Effects of Peritoneal Fluid from Endometriosis Patients on Interferon-γ-Induced Protein-10 (CXCL10) and Interleukin-8 (CXCL8) Released by Neutrophils and CD4+ T Cells

Problem  Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-γ-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4⁺ T cells, and monocytes.
Method of study  Neutrophils, CD4⁺ T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay.
Results  The addition of ePF to cultures of CD4⁺ T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF ( R  = 0.89, P  =   0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4⁺ T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils ( R  = 0.89, P  <   0.001), CD4⁺ T cells ( R  = 0.93, P  <   0.001), and monocytes ( R  = 0.70, P  =   0.01). Moreover, the addition of ePF significantly enhanced the interferon-γ-induced release of IP-10 by nuetrophils and CD4⁺ T cells.
Conclusion  These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.     

Downregulation of CXCR6 and CXCR3 in lymphocytes from birch-allergic patients

Preferential expression of chemokine receptors on Th1 or Th2 T-helper cells has mostly been studied in cell lines generated in vitro or in animal models; however, results are less well characterized in humans. We determined T-cell responses through chemokine receptor expression on lymphocytes, and cytokine secretion in plasma from birch-allergic and healthy subjects. The expression of CCR2, CCR3, CCR4, CCR5, CCR7, CXCR3, CXCR4, CXCR6, IL-12 and IL-18R receptors was studied on CD4⁺ and CD8⁺ cells from birch-allergic ( n  = 14) and healthy ( n  = 14) subjects by flow cytometry. The concentration of IL-4, IL-5, IL-10, IL-12, IFN-γ and TNF-α cytokines was measured in plasma from the same individuals using a cytometric bead array human cytokines kit. The similar expression of CCR4 in T cells from atopic and healthy individuals argues against the use of the receptor as an in vivo marker of Th2 immune responses. Reduced percentages of CD4⁺ cells expressing IL-18R, CXCR6 and CXCR3 were found in the same group of samples. TNF-α, IFN-γ, IL-10, IL-5, IL-4 and IL-12 cytokines were elevated in samples from allergic individuals. Reduced expression of Th1-associated chemokine receptors together with higher levels of Th1, Th2 and anti-inflammatory cytokines in samples from allergic patients indicate that immune responses in peripheral blood in atopic diseases are complex and cannot be simplified to the Th1/Th2 paradigm. Not only the clinical picture of atopic diseases but also the clinical state at different time points of the disease might influence the results of studies including immunological markers associated with Th1- or Th2-type immune responses.     

ASRI2005-88 Comparable levels of CD4+CD25+ CTLA4+ Treg cells in peripheral blood of pre-eclampsia and normal pregnant patients

The acceptance of the semiallogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, T-regulatory cells were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Tregulatory cells (Treg). The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. Therefore, we measured the surface antigens CD4, CD25, CD8, CTLA4 (as well as the secretion of IL-10) in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by 4-colour flow-cytometry. We were not able to find any significant differences in the levels of CD4⁺, CD25⁺, CD8⁺, CTLA4, CD4⁺/CD25⁺, CD4⁺/CD25^bright, CD4⁺/CTLA4, CD25⁺/CTLA4, CD4⁺/CD25⁺/CTLA4, CD8⁺/CD25⁺, CD8⁺/CTLA4 or CD8⁺/CD25⁺/CTLA4 cell subsets. Our data suggest that Treg may not participate in the onset of pre-eclampsia and suggest other regulatory mechanisms during late pregnancy.     

