CD4+ CD25+ [corrected] regulatory T cells render naive CD4+ CD25- T cells anergic and suppressive

CD4(+) CD25(+) Foxp3(+) naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4(+) CD25(-) T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG(-/-)) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4(+) CD25(-) T cells and CD8(+) T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-beta partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4(+) CD25(-) responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.     

Naturally anergic and suppressive CD25(+)CD4(+) T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation

A CD4(+) T cell subpopulation defined by the expression levels of a particular cell surface molecule (e.g. CD5, CD45RB, CD25, CD62L or CD38) bears an autoimmune-preventive activity in various animal models. Here we show that the expression of CD25 is highly specific, when compared with other molecules, in delineating the autoimmune-preventive immunoregulatory CD4(+) T cell population. Furthermore, although CD25 is an activation marker for T cells, the following findings indicate that immunoregulatory CD25(+)CD4(+) T cells are functionally distinct from activated or anergy-induced T cells derived from CD25(-)CD4(+) T cells. First, the former are autoimmune-preventive in vivo, naturally unresponsive (anergic) to TCR stimulation in vitro and, upon TCR stimulation, able to suppress the activation/proliferation of other T cells, whereas the latter scarcely exhibit the in vivo autoimmune-preventive activity or the in vitro suppressive activity. Second, such activated or anergy-induced CD25(-) spleen cells produce various autoimmune diseases when transferred to syngeneic athymic nude mice, whereas similarly treated normal spleen cells, which include CD25(+)CD4(+) T cells, do not. Third, upon polyclonal T cell stimulation, CD25(+)CD4(+) T cells express CD25 at higher levels and more persistently than CD25(-)CD4(+) T cell-derived activated T cells; moreover, when the stimulation is ceased, the former revert to the original levels of CD25 expression, whereas the latter lose the expression. These results collectively indicate that naturally anergic and suppressive CD25(+)CD4(+) T cells present in normal naive mice are functionally and phenotypically stable, distinct from other T cells, and play a key role in maintaining immunologic self-tolerance.     

Daily subcutaneous injections of peptide induce CD4+ CD25+ T regulatory cells

Peptide immunotherapy is being explored to modulate varied disease states; however, the mechanism of action remains poorly understood. In this study, we investigated the ability of a subcutaneous peptide immunization schedule to induce of CD4(+) CD25(+) T regulatory cells. DO11.10 T cell receptor (TCR) transgenic mice on a Rag 2(-/-) background were injected subcutaneously with varied doses of purified ovalbumin (OVA(323-339)) peptide daily for 16 days. While these mice have no CD4(+) CD25(+) T regulatory cells, following this injection schedule up to 30% of the CD4(+) cells were found to express CD25. Real-time quantitative polymerase chain reaction (QPCR) analysis of the induced CD4(+) CD25(+) T cells revealed increased expression of forkhead box P3 (FoxP3), suggesting that these cells may have a regulatory function. Proliferation and suppression assays in vitro utilizing the induced CD4(+) CD25(+) T cells revealed a profound anergic phenotype in addition to potent suppressive capability. Importantly, co-injection of the induced CD4(+) CD25(+) T cells with 5,6-carboxy-succinimidyl-fluorescence-ester (CFSE)-labelled naive CD4(+) T cells (responder cells) into BALB/c recipient mice reduced proliferation and differentiation of the responder cells in response to challenge with OVA(323-339) peptide plus adjuvant. We conclude that repeated subcutaneous exposure to low-dose peptide leads to de novo induction of CD4(+) CD25(+) FoxP3(+) T regulatory cells with potent in vitro and in vivo suppressive capability, thereby suggesting that one mechanism of peptide immunotherapy appears to be induction of CD4(+) CD25(+) Foxp3(+) T regulatory cells.     

Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state

Elimination of CD25+ T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, leads to spontaneous development of various autoimmune diseases. These immunoregulatory CD25+CD4+ T cells are naturally unresponsive (anergic) in vitro to TCR stimulation, and, upon stimulation, suppress proliferation of CD25-CD4+ T cells and CD8+ T cells. The antigen concentration required for stimulating CD25+CD4+ T cells to exert suppression is much lower than that required for stimulating CD25-CD4+ T cells to proliferate. The suppression, which results in reduced IL-2 production by CD25-CD4+ T cells, is dependent on cellular interactions on antigen-presenting cells (and not mediated by far-reaching or long-lasting humoral factors or apoptosis-inducing signals) and antigen non-specific in its effector phase. Addition of high doses of IL-2 or anti-CD28 antibody to the in vitro T cell stimulation culture not only breaks the anergic state of CD25+CD4+ T cells, but also abrogates their suppressive activity simultaneously. Importantly, the anergic/suppressive state of CD25+CD4+ T cells appeared to be their basal default condition, since removal of IL-2 or anti-CD28 antibody from the culture milieu allows them to revert to the original anergic/suppressive state. Furthermore, transfer of such anergy/suppression-broken T cells from normal mice produces various autoimmune diseases in syngeneic athymic nude mice. These results taken together indicate that one aspect of immunologic self-tolerance is maintained by this unique CD25+CD4+ naturally anergic/suppressive T cell population and its functional abnormality directly leads to the development of autoimmune disease.      

A clonal model for human CD8+ regulatory T cells: unrestricted contact-dependent killing of activated CD4+ T cells

Previous studies in murine systems have demonstrated that CD8(+) Treg cells down-regulate immune responses in vivo through suppressing activated CD4(+) T cells. Here we describe novel regulatory CD8(+) T-cell clones isolated from healthy human peripheral blood following in vitro stimulation with autologous Epstein-Barr virus (EBV)-specific CD4(+) T cells. TCR activation of CD4(+) target T cells was required for CD8(+) Treg cells to exert suppressive activity, which was mediated through lysis of CD4(+) targets in a cell contact-dependent manner. Suppression was independent of Foxp3 expression in CD8(+) Treg cells, HLA compatibility between CD8(+) Treg cells and CD4(+) target cells and antigen-specificity of CD4(+) target T cells. CD8(+) Treg clones expressed CD3 and a variety of TCR V(β) chains as well as CD56, CD69, CD62L and CD95 but did not express CD16, CD161, CXCR4 and CCR7. When used together, antibodies specific for CD11a/CD18 and CD8 inhibited suppressive activity of CD8(+) Treg clones. The ability to establish clonal CD8(+) T cells that maintain regulatory function in vitro will facilitate further studies to define this population in vivo and to identify the mechanisms used for recognition and suppression of activated target cells.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Alloantigen-induced regulatory CD8+CD103+ T cells

Regulatory T cells (Tregs) appear of great importance in the balance between alloreactivity and tolerance and subsets of both CD4(+) and CD8(+) T cells have been recognized to function as regulatory T cells after allogenic transplantation. Among the CD8(+) T-cell subsets, the CD103(+) cells were most recently identified as regulatory. In this review, we describe their phenotypical and functional properties, as well as their relevance for the alloimmune response in vivo. These CD8(+)CD103(+) Tregs are generated within mixed lymphocyte cultures (MLCs) and are elevated by additional transforming growth factor-beta. Interestingly, myeloid dendritic cells are the responsible cell type for induction of CD103(+) Tregs. Allostimulated CD8(+)CD103(+) Tregs display an antigen-experienced effector phenotype with limited effector functions such as cytotoxicity and interferon-gamma production and show a reduced proliferation capacity after restimulation. Beside this anergic phenotype, CD8(+)CD103(+) Tregs are able to suppress alloreactive effector T cells. Through intracellular cytokine staining and transwell assays, we showed that the mechanism of suppression is cytokine independent, but close cell-cell contact is required for suppression.     

Human anergic/suppressive CD4(+)CD25(+) T cells: a highly differentiated and apoptosis-prone population

Anergic/suppressive CD4(+)CD25(+) T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4(+)CD25(+) T cell subset, which constitutes 7-10 % of CD4(+) T cells in peripheral blood and tonsil. These cells are a CD45RO(+)CD45RB(low) highly differentiated primed T cell population that is anergic to stimulation. Depletion of this small subset from CD4(+) T cells significantly enhances proliferation by threefold in the remaining CD4(+)CD25(-) T cells, while the addition of isolated CD4(+)CD25(+) T cells to CD4(+)CD25(-) T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL-4, IL-10 or TGF-beta and is cell-contact dependent. Isolated CD4(+)CD25(+) T cells are susceptible to apoptosis that is associated with low Bcl-2 expression, but this death can be prevented by IL-2 or fibroblast-secreted IFN-beta. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis-prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.     

