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.     

Functional alteration of peripheral CD25(+)CD4(+)immunoregulatory T cells in a transgenic rat model of autoimmune diseases

Transgenic rats carrying the env-pX gene of human T cell leukemia virus type-I (env-pX rats) develop various collagen vascular diseases. Since autoantibodies are present in their sera, env-pX rats are considered to be a prototype model for autoimmune diseases. Adoptive transfers of spleen cells from syngenic non-transgenic rats decreased the incidence of diseases in env-pX rats, thus suggesting that normal spleen contains cells, which suppress autoimmune diseases. Murine peripheral CD25(+)CD4(+)T cells play roles in maintaining immunological self-tolerance. To examine if alterations of immunoregulatory cells may be evident in env-pX rats, quantitative and qualitative analyses of splenic CD25(+)CD4(+)T cells were done before these rats developed autoimmune diseases. Env-pX and non-transgenic rats had equivalent number of CD25(+)CD4(+)T cells. However, CD25(+)CD4(+)T cells from env-pX rats did not suppress proliferation of T cells stimulated by anti-CD3 antibodies (Ab) in vitro, whereas those from non-transgenic rats did. Additionally, env-pX CD25(+)CD4(+)T cells showed autologous and anti-CD3 Ab-mediated proliferation, in contrast to the anergic features in non-transgenic rats. These findings appear to be the first evidence that CD25(+)CD4(+)immunoregulatory T cells are altered in animal models, which naturally develop autoimmune diseases.     

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.     

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.     

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.      

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.     

CD5 plays an inhibitory role in the suppressive function of murine CD4(+) CD25(+) T(reg) cells

A subset of CD4(+) T cells, the CD4(+) CD25(+) regulatory T (T(reg)) cells in the lymphoid organs and peripheral blood are known to possess suppressive function. Previous in vitro and in vivo studies have indicated that T cell receptor (TCR) signal is required for development of such 'natural regulatory (T(reg)) cells' and for activation of the effector function of CD4(+) CD25(+) regulatory T cells. CD5 is a cell surface molecule present on all T cells and a subtype of B lymphocytes, the B-1 cells, primarily localized to coelomic cavities, Peyer's patches, tonsils and spleen. CD5 acts as a negative regulator of T cell and B cell signaling via recruitment of SHP-1. Here, we demonstrate that T(reg) cells obtained from CD5(-/-) mice are more potent than those from wild type mice in suppressing the in vitro cell proliferation of anti-CD3 stimulated CD4(+) CD25(-) responder T cells. This phenomenon was cell contact and GITR dependent. Lack of CD5 expression on T(reg) cells (from spleen, lymph node and thymus) did not affect the intracellular levels of Foxp3. However, CD5(-/-) T(reg) thymocytes were able to elicit a higher Ca(2+) response to TCR + co-stimulatory signals than the wild type cells. CD5(-/-) mice expressed more Foxp3 mRNA in the colon than wild type mice, and additionally, the severity of the dextran sulfate sodium (DSS)-induced colitis in CD5(-/-) mice was less than the wild type strain. We suggest that manipulation of CD5 expression or the downstream signaling components of CD4(+) CD25(+) T(reg) cells as a potential strategy for therapeutic intervention in cases of auto-immune disorders.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

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.     

Selective expansion of memory CD4(+) T cells by mitogenic human CD28 generates inflammatory cytokines and regulatory T cells

Costimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAb reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMC without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4(+)CD25(+)FOXP3(-) (Teff) and CD4(+)CD25(+)FOXP3(+) (Treg) cells. ANC28 stimulated the CD45RO(+) CD4(+) (memory) population, whereas CD45RA(+)CD4(+) (naive) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than costimulated Treg. Treg induced by ANC28 suppressed CD25(-) T cells through a contact-dependent mechanism. Purity influenced the response of CD4(+)CD25(+ )cells because bead-purified CD4(+)CD25(+ )cells (85-90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4(+)CD25(bright) (Treg) did not respond. Purified CD4(+)CD25(int) cells responded similarly to the bead-purified CD4(+)CD25(+) cells. Thus, pre-activated CD4(+) T cells (CD25(int)) respond to ANC28 rather than Treg (CD25(bright)). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells.     

