CCR4(+) Tregs express FasL to primarily regulate T cell proliferation via a contact-mediated process involving FasL/Fas signaling, a major regulatory pathway of T cell homeostasis. Finally, we also demonstrate that the depletion of CCR4(+) T cells leads to Th1-type polarization of CD4(+) T cells and augmentation of CD8(+) T cell responses to tumor Ags.