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.     

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.     

CD4+CD25+ T cells as immunoregulatory T cells in vitro

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4(+)25(+) T cells. Following TCR ligation, DO.11.10 CD4(+)25(+) T cells suppress the activation of OT-1 CD8(+)25(-) T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4(+)25(+) T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4(+)25(+) T cell regulation. CTLA-4 blockade failed to abrogate suppression by CD4(+)25(+) T cells in mixing experiments. Although CD4(+)25(+) T cells failed to respond following cross-linking of TCR, they could be induced to proliferate following the addition of exogenous IL-2, allowing the generation of a T cell line from CD4(+)25(+) T cells. After the first in vitro restimulation, CD4(+)25(+) T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.     

Interleukin-7 matures suppressive CD127(+) forkhead box P3 (FoxP3)(+) T cells into CD127(-) CD25(high) FoxP3(+) regulatory T cells

We have identified a novel interleukin (IL)-7-responsive T cell population [forkhead box P3 (FoxP3(+) ) CD4(+) CD25(+) CD127(+) ] that is comparably functionally suppressive to conventional FoxP3(+) CD4(+) CD25(+) regulatory T cells (T(regs) ). Although IL-2 is the most critical cytokine for thymic development of FoxP3(+) T(regs) , in the periphery other cytokines can be compensatory. CD25(+) CD127(+) T cells treated with IL-7 phenotypically 'matured' into the known 'classical' FoxP3(+) CD4(+) CD25(high) CD127(-) FoxP3(+) T(regs) . In freshly isolated splenocytes, the highest level of FoxP3 expression was found in CD127(+) CD25(+) T cells when compared with CD127(-) CD25(+) or CD127(+) CD25(-) cells. IL-7 treatment of CD4(+) CD25(+) T cells induced an increase in the accumulation of FoxP3 in the nucleus in vitro. IL-7-mediated CD25 cell surface up-regulation was accompanied by a concurrent down-regulation of CD127 in vitro. IL-7 treatment of the CD127(+) CD25(+) FoxP3(+) cells also resulted in up-regulation of cytotoxic T lymphocyte antigen 4 without any changes in CD45RA at the cell surface. Collectively, these data support emerging evidence that FoxP3(+) T cells expressing CD127 are comparably functionally suppressive to CD25(+) CD127(-) FoxP3(+) T cells. This IL-7-sensitive regulation of FoxP3(+) T(reg) phenotype could underlie one peripheral non-IL-2-dependent compensatory mechanism of T(reg) survival and functional activity, particularly for adaptive T(regs) in the control of autoimmunity or suppression of activated effector T cells.     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

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.     

Human CD4(+)CD25(+) thymocytes and peripheral T cells have immune suppressive activity in vitro

CD4(+)CD25(+) T cells in mice and rats are capable of transferring protection against organ-specific autoimmune disease and colitis and suppressing the proliferation of other T cells after polyclonal stimulation in vitro. Here we describe the existence in humans of CD4(+)CD25(+) T cells with the same in vitro characteristics. CD4(+)CD8(-)CD25(+) T cells are present in both the thymus and peripheral blood of humans ( approximately 10 % of CD4(+)CD8(-) T cells), proliferate poorly in response to mitogenic stimulation and suppress the proliferation of CD4(+)CD25(-) cells in co-culture. This suppression requires cell contact and can be overcome by the addition of exogenous IL-2. CD4(+)CD25(+) cells from thymus and blood were poor producers of IL-2 and IFN-gamma, and suppressed the levels of these cytokines produced by CD4(+)CD25(-) cells. However, CD4(+)CD25(+) PBL produced higher levels of IL-4 and similar amounts of IL-10 as CD4(+)CD25(-) cells. Regulatory CD4(+)CD25(+) T cells have an activated phenotype in the thymus with expression of CTLA-4 and CD122 (IL-2Rbeta). The fact that CD4(+)CD25(+) regulatory T cells are present with a similar frequency in the thymus of humans, rats and mice, suggests that the role of these cells in the maintenance of immunological tolerance is an evolutionarily conserved mechanism.     

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.     

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.      

Cat allergen peptide immunotherapy reduces CD4(+) T cell responses to cat allergen but does not alter suppression by CD4(+) CD25(+) T cells: a double-blind placebo-controlled study

BACKGROUND: We have previously described both modification of allergen immunotherapy using peptide fragments, and reduced regulation of allergen stimulated T cells by CD4(+) CD25(+) T cells from allergic donors when compared with nonallergic controls. It has been suggested that allergen immunotherapy induces regulatory T cell activity: we hypothesized that allergen peptide immunotherapy might increase suppressive activity of CD4(+) CD25(+) T cells. OBJECTIVE: To examine cat allergen-stimulated CD4 T cell responses and their suppression by CD4(+) CD25(+) T cells before and after cat allergen peptide immunotherapy in a double-blind placebo-controlled study. METHODS: Peripheral blood was obtained and stored before and after peptide immunotherapy or placebo treatment. CD4(+) and CD4(+) CD25(+) were then isolated by immunomagnetic beads and cultured with allergen in vitro. RESULTS: Comparing cells from blood taken before with that after peptide immunotherapy there was a significant reduction in both proliferation and IL-13 production by allergen-stimulated CD4+ T cells, whereas no change was seen after placebo. CD4(+) CD25(+) T cells suppressed both proliferation and IL-13 production by CD4(+) CD25(-) T cells before and after therapy but peptide therapy was not associated with any change in suppressive activity of these cells. CONCLUSION: Allergen peptide immunotherapy alters T cell response to allergen through mechanisms other than changes in CD4(+) CD25(+) T cell suppression.     

