Integration and independent acquisition of specialized skin- versus gut-homing and Th1 versus Th2 cytokine synthesis phenotypes in human CD4+ T cells

CD4+)T helper cells are heterogeneous in terms of tissue-specific homing and cytokine synthesis phenotypes. The mechanisms for the acquisition of tissue-specific homing phenotypes and their relationship with the attainment of polarized cytokine synthesis profiles of T cells are critically important but poorly understood. Here, we analyze the coordinate acquisition of Th1 versus Th2 cytokine (IFN-gamma vs. IL-4) and skin- versus gut-homing (CLA vs. integrin beta7) phenotypes in human CD4+ T cells. We show that the acquisition of skin- versus gut-homing T cell phenotypes is independent of Th1 versus Th2 cell fate determination and that it occurs in relation to cell cycle progression following instructive mechanisms and distinct kinetics. Expression of chemokine receptors CXCR3 and CCR4 correlates with the acquisition of Th1 versus Th2 rather than skin- versus gut-homing phenotypes. These findings, together with the skewed overlap observed in vivo between CLA vs. integrin beta7 and IL-4 vs. IFN-gamma expression, suggest a novel interpretation to the complex patterns of chemokine receptor expression on memory T cells.     

Skin-homing CLA+ T cells and regulatory CD25+ T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309

Functionally distinct T cell subsets exhibit specific chemokine receptor profiles that regulate their tissue localization. Here, we show that human peripheral blood CD4(+) and CD8(+) cutaneous (CLA(+)), but not intestinal memory (integrin beta(7) (+)) nor IL-4-producing T cells, represent major subpopulations of circulating T cells that specifically migrate in response to the chemokine I-309/CCL1 by virtue of CCR8 expression. Expression of CCR8 is markedly up-regulated upon activation and in vitro culture of human CLA(+) T cells, suggesting the involvement of CCR8 in localization of cutaneous memory T cells to the skin. Interestingly, amongst circulating memory CD4(+)CD45RO(+) T cells, chemotactic responsiveness to CCL1 is restricted to cells expressing CD25 and/or CLA surface markers for regulatory T cells (Treg) and skin-homing T cells and maximal responsiveness is observed on CLA(+)CD25(+)T cells. Such pattern of CCL1 responsiveness suggests that the CCR8/CCL1 axis may regulate trafficking of cutaneous Treg and memory T cells into the skin.     

Interleukin-4 promotes human CD8 T cell expression of CCR7

Despite strong evidence supporting a pathway of human T cell differentiation characterized by changes in the expression of CCR7, CD28, CD27 and CD62L, few studies have addressed the mechanisms of pathway regulation. Cutaneous lymphocyte-associated antigen (CLA)-positive skin-homing CD8(+) T cells expressed significantly elevated levels of activation markers compared with CLA(-) CD8(+) T cells in individuals (n = 27) with cutaneous atopic disease. Despite such an activated phenotype, CLA(+) T cells expressed significantly higher levels of CCR7 than a CLA(-) T cell subset. Interleukin (IL)-4 was found to dramatically promote CCR7 expression by antigen-specific CD8(+) cells. Furthermore, skin-homing CD8(+) T cells from individuals with severe disease produced significantly less IL-10 than those derived from mildly affected atopic subjects. Thus in a T-helper 2 dominated disease, tissue-specific CD8(+) T cells show altered CCR7 expression and cytokine production, which may contribute to continued lymph node homing, antigen presentation and disease. IL-4 promotes expression of CCR7, a marker linked to existing models of CD8(+) T cell differentiation.     

Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation

Multi-color flow cytometric analysis on human CD8(+) T cell subsets revealed that CXCR4 is predominantly expressed on CD8(+) T cells with the naive CD27(+)CD28(+)CD45RA(+) phenotype, and is down-regulated during differentiation into those with an effector phenotype. The down-regulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8(+) T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co-expression further showed that CD8(+) T cells expressing a high level of CXCR4 are CCR7(+)CCR5(-) naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7(-)CCR5(+/-) memory/effector and effector subsets. Epstein Barr virus-specific CD8(+) T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus-specific CD8(+) T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down-regulated during differentiation of viral antigen-specific CD8(+) T cells. The classification of human CD8(+) T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8(+) T cells.     