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.     

Identification of a CD4+CD25+ T cell subset committed in vivo to suppress antigen-specific T cell responses without additional stimulation

Naturally occurring CD4+ regulatory T cells can be identified on the basis of expression of CD25 and suppression of T cell responses in vitro after TCR triggering. Here, we demonstrate that a CD134+ subset of CD4+CD25+ T cells in naive rats suppresses antigen-specific T cell responses in vitro without additional TCR stimulation. In contrast, CD4+CD25+CD134- regulatory T cells and total CD4+CD25+ regulatory T cells have suppressive activity only during simultaneous activation of responder and regulatory T cells or after in vitro pre-activation. Furthermore CD4+CD25+CD134+ T cells have a more activated phenotype than CD4+CD25+CD134- T cells, as based on the expression of CD62L, CD45RC, and MHC class II. We propose that the CD134+ regulatory T cells contain an in vivo activated and highly suppressive regulatory T cell subset. CD4+CD25+CD134+ T cells can be found in several compartments of the immune system, including spleen, lymph nodes, and blood. Interestingly though, the relative amounts of these cells within the CD4+ population and their CD134 expression levels are highest in mucosa-draining lymph nodes and lowest in blood. This suggests that the presence of CD4+CD25+CD134+ T cells indicates sites of active immune suppression.     

CD25(+)CD4(+) regulatory T cells exert in vitro suppressive activity independent of CTLA-4

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is constitutively expressed on CD25(+)CD4(+) regulatory T cells (Treg) and is suggested to play a role in Treg-mediated suppression. However, the results of analysis with anti-CTLA-4 have been controversial. We addressed this issue by analyzing mice over-expressing or deficient in CTLA-4. For over-expression, CTLA-4 transgenic mice expressing a full-length (FL) or a truncated (TL) mutant of CTLA-4 were analyzed. FL T cells expressed similar levels of CTLA-4 to Treg, whereas TL T cells expressed much higher levels on the cell surface. The number of Treg in both mice was decreased, although Foxp3 expression was not altered. Treg from both mice exerted suppressive activity, whereas CD25(-) T cells from FL mice showed no suppression. Furthermore, CD25(+)CD4 thymocytes from young CTLA-4-deficient mice were analyzed and found to exhibit suppressive activity. These results indicate that Treg exert in vitro suppressive activity independent of CTLA-4 expression.     

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.     

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.     

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.     

Defect in TCR-CD3zeta signaling mediates T cell hypo-responsiveness in mesenteric lymph node

Mesenteric lymph node (MLN) in gut-associated lymphoid tissue plays obligatory roles in the induction of oral tolerance and ignorance to commensals. However, little is known about its immunological characteristics. In this study, we investigated the hypo-responsiveness of MLN CD4(+) T cells, comparing them with spleen CD4(+) T cells. MLN CD4(+) T cells were hypo-proliferative and expressed low levels of Th1-type cytokines in response to antigen or CD3/T cell receptor (TCR) stimulation. The hypo-responsiveness of MLN CD4(+) T cells is linked neither with changes in the regulatory T cell population (CD4(+)CD25(+), CD4(+)Foxp3(+)) nor the apoptotic population. Rather, MLN CD4(+) T cells showed deformity of T cell:APC conjugation and reduced expression of TCR signaling molecules such as CD3zeta, PLC-gamma1, PKC-theta, Zap70, with reduced phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). Among the alterations in TCR signaling molecules, defective CD3zeta expression is the most evident, and reversal of the anergic state by CD3/CD28 costimulation restored CD3zeta expression levels. Collectively, we suggest that reduced CD3zeta expression and defects in TCR signaling mediate the anergy state of MLN CD4(+) T cells, which play a critical role in maintenance of mucosal tolerance in gut-associated lymphoid tissue.