Distribution of immunocompetent cells in decidua of controlled and uncontrolled (choriocarcinoma/hydatidiform mole) trophoblast invasion

Problem:  Pregnancy has been considered as a model of successfully controlled tissue invasion where trophoblast cells infiltrate the maternal decidua without being rejected or without destroying the tissue. In choriocarcinoma (CC) and hydatidiform mole (HM), a dysregulation of invasive (malignant/benign) trophoblast cells is present. Immunocompetent cells (IC) are known to be involved in rejection pathways of malignant cells and can also be identified in early pregnancy decidua. The aim of the present study was to identify the phenotype of IC in decidua of women with normal pregnancy (NP), CC and HM.
Methods:  Immunocompetent cells were detected by immunohistochemistry in decidual tissue from first trimester NP ( n  = 10), CC ( n  = 12) and HM ( n  = 11) using antibodies against CD8⁺, CD3⁺, CD56⁺, CD68⁺ cell surface markers and mast cell tryptase (MCT). A scaled eye piece was used for cell counting to obtain semiquantitative results. Statistical analysis was performed using Wilcoxon rank/Mann–Whitney tests.
Results:  We observed a significantly increased number of lymphocytes positive for CD8, CD3 and MCT positive granulocytes in CC and HM compared with the samples from NP (all P  ≤ 0.001). Lymphocytes positive for natural killer (NK) cell marker CD56 were significantly decreased in CC and HM versus NP ( P  ≤ 0.001). The number of CD68 positive cells (macrophages) were not significantly different among the tissue pools.
Conclusion:  The increase of CD8/CD3 T cells and mast cells in CC and HM and the decrease of CD56 cells, compared with NP, suggests the necessity of a balance between T and NK cells in controlling trophoblast invasion.     

Low-dose cyclosporine A therapy increases the regulatory T cell population in patients with atopic dermatitis

Background:  Atopic dermatitis (AD) is a T cell dependent chronic relapsing inflammatory skin disorder successfully treated with cyclosporine A (CsA). Clinical observations indicate that even low-dose CsA therapy is successful in severely affected AD patients. We studied the impact of low-dose CsA therapy on the ability of T helper cells to be activated, and examined whether regulatory T (Treg) cells are increased in these patients.
Methods:  Peripheral T cells were activated in a whole blood sample and interleukin-2 producing cells were measured by intracellular cytokine staining. Regulatory T cells were analyzed by intracellular FoxP3 staining. Regulatory T cells (CD4⁺CD25⁺CD127^low) and effector T cells (CD4⁺CD25⁻CD127⁺) were sorted by flow cytometry and used for suppression assays.
Results:  A group of AD patients treated with low-dose CsA had a significantly larger Treg cell population than a healthy control subject group. In individual patients, onset of low-dose CsA therapy reduced the ability of T cells to be activated to 42 ± 18% ( P  <   0.005) and significantly increased Treg cells, both in absolute numbers (1.6-fold change) and frequencies (1.7-fold change). Treg cells from AD patients showed similar suppressive capacities as Treg cells from healthy donors. Furthermore, Treg cells from AD patients had skin homing properties.
Conclusion:  Our results indicate that the therapeutic effect of low-dose CsA therapy in AD patients might be not only mediated by the inhibition of T cell hyperactivity but also by an increased population of Treg cells.     

The Effects of Mitogens, IL-2 and Anti-CD3 Antibody on the T-Cell Receptor Vβ Repertoire

Phytohaemagglutinin (PHA), Concanavalin A (Con A), interleukin-2 (IL-2), and monoclonal antibodies to CD3 (CD3MoAbs) are used for the assessment of the T-cell receptor (TCR) BV gene family expression in autoimmune disorders and multiple sclerosis, and to produce clones for assessment of cytokine profiles in progressive human immunodeficiency virus infection. The authors examined the effects of these stimulants on the TCR Vβ repertoire of resting and blastic CD4⁺ and CD8⁺ normal human peripheral blood lymphocytes, using three-colour cytofluorometry and a panel of anti-TCR Vβ monoclonal antibodies. IL-2 was associated with an increased percentage of blastic CD4⁺ cells expressing V_β5.1 (from median of 3.7% to 8.0%, P  = 0.0002) and blastic CD8⁺ cells expressing V_β5.3 (1.0 to 1.5%, P  = 0.0039). CD3MoAb caused a slight increase in V_β6.7 + blastic CD4⁺ cells (4.5 to 6.9%, P  = 0.0078). PHA did not alter the Vβ repertoire of blastic cells. Con A caused skewing in CD8⁺ blastic cells, toward expression of V_β5.2/5.3 (3.1 to 8.1%) and V_β5.3 (0.8 to 4.8%) ( P  = 0.0020). Thus, IL-2 stimulation causes slight alterations in the Vβ repertoire that should be taken into account in certain research settings. Con A produced skewing in CD8⁺ blastic cells suggesting that, in the presence of CD8, either Con A binds selectively to certain Vβ or the three-dimensional complex created by Con A's binding to other T-cell surface molecules induces preferential V_β5 stimulation.     

