CD4+CD25- T cells in aged mice are hyporesponsive and exhibit suppressive activity

Studies on humans and rodents have established that functional deterioration of CD4 T cells occurs with aging. We report in this study that approximately 70% of CD4(+)CD25(-) T cell preparations from individual 24-mo-old mice are hyporesponsive to in vitro stimulation with anti-CD3 Ab. The remaining 30% of CD4(+)CD25(-) T cell preparations showing the intermediate or normal responsiveness in the primary stimulation also exhibit the hyporesponsive properties after primary stimulation. Both of these hyporesponsive aged CD4(+)CD25(-) T cells could inhibit the proliferation of cocultured CD4(+)CD25(-) T cells from young mice, like CD4(+)CD25(+) T cells, which have recently been demonstrated as an immune regulator in young mice. Another experiment revealed that hyporesponsive aged CD4(+)CD25(-) T cells arrest the cell division of cocultured young CD4(+)CD25(-) T cells. The suppressive activity observed in aged CD4(+)CD25(-) T cells is aging-dependent, not mediated by humoral factors, cell-contact dependent, and broken by the addition of IL-2 or anti-GITR Ab, but not by anti-CTLA-4 Ab. These studies show that aging not only leads to a decline in the ability to mount CD4(+)CD25(-) T cell responses, but at the same time, also renders these aged CD4(+)CD25(-) T cells suppressive.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

Superantigen-induced regulatory T cells display different suppressive functions in the presence or absence of natural CD4+CD25+ regulatory T cells in vivo

Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4(+)CD25(+) regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4(+)CD25(+) regulatory T cells. In this study, we demonstrate that CD4(+)CD25(+) as well as CD4(+)CD25(-) T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4(+)CD25(+) regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4(+) T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4(+)CD25(-) T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.     

Differential control of T regulatory cell proliferation and suppressive activity by mature plasmacytoid versus conventional spleen dendritic cells

Anergy and suppression are cardinal features of CD4(+)CD25(+)Foxp3(+) T cells (T regulatory cells (Treg)) which have been shown to be tightly controlled by the maturation state of dendritic cells (DC). However, whether lymphoid organ DC subsets exhibit different capacities to control Treg is unclear. In this study, we have analyzed, in the rat, the role of splenic CD4(+) and CD4(-) conventional DC and plasmacytoid DC (pDC) in allogeneic Treg proliferation and suppression in vitro. As expected, in the absence of exogenous IL-2, Treg did not expand in response to immature DC. Upon TLR-induced maturation, all DC became potent stimulators of CD4(+)CD25(-) T cells, whereas only TLR7- or TLR9-matured pDC induced strong proliferation of CD4(+)CD25(+)Foxp3(+) T cells in the absence of exogenous IL-2. This capacity of pDC to reverse Treg anergy required cell contact and was partially CD86 dependent and IL-2 independent. In suppression assays, Treg strongly suppressed proliferation and IL-2 and IFN-gamma production by CD4(+)CD25(-) T cells induced by mature CD4(+) and CD4(-) DC. In contrast, upon stimulation by mature pDC, proliferating Treg suppressed IL-2 production by CD25(-) cells but not their proliferation or IFN-gamma production. Taken together, these results suggest that anergy and the suppressive function of Treg are differentially controlled by DC subsets.     

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.     

CD4+CD25- T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-beta-dependent mechanism

Murine CD4(+)CD25(+) regulatory cells have been reported to express latency-associated peptide (LAP) and TGF-beta on the surface after activation, and exert regulatory function by the membrane-bound TGF-beta in vitro. We have now found that a small population of CD4(+) T cells, both CD25(+) and CD25(-), can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP(+) cells are also positive for thrombospondin, which has the ability to convert latent TGF-beta to the active form. In the CD4(+)CD45RB(high)-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25(+)LAP(+) and CD25(+)LAP(-) cells, but also by CD25(-)LAP(+) cells. CD4(+)CD25(-)LAP(+) T cells were part of the CD45RB(low) cell fraction. CD4(+)CD25(-)LAP(-)CD45RB(low) cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25(-)LAP(+) cells was abrogated in vivo by anti-TGF-beta mAb. These results identify a new TGF-beta-dependent regulatory CD4(+) T cell phenotype that is CD25(-) and LAP(+).     

Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10

Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.     