Alpha4beta1 and alpha4beta7 CD4 T cell numbers increase and CLA CD4 T cell numbers decrease in systemic sclerosis

We studied the expression of adhesion molecules affecting recirculation and homing on peripheral blood CD4(+) T cells of patients with systemic sclerosis (SSc), in order to evaluate whether the distribution of tissue targeted subsets could reflect the participation of internal organs or the extent of cutaneous involvement [i.e. limited cutaneous (lc) and diffuse cutaneous (dc)]. Peripheral blood mononuclear cells (PBMC) from 51 patients with SSc and 19 sex- and age-matched controls were investigated by cytofluorimetric analysis for lymphocyte subpopulations carrying the following surface molecules: CD3, CD4, CLA, alpha4beta7 and alpha4beta1. Standard routine biochemistry and clinical examinations were also performed in all patients. We found that both alpha4beta1(+) and alpha4beta7(+) cells within the CD4(+) T cell population were significantly increased, while CLA(+) CD4(+) T cells were significantly reduced in SSc, compared to healthy donors. Significantly lower absolute numbers of alpha4beta7(+) cells were found in lc- compared to dc-SSc. Patients with oesophageal involvement had high numbers of alpha4beta7(+) cells, while those with nephritis also showed low levels of CLA(+) cells. Lung involvement was related directly to alpha4beta1(+) cell numbers and inversely to alpha4beta7(+) CD4 cell numbers. Taken together, our findings demonstrate that distinct CD4(+) T cell populations with selective homing properties show changes from normal distribution in SSc, and such changes are related to clinical expression and organ involvement in the course of the disease.     

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.     

Interleukin-4 induced down-regulation of skin homing receptor expression by human viral-specific CD8 T cells may contribute to atopic risk of cutaneous infection

Factors controlling the expression of cutaneous lymphocyte-associated antigen (CLA) by T cells are poorly understood, but data from murine and human CD4(+) T cell systems have suggested that cytokines play an important role. However, there are no data examining the influence of cytokines on the expression of CLA by human antigen-specific CD8(+) T cells. Peripheral blood mononuclear cells (PBMC) were isolated from 10 HLA-A*0201-positive healthy individuals. Using HLA-peptide tetrameric complexes refolded with immunodominant peptides from Epstein-Barr virus (EBV), cytomegalovirus (CMV) and influenza A virus, we investigated the temporal associations of CLA expression by viral-specific CD8(+) T cells following stimulation with antigen. Ex vivo influenza matrix-specific CD8(+) T cells expressed significantly (P < 0.05) greater levels of CLA than EBV BMLF1 and CMV pp65-specific CD8(+) T cells (mean 9.7% influenza matrix versus 1.4% BMLF1 versus 1.1% pp65) and these differences were sustained on culture. However, regardless of viral specificity, interleukin (IL)-12 and IL-4 induced significant (P < 0.05) dose-dependent up-regulation and down-regulation of CLA expression, respectively, with IL-4 showing a dominant negative effect. In many cases, IL-4 resulted in complete abrogation of detectable CLA expression by the viral-specific CD8(+) T cells. Overall these data demonstrate that CLA expression by human viral-specific CD8(+) T cells is highly dynamic and that IL-4 causes significant down-regulation. Disorders associated with a type 2 cytokine shift may reduce the efficiency of skin homing by viral-specific CD8(+) T cells. Furthermore, the ability to modify the local and systemic microenvironment may offer novel therapeutic strategies that influence tissue-specific T cell homing.     

Oral immunization with a Salmonella enterica serovar typhi vaccine induces specific circulating mucosa-homing CD4(+) and CD8(+) T cells in humans

The kinetics and homing characteristics of T-cell responses in humans after mucosal immunizations have not been well characterized. Therefore, we have investigated the magnitude and duration of such responses as well as the homing receptor expression of antigen-specific peripheral blood T cells by using an oral model vaccine, i.e., the live, attenuated Salmonella enterica serovar Typhi vaccine (Ty21a). Eight volunteers were each given three doses of the vaccine 2 days apart, and blood samples, from which CD4(+) and CD8(+) T cells were selected by the use of magnetic beads, were collected before vaccination and at regular intervals thereafter. To purify the potentially antigen-specific gut-homing T cells, CD45RA(-) integrin beta(7)(+) cells were further sorted by flow cytometry. The sorted cells were then stimulated in vitro with the serovar Typhi vaccine strain, and the proliferation of cells and the cytokine production were measured. Following vaccination, there was a large increase in both the proliferation of and the gamma interferon (IFN-gamma) production by blood T cells stimulated with the vaccine strain. The responses were seen among both CD4(+) and CD8(+) T cells, although the CD8(+) cells produced the largest amounts of IFN-gamma. Peak responses were seen 7 to 14 days after the onset of vaccination. Furthermore, most of the IFN-gamma produced by both CD4(+) and CD8(+) cells emanated from cells with the potential to home to mucosal tissues, as the integrin beta(7)-expressing memory T cells produced around 10-fold more IFN-gamma than the remaining populations. In conclusion, we demonstrate that oral vaccination with a live oral bacterial vaccine induces antigen-specific CD4(+) and CD8(+) memory T cells, almost all of which express the gut-homing integrin beta(7).     

