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.     

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.     

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.     

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.     

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.     

大鼠CD4+ CD25+ T调节细胞的分离培养及其功能分析

目的:研究大鼠CD4 CD25 T调节细胞(Tr)的分离培养,并对其功能进行初步分析。方法:无菌条件下切取大鼠脾脏分离脾淋巴细胞。用免疫磁珠细胞分离系统(MACS)分选CD4 CD25 T细胞,并以流式细胞术检测其纯度后,对其进行扩增。采用混合淋巴细胞反应研究CD4 CD25 Tr细胞对CD4 CD25-T细胞的免疫抑制作用。用ELISA法检测培养上清中IL-2、IFN-γ及IL-10水平的差异。结果:MACS分离的CD4 CD25 T细胞的纯度达86%~93%。该细胞与CD4 CD25-T细胞相比能特异性地表达Foxp3基因。体外培养中能明显抑制效应T细胞增殖及其分泌IFN-γ、IL-2,但其自身能分泌Th2型细胞因子IL-10。结论:采用MACS系统阴性加阳性分选,可高效快速的获得理想纯度和免疫抑制功能的大鼠CD4 CD25 T调节细胞,该细胞对CD4 CD25-T细胞具有明显的免疫抑制作用,并能特异性的表达Foxp3基因。     

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.     

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.     

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.      

CD8+ Foxp3+ T cells share developmental and phenotypic features with classical CD4+ Foxp3+ regulatory T cells but lack potent suppressive activity

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.     

人外周血CD4+CD25+调节性T细胞的分离、鉴定和功能特征

目的： 分离人外周血CD4＋ CD25＋ Treg细胞, 并检测其功能.方法： RT-PCR技术检测CD4＋ CD25＋ Treg细胞中Foxp3的mRNA表达;与CD8＋ T细胞和CD4＋ CD25- T细胞共同培养, 或加入外源性IL-2及IL- 4, 检测其抑制功能;流式细胞术检测IFN-γ、 IL- 4和IL-10.结果： CD4＋ CD25＋ Treg细胞高表达Foxp3, 主要分泌IL-10, 能够抑制CD8＋ T细胞和CD4＋ CD25- T细胞的增殖, 高浓度IL-2能够阻断CD4＋ CD25＋ Treg细胞的抑制功能.结论： CD4＋ CD25＋ Treg细胞是一群具有免疫抑制功能的调节性T细胞, 这种抑制作用能够被高浓度IL-2阻断.     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

Activation requirements for the induction of CD4+CD25+ T cell suppressor function

The in vivo differentiation/survival of CD4(+)CD25(+) T suppressor cells is dependent on IL-2 and CD28-mediated costimulatory signals. To determine the cytokine and costimulatory requirements for CD25(+) T cells in vitro, we established a two-stage culture system where CD25(+) T cells were activated in a primary culture. In the subsequent culture, activated CD4(+)CD25(+) cells were then mixed with responders in order to assess their suppressor function. Pre-culture of CD25(+) T cells with anti-CD3 alone resulted in poor survival and minimal induction of suppressor activity. Pre-culture in the presence of anti-CD3 and IL-2 or IL-4, but not IL-6, IL-7, IL-9, IL-10 or IL-15, resulted in proliferation of the CD25(+) cells and induction of potent suppressor function. Inhibition of the interaction of CD28 or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) with CD80/CD86 in the pre-culture of CD4(+)CD25(+) cells did not prevent the induction of suppressor function. Furthermore, the inhibition of costimulatory signals did not inhibit the ability of fresh CD25(+) T cells to inhibit CD8(+) responders under conditions where activation of the responders was independent of CD80/CD86. These studies support the view that activation of CD25(+) T cells requires IL-2/IL-4 for their survival/differentiation into effector cells, but is independent of CD28/CTLA-4-mediated costimulation.     

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.     

Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells

Naturally arising CD4(+)CD25(+) regulatory T (T(R)) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4(+)CD25(-) T cells also possesses regulatory activity. Programmed death-1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4(+)CD25(-)PD-1(+) T cells in the spleen of naive mice that constitutively expressed CTLA-4 and FoxP3 and was hypoproliferative in response to anti-CD3 antibody stimulation in vitro. However, the CD4(+)CD25(-)PD-1(+) T cells uniquely produced large amounts of IL-4 and IL-10 in response to anti-CD3 and anti-CD28 mAb stimulation, unlike the CD4(+)CD25(+) T(R) cells. The CD4(+)CD25(-)PD-1(+) T cells exhibited a suppressor activity against the proliferation of anti-CD3 antibody-stimulated CD4(+)CD25(-)PD-1(-) T cells in vitro, which was partially abrogated by anti-CTLA-4 mAb, but not by anti-IL-10 or anti-PD-1 mAb. Remarkably, the CD4(+)CD25(-)PD-1(+) T cells inhibited the development of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into C.B17-scid/scid mice, albeit to a lesser extent than CD4(+)CD25(+) T(R) cells, in a CTLA-4-dependent manner. These results indicate that the CD4(+)CD25(-)PD-1(+) T cells contain substantial amounts of T(R) cells that are involved in the maintenance of peripheral tolerance.     

CD4+CD25+CD127dimFoxp3+ T cells are cytotoxic for human neurons

MS lesions are characterized by destruction of myelin and significant neuronal and axonal loss. Preliminary studies with the use of T(regs) in the mouse model of MS have been extremely encouraging. However, recent studies with human cells have shown the presence of different subpopulations of T cells within the CD4(+)CD25(+)Foxp3(+) T cell phenotype, some of which do not have regulatory functions. These findings suggest a potential difference between mouse and human in the regulatory phenotype. Here, we show that human activated CD4(+)CD25(+)Foxp3(+) T cells are neurotoxic in vitro. These cells expressed high levels of the cytotoxic molecule GrB and had no suppressive effect. On the contrary, they produced IFN-γ and low IL-17, suggesting a shift toward a T(H)1 phenotype. Thus, our data confirm the presence of a nonregulatory cytotoxic subpopulation within the human CD4(+)CD25(+)Foxp3(+) T cells and suggest further studies on the human regulatory phenotype prior to any potential therapeutic application.     

小鼠脾细胞CD4~+ CD25~+调节性T细胞的表型特征

观察CD4+CD25+T和CD4+CD25-T细胞的表型和细胞因子的表达。自小鼠脾脏制备单个细胞悬液,分离CD4+T细胞、CD4+CD25+和CD4+CD25-T细胞,进行细胞表面标记,激活后进行细胞内细胞因子染色,利用流式细胞仪在单个细胞水平上分析细胞表面分子、转录因子和细胞因子表达之间的关系。结果:在CD4+T细胞中,约有7.8%的细胞同时表达CD25分子。与CD4+CD25-T细胞相比,CD4+CD25+T细胞CD44的表达略有增加,CD45RB的表达明显下降,CTLA-4和Foxp3明显增加。以同时表达CTLA-4和Foxp3的细胞为主,其次为单独表达Foxp3的细胞。细胞因子的研究结果表明,与CD4+CD25-T细胞相比,CD4+CD25+T细胞IL-2、IFN-γ明显减少,而只产生IL-10的细胞略有增加。CD4+CD25+调节性T细胞无论在表型、转录因子的表达以及细胞因子表达方面均于非调节性T细胞不同。     

Suppressive properties of human CD4+CD25+ regulatory T cells are dependent on CTLA-4 expression

It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4(+)CD25(+) T cell compartment. Here, we describe an original method to purify human CD4(+)CD25(+)CD152(+) T lymphocytes as living cells by forcing the exportation of CTLA-4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4(+)CD25(+) T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4(+)CD25(+)CD152(+) T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen-specific T cells than CD4(+)CD25(+)CD152(-) T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA-4, we demonstrate that indeed CTLA-4 is involved in CD4(+)CD25(+)CD152(+) T cell regulatory activity, while suppressive cytokines are not.