Increased CD45RA+ FoxP3(low) regulatory T cells with impaired suppressive function in patients with systemic lupus erythematosus

BACKGROUND: The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4(+)FoxP3(+) T cells into subpopulations during the dynamic SLE development. METHODLOGY/PRINCIPAL FINDINGS: To evaluate the proliferative and suppressive capacities of different CD4(+) T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA(+)FoxP3(low) naive Treg cells (nTreg cells) and CD45RA(-)FoxP3(low) (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA(+)FoxP3(low) nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25(+)CD45RA(+) can be used to defined CD45RA(+)FoxP3(low) nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA(+)FoxP3(low) nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion. CONCLUSIONS/SIGNIFICANCE: Our results indicate that impaired numbers of functional CD45RA(+)FoxP3(low) naive Treg cell and CD45RA(-)FoxP3(low) non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3(+) T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3(+) T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

Intestinal lamina propria retaining CD4+CD25+ regulatory T cells is a suppressive site of intestinal inflammation

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (T(REG)) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4(+)CD25(+) T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4(+) T cells in vitro. Furthermore, cotransfer of intestinal LP CD4(+)CD25(+) T cells prevents the development of chronic colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into SCID mice. When lymphotoxin (LT)alpha-deficient intercrossed Rag2 double knockout mice (LTalpha(-/-) x Rag2(-/-)), which lack mesenteric lymph nodes and Peyer's patches, are transferred with CD4(+)CD45RB(high) T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTalpha(+/+) x Rag2(-/-) mice transferred with CD4(+)CD45RB(high) T cells. Of note, when a mixture of splenic CD4(+)CD25(+) T(REG) cells and CD4(+)CD45RB(high) T cells are transferred into LTalpha(-/-) x Rag2(-/-) recipients, CD4(+)CD25(+) T(REG) cells migrate into the colon and prevent the development of colitis in LTalpha(-/-) x Rag2(-/-) recipients as well as in the control LTalpha(+/+) x Rag2(-/-) recipients. These results suggest that the intestinal LP harboring CD4(+)CD25(+) T(REG) cells contributes to the intestinal immune suppression.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

CD4+CD25high T cell numbers are enriched in the peripheral blood of patients with rheumatoid arthritis

Accumulating evidences support that CD4(+)CD25(high) T regulatory (Treg) cells play an essential role in controlling and preventing autoimmunity. Paradoxically, RA patients have elevated numbers of circulating CD4(+)CD25(high) T cells, however, the inflammation is still ongoing. Further identification of these CD4(+)CD25(high) T cells may contribute to a better understanding of underlying mechanisms. We show here that these CD4(+)CD25(high) T cells were composed of CD4(+)CD25(high)FoxP3(+) Treg cells and activated CD4(+)CD25(high)FoxP3(-) effector cells. Moreover, there were significantly more Treg cells and effector T cells expressing GITR, and more monocytes expressing GITR-L. Thus, although RA patients have elevated numbers of CD4(+)CD25(high) T cells, the suppressive function is not increased, because of the increased number of activated effector T cells. In addition, the GITR-GITR-L system was activated in RA patients, which might lead to diminish suppressive activity of Treg cells and/or lead to resistance of activated effector T cells to suppression by Treg cells, thus, contributing to the ongoing inflammation in RA patients.     

Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.     

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.     

Prevalence of newly generated naive regulatory T cells (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis

The suppressive function of regulatory T cells (T(reg)) is impaired in multiple sclerosis (MS) patients. The mechanism underlying the T(reg) functional defect is unknown. T(reg) mature in the thymus and the majority of cells circulating in the periphery rapidly adopt a memory phenotype. Because our own previous findings suggest that the thymic output of T cells is impaired in MS, we hypothesized that an altered T(reg) generation may contribute to the suppressive deficiency. We therefore determined the role of T(reg) that enter the circulation as recent thymic emigrants (RTE) and, unlike their CD45RO(+) memory counterparts, express CD31 as typical surface marker. We show that the numbers of CD31(+)-coexpressing CD4(+)CD25(+)CD45RA(+)CD45RO(-)FOXP3(+) T(reg) (RTE-T(reg)) within peripheral blood decline with age and are significantly reduced in MS patients. The reduced de novo generation of RTE-T(reg) is compensated by higher proportions of memory T(reg), resulting in a stable cell count of the total T(reg) population. Depletion of CD31(+) cells from T(reg) diminishes the suppressive capacity of donor but not patient T(reg) and neutralizes the difference in inhibitory potencies between the two groups. Overall, there was a clear correlation between T(reg)-mediated suppression and the prevalence of RTE-T(reg), indicating that CD31-expressing naive T(reg) contribute to the functional properties of the entire T(reg) population. Furthermore, patient-derived T(reg), but not healthy T(reg), exhibit a contracted TCR Vbeta repertoire. These observations suggest that a shift in the homeostatic composition of T(reg) subsets related to a reduced thymic-dependent de novo generation of RTE-T(reg) with a compensatory expansion of memory T(reg) may contribute to the T(reg) defect associated with MS.     

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.     

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.