Generation and characterization of antigen-specific CD4+, CD8+, and CD4+CD8+ T-cell clones from patients with carbamazepine hypersensitivity

BACKGROUND: Hypersensitivity is a serious manifestation of anticonvulsant therapy characterized by infiltration of the epidermis and dermis by activated CD8(+) and CD4(+) T-cells, respectively. Attempts to characterize drug-specific CD8(+) T cells have been largely unsuccessful. OBJECTIVES: The aim of these studies was to generate and characterize CD4(+), CD8(+), and CD4(+)CD8(+) T cells in patients with carbamazepine hypersensitivity. METHODS: Carbamazepine-specific T-cell clones were generated from 5 patients by using modified cloning methodologies. Cell surface receptor phenotype, functionality, and mechanisms of antigen presentation were then compared. RESULTS: Ninety CD4(+), 23 CD8(+), and 14 CD4(+)CD8(+) carbamazepine-specific T-cell clones were generated. CD4(+) T-cell clones proliferated vigorously with carbamazepine associated with MHC class II but exhibited little cytotoxic activity. In contrast, most CD8(+) T cells proliferated weakly but effectively killed target cells via an MHC class I or MHC class II restricted, perforin-dependent pathway. CD4(+)CD8(+) T cells displayed characteristics similar to those of CD4(+) T cells; however, drug stimulation was demonstrable in the absence of antigen-presenting cells. Carbamazepine was presented to CD4(+), CD8(+), and CD4(+)CD8(+) T cells in the absence of antigen processing. Drug stimulation resulted in the secretion of IFN-gamma and IL-5. A panel of CD11a(+)CD27(-) clones differentially expressed the receptors CXCR4, CCR4, CCR5, CCR8, CCR9, and CCR10. CONCLUSION: Carbamazepine-specific CD4(+), CD8(+), and CD4(+)CD8(+) T cells exist in the peripheral circulation of hypersensitive patients, often many years after the resolution of clinical manifestations. CLINICAL IMPLICATIONS: Carbamazepine-specific CD4(+), CD8(+), and CD4(+)CD8(+) T cells displaying different effector functions and homing characteristics persist in hypersensitive patients' blood for many years after resolution of clinical symptoms.     

SLE患者外周血中Foxp3+CD4+CD25+调节性T细胞的分析

目的分析SLE患者外周血中Foxp3 CD4 CD25 调节性T细胞和T细胞亚群上GITR的表达,以初步阐述其在SLE患者免疫稳态调节中的作用和意义。方法以流式细胞术检测SLE患者外周血中Foxp3 CD4 CD25 调节性T细胞和T细胞亚群上GITR的表达。结果稳定期及活动期SLE患者外周血中Foxp3 CD4 CD25 调节性T细胞比例显著低于健康对照(P<0.05),且活动期SLE患者Foxp3 CD4 CD25 调节性T细胞比例高于稳定期SLE患者(P>0.05)。SLE患者外周血CD3 CD4 T细胞和CD3 CD8 T细胞上GITR表达显著增加(P<0.05);随着疾病活动性增加,CD3 CD4 T细胞上GITR表达降低(P>0.05),CD3 CD8 T细胞上GITR表达增加(P>0.05)。结论SLE患者外周血中Foxp3 CD4 CD25 调节性T细胞表达降低,而患者T细胞亚群上GITR表达增加,其共同作用在诱导SLE患者外周耐受障碍中具有重要意义。     

Delayed allergic reaction to suxamethonium driven by oligoclonal Th1-skewed CD4+CCR4+IFN-gamma+ memory T cells