Immunoregulatory T cells in autoimmunity

The aim of this work was to discuss the current knowledge concerning regulatory T cells in the pathogenesis of autoimmune diseases. CD4(+) T cells that constitutively express CD25 exhibit powerful suppressive properties. Such cells have been denominated regulatory T cells (T(R)). Alterations in T(R) cells are known to cause organ-specific autoimmune disease in animal models. These cells are anergic when stimulated via their TCR but proliferate when co-stimulated with IL-2. A particular characteristic is that CD4(+)CD25(+) T cells inhibit the proliferative responses of CD4(+)CD25(-) T cells by suppressing the capacity of the responders to transcribe IL-2. The survival and/or expansion of this regulatory subset in the periphery appears to need the availability of IL-2, the components of the IL-2R, as well as cell surface costimulatory molecules. Cytokine participation has been shown in many of the in vivo models of autoimmunity where regulatory cells participate, providing evidence in favour of a role for IL-10, transforming growth factor-beta and IL-4. The behavior and possible participation of regulatory T cells in human disease is still a poorly explored topic but their pathogenic role is warranted.     

Preferential replication of FIV in activated CD4(+)CD25(+)T cells independent of cellular proliferation

Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4(+) cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4(+) cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4(+)CD25(+) and CD4(+)CD25(-) cells are susceptible to FIV infection in vitro and in vivo, only CD4(+)CD25(+) cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4(+)CD25(-) cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4(+)CD25(-) cells, CD4(+)CD25(+) cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4(+)CD25(+) cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4(+)CD25(-) cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation.     

Antigen-specific,CD4+CD25+ regulatory T cell clones induced in Peyer's patches

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.     

Generation of highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4(+)CD25(-) T cells leads to generation of adaptive CD4(+)CD25(+)FOXP3(+) regulatory T (T(R)) cells. Here, we show that highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14(+) monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8(+) T(R) cells inhibit CD4(+) and CD8(+) T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8(+) effector T cells. Although, the CD8(+) T(R) cells express prostaglandin E(2), IL-10 and TGF-beta, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4(+) T(R) cells, the CD8(+) T(R) cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8(+) T(R)-mediated suppression, indicating that other mechanism(s) must be employed by these cells.     

CD4-mediated functional activation of human CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells (CD25(+) Tregs) constitute a specialized population of T cells that is essential for the maintenance of peripheral self-tolerance. The immune regulatory function of CD25(+) Tregs depends upon their activation. We found that anti-CD4 antibodies activate the suppressive function of human CD25(+) Tregs in a dose-dependent manner. We demonstrate that CD4-activated CD25(+) Tregs suppress the proliferation of CD4(+) and CD8(+) T cells, their IL-2 and IFN-gamma production as well as the capacity of CD8(+) T cells to re-express CD25. By contrast, anti-CD4 stimulation did not induce suppressive activity in conventional CD4(+) T cells. These results identify CD4 as a trigger for the suppressive function of CD25(+) Tregs and suggest a possible CD4-mediated exploitation of these cells.     

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.     

Expansion and activation of CD4(+)CD25(+) regulatory T cells in Heligmosomoides polygyrus infection

Regulatory T cell responses to infectious organisms influence not only immunity and immunopathology, but also responses to bystander antigens. Mice infected with the gastrointestinal nematode parasite Heligmosomoides polygyrus show an early Th2-dominated immune response (days 7-14), but by day 28 a strongly regulatory profile is evident with antigen-specific IL-10 release and elevated frequency of CD4(+) T cells bearing surface TGF-beta. CD4(+)CD25(+) T cells from infected mice show enhanced capacity to block in vitro effector T cell proliferation. CD4(+)CD25(+) cell numbers expand dramatically during infection, with parallel growth of both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets. CTLA-4 and glucocorticoid-induced tolerance-associated receptor, also associated with regulatory T cell function, become more prominent, due to both expanded CD25(+) cell numbers and increased expression among the CD25(-) population. Both intensity and frequency of CD103 expression by CD4(+) T cells rise significantly, with greatest expansion among CD25(+)Foxp3(+) cells. While TGF-beta expression is observed among both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets, it is the latter population which shows higher TGF-beta staining following infection. These data demonstrate in a chronic helminth infection that Foxp3(+) regulatory T cells are stimulated, increasing CD103 expression in particular, but that significant changes occur to other populations including expansion of CD25(+)TGF-beta(+)Foxp3(-) cells, and induction of CTLA-4 on CD25(-) non-regulatory lymphocytes.