Aging-dependent generation of suppressive CD4+CD25-R123loCD103+ T cells in mice

Advancing age is associated with significant alterations in immune functions, including a decline in CD4 T cell function, in both mice and humans. In our previous report, we showed that CD4(+)CD25(-) T cells in aged (24-month-old) mice, especially after in vitro pre-stimulation of these cells, exhibit hyporesponsive and suppressive properties. We examined here whether the suppressive activity of aged CD4(+)CD25(-) T cells is ascribable to a particular population within these cells. In vitro analyses revealed that cell populations rapidly extruding Rhodamine-123 (R123) (referred to as R123(lo) cells) in aged CD4(+)CD25(-) T cells have a more potent suppressive function compared with R123(hi) populations.In addition, CD103(+) cells in freshly prepared aged CD4(+)CD25(-)R123(lo) T cells had a most potent suppressive activity. Both R123(hi) and R123(lo) populations had individually stronger suppressive activity after pre-stimulation than before pre-stimulation. Furthermore, the R123(lo) population in young CD4(+)CD25(-) T cells also had different properties from R123(hi) T cells: low responsiveness, no additive effect in proliferation assays, and the gain of a suppressive function after in vitro pre-stimulation. Taken together, these results suggest that CD4(+)CD25(-)R123(lo) T cells are a unique population within whole CD4(+)CD25(-) T cells. This population exists in the early stage of the life span, and the properties in this population become obvious with aging, that is the gain of their suppressive activity.     

Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells

CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3(-) T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3(-) T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy.     

TGF-beta1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets

Although forkhead box p3 (Foxp3) expression is restricted to naturally occurring CD4(+) regulatory T cells (T(REG)), little is known about the various signals that regulate it in T cells. As TGF-beta has been reported to modulate Foxp3 expression in T cells, we investigated its effects on the induction or maintenance of regulatory functions in different CD4(+) T cell subsets. TGF-beta1 priming was able to promote differentiation of T(REG) cells from nonregulatory CD4(+)CD25(-) T cells in a concentration-dependent manner through Foxp3 induction. As CD4(+)CD25(-) T cells remain a highly heterogeneous population with variable degrees of antigen experience, we then examined the effect of TGF-beta1 on naive CD4(+)CD25(-)CD45RB(HIGH) T cells. Freshly isolated or TGF-beta1-treated CD4(+)CD25(-)CD45RB(HIGH) T cells never displayed any regulatory functions or significant Foxp3 expression following TCR activation. In stark contrast, freshly isolated CD4(+)CD25(-)CD45RB(LOW) cells, albeit expressing low levels of Foxp3 mRNA and protein, were unable to suppress CD4(+) effector T cell proliferation but acquired regulatory activity and de novo Foxp3 expression following TGF-beta1 exposure. Furthermore, suppression was IL-10-dependent, as anti-IL-10 receptor antibody treatment abrogated this suppression completely, consistent with the ability of TGF-beta1-treated CD4(+)CD25(-)CD45RB(LOW) to synthesize IL-10 upon restimulation in vitro. Last, we show that TGF-beta1 treatment or blockade did not lead to enhanced expansion or function of naturally occurring CD4(+)CD25(+) T(REG) cells, although it maintained Foxp3 mRNA and protein expression. Altogether, TGF-beta1 promotes the induction of IL-10-secreting CD4(+) T(REG) cells from CD4(+)CD25(-)CD45RB(LOW) precursors through de novo Foxp3 production and maintains natural T(REG) cell peripheral homeostasis by sustaining Foxp3 expression.     

Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts

CD4(+)CD25(+) regulatory T cells (Tregs) are well known to suppress immunopathology induced in lymphopenic animals following T cell reconstitution, including acute graft-versus-host disease (GVHD) post-bone marrow transplantation. The regulatory potential of this subset in nonlymphopenic hosts and in chronic, Th2-mediated GVHD is less clear. We have generated alloantigen-specific cells from CD4(+)CD25(+) populations stimulated with MHC-disparate dendritic cells and found them to express a stable Treg forkhead box p3(+) phenotype with enhanced suppressive activity mediated by cell contact. When transferred into nonlymphopenic F1 hosts, nonspecific Tregs proliferated as rapidly as CD4(+)CD25(-) cells but displayed distinct growth kinetics in vitro. Tregs, expanded in response to alloantigen in vitro, displayed greatly enhanced suppressive activity, which was partially antigen-specific. They were effective inhibitors of chronic GVHD, blocking donor cell engraftment, splenomegaly, autoantibody production, and glomerulonephritis. CD25(+) and CD25(-) cells were equally susceptible to inhibition by immunosuppressive drugs targeting TCR signaling and rapamycin, but Tregs were resistant to inhibition by dexamethasone. The data indicate that alloantigen-driven expansion, rather than homeostatic proliferation, is key to the effectiveness of CD4(+)CD25(+) Tregs in GVHD and suggest that cellular therapy with alloantigen-induced Tregs in combination with glucocorticoid treatment would be effective in prevention of chronic GVHD after immune reconstitution.     

P59(fyn) is upregulated in anergic CD8+ T cells

The ultimate goal in clinical transplantation is achievement of graft tolerance. Despite long-term immunosuppression, alloantigens on transplants elicit alloresponses that can initiate organ rejection. Acute rejection is mediated by CD8(+) cytotoxic T cells, whereas chronic rejection is a result of many factors including non-immunological events. The aim of this study was to examine the molecular requirements of T cell anergy, a cellular state that is an integral component of tolerance in vivo. In vitro, the tolerant state is usually best represented by T cell anergy, which is defined by loss of the ability of T cells to produce and secrete interleukin-2 upon restimulation. In the literature, molecular changes in anergic CD4(+) T cells have been studied in great detail, but only little is known about functional and biochemical characteristics of anergic CD8(+) T lymphocytes. In this study, we demonstrate, that CD8(+) T cells are rendered anergic by TCR stimulation without costimulation. They exhibit impaired interleukin-2 production and tyrosine-phosphorylation, but markedly upregulated p59(fyn) expression, which could be shown to be an early event during anergization. Anergic CD8(+) T lymphocytes show elevated surface expression of early activation markers as well as costimulatory molecules, especially that of CTLA4. These results, are an important component for the discovery of potential molecular targets, which contribute to the development and maintenance of tolerance.     

A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.     

CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor

CD4(+)CD25(+) immunoregulatory T cells represent a unique lineage of thymic-derived cells that potently suppress both in vitro and in vivo effector T cell function. We analyzed CD4(+)CD25(+) and CD4(+)CD25(-) T cells by DNA microarray, identifying 29 genes differentially expressed in the resting subpopulations, and 77 that were differentially expressed following activation. Most of these genes were elevated in the CD4(+)CD25(+) population, suggesting a previously activated phenotype. Among these were a number of genes that antagonize signaling, including members of the SOCS family, which may contribute to their anergic phenotype. Multiple cell surface receptors also had increased expression in CD4(+)CD25(+) cells, including GITR, a member of the TNF receptor superfamily. Importantly, antibodies to GITR abrogated suppression, demonstrating a functional role for this receptor in regulating the CD4(+)CD25(+) T cell subset.     

Differential response of CD4+ V7+ and CD4+ V7− T cells to T cell receptor-dependent signals: CD4+ V7+ T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen

V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells. In the current study, CD4⁺ V7+ and CD4⁺ V7? T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4⁺ V7+ T cells alone produced IL-2 and proliferated vigorously. By contrast, CD4⁺ V7? cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28 antibody were added to the cultures. The enhancement of the V7? T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up-regulate such signals through their interaction with counter-receptors on T cells. To assess the role of V7 in a system independent of co-stimulation, CD4⁺ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.     

CTLA-4 x Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells

CTLA-4 x Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4 x Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4+CD25- T cells into Foxp3+ regulatory T (T(reg)) cells, as well as to expand their numbers. The CD4+CD25+Foxp3+ T(reg) generated by CTLA-4 x Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4 x Ig increases the percentage of CD4+CD25(hi)Foxp3+ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4+CD25- T cells into T(reg) cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4 x Ig-driven T(reg) induction may be predicated upon active CTLA-4 x Ig to B7-2 signaling within APC, which elicits from them T(reg)-inducing potential. These findings extend CTLA-4 x Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.