BACKGROUND: Muscle relaxants represent the drugs most frequently involved in intraoperative anaphylaxis during surgical procedures. Our aim was to report the case of a delayed reaction to suxamethonium and analyze specific T cell lines with regard to their specificity, phenotype and cytokine profile. METHODS: We generated a drug-specific T cell line from a biopsy at the site of positive intradermal reactions and analyzed the immunophenotype, T cell receptor Vbeta domain expression and cytokine profile. RESULTS: T cells isolated from positive intradermal test reactions to suxamethonium showed a strict dose-dependent proliferation in response to drug-pulsed autologous antigen-presenting cells. The drug-specific CD4+ T cells were oligoclonal memory CD3+CD4+ T cells and expressed the skin homing receptors cutaneous lymphocyte antigen (CLA) and CCR4. Furthermore CD4+ suxamethonium-reactive T cell lines were IFN-gamma-positive and synthesized high levels of IFN-gamma and TNF-alpha. CONCLUSION: The study describes a delayed hypersensitivity to suxamethonium, driven by an oligoclonal T helper cell 1-skewed CD4+ memory T cell population, expressing the skin homing receptors CLA and CCR4.     

Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25− T cells from atopic and nonatopic donors

Background:  Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.
Objective:  To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25− T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro .
Methods:  Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25− T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.
Results:  CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25− effector cells, which downregulated CCR7 and upregulated CCR4.
Conclusions:  CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25− effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.     

Higher proportions of circulating FOXP3+ and CTLA-4+ regulatory T cells are associated with lower fractions of memory CD4+ T cells in infants

In adults, a majority of FOXP3(+) T(regs) expresses CTLA-4, and this costimulatory molecule is essential to control the expansion of other T cells. However, it remains to be investigated whether FOXP3(+) and/or CTLA-4(+) T(regs) are associated with the expression of memory markers and homing receptors on CD4(+) T cells. Thus, in a prospective newborn-infant cohort study, we examined the proportions of FOXP3(+) and CTLA-4(+) T(regs) within the CD4(+)CD25(+) T cell population and the fractions of CD4(+) T cells that expressed CD45RA, CD45RO, HLA-DR, α(4)β(7), CD62L, and CCR4 at several time-points during the first 3 years of life using flow cytometry. With the use of multivariate factor analysis, we found that a high proportion of FOXP3(+) or CTLA-4(+) T(regs) during the first 18 months of life was associated positively with the fraction of T cells that expressed a na?ve phenotype (CD45RA and α(4)β(7)) and inversely related to the fraction of T cells that expressed a memory phenotype (CD45RO and CCR4) later in childhood. In conclusion, FOXP3(+) or CTLA-4(+) T(regs) may modulate CD4(+) T cell activation and homing receptor expression in children.     

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)-induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4(+) phenotype. In particular, the T helper 1-type (Th1) subset, as evaluated by chemokine receptor-5 (CCR5) expression, is involved in this process. Cell death in Th1-type uses a CD95-mediated mechanism. Furthermore, Th1-type CCR5(+) cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1-type cells by apoptosis was confirmed by the decrease in interferon-gamma secretion. In conclusion, apoptosis of monocytes, CD4(+) and CD4(+) CCR5(+) T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune-depression in VL.     

In vitro exposure to highly cytopathic HIV-1 X4 strains increases expression of mucosa-associated integrins on CD4(+) T cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins alpha 4 beta 7 and alpha E beta 7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1(IIIB) strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4(+) T cells from cultures infected with HIV-1(IIIB) and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of alpha 4 beta 7 and alpha E beta 7 12 days after infection. L-selectin expression was not significantly affected on CD4(+) T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4(+) T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4(+) T cells may result as a "bystander" effect after infection with some cytopathic isolates of HIV-1.     

Detectable expression of IL-35 in CD4+ T cells from peripheral blood of chronic hepatitis B patients

Epstein-Barr virus-induced gene 3 (Ebi3) and the p35 subunit of IL-12 have been reported to form a heterodimeric cytokine, named IL-35, in human and mouse. In mice, IL-35 has been shown to be constitutively expressed by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) and suggested to contribute to their suppressive activity. However, human CD4(+)CD25(+)Foxp3(+) Tregs do not constitutively express detectable amounts of IL-35 in both mRNA and protein levels. Circulating CD4(+)CD25(+) Treg frequency of chronic Hepatitis B patients significantly correlates with serum viral load. In this study, we investigated whether IL-35 expression could be detected in CD4(+) T cells from peripheral blood of chronic Hepatitis B patients. Using both RT-PCR and immunoprecipitation plus Western blot analysis, we demonstrated that IL-35 expression could be detected in the CD4(+) T cells from peripheral blood of Chronic Hepatitis B patients.     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-β expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-β-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.