Suppression of Experimental Autoimmune Myasthenia Gravis After CD8 Depletion is Associated with Decreased IFN-γ and IL-4

CDS⁺ T cells can perform both Th1 - and Th2-like functions by producing cytokines such as interferonγ (IFN-γ) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-β (TGF-β), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8⁺ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-γ secreting Th1-like cells. To identify the involvement of IFN-γ, IL-4 and TGF-β in the development of EAMG after CD8⁺ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8⁺ T cells resulted in decreased levels of IFN-7 and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-β mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8⁺ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).     

Culture of Regulatory T-Cell Lines from Bronchial Mucosa

T lymphocytes play a major role in many immune responses. In the last decade, special focus has been on the function of Th1 and Th2 effector cells. Now the importance of regulatory CD4⁺CD25⁺ T cells in maintenance of the immunological homeostasis emerges. Sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. The typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly CD4⁺ T cells of Th1 phenotype. We have cultured T cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (IL-2) and IL-4 and demonstrate spontaneously arising CD4⁺ CD25⁺ populations and high concentrations of IL-10 in these cultures. The main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines IL-6 and TNF-α in cultures of sarcoid origin.     

Vβ Usage and T Regulatory Cells in Children Following Partial or Total Thymectomy after Open Heart Surgery in Infancy

During open heart surgery in infants the thymus was usually removed, partly or completely. Our previous studies on 16 such children indicated reduced T-cell output later in life with signs of extrathymic maturation of the T cells, but no reduction in T regulatory cells (CD4⁺CD25⁺). The diversity of the T-cell repertoire in these children was examined to test if the extrathymic microenvironment could alter Vβ usage. The expression of Foxp3 and CD127 in CD4⁺CD25^high T cells was measured in order to determine whether the T regulatory cells had the phenotype of natural T regulatory cells. There was a wide distribution of Vβ usage in both study and control groups. Significant variability was found in Vβ usage for CD4⁺ and CD8⁺ T cells when the distribution of the percentage of T cells expressing each Vβ family was analysed between individuals within each group ( P  < 0.001; Kruskal–Wallis). Significant difference was also found in average usage of Vβ2, Vβ5.1 and Vβ14 chains within CD4⁺ T cells and Vβ2, Vβ8 and Vβ21.3 chains within CD8⁺ cells between the groups ( P  < 0.05; Student's t -test). There was no difference between the two groups with regard to the proportion of CD4⁺CD25^high T cells and no difference in the average expression of Foxp3 or CD127 within the CD4⁺CD25^high population. Our data provide evidence that cardiothoracic surgery in infants and total or partial thymectomy alters Vβ usage, suggesting more limited selection in such children than in the control group. The frequency of natural T regulatory cells seems to be unimpaired.     

Participation of the CD69 Antigen in the T-Cell Activation Process of Patients with Systemic Lupus Erythematosus

Accumulating evidence has implicated T cells in the pathogenesis of systemic lupus erythematosus (SLE). The CD69 antigen is an integral membrane protein rapidly induced on the surface of activated lymphocytes. We obtained CD4⁺ and CD8⁺ T cells from normal subjects and patients with SLE. The percentage of CD69 expression in freshly isolated cells and after in-vitro incubation with mitogens was quantified by three-colour immunofluorescent staining. Expression of this protein was increased in both CD4⁺ and CD8⁺ T-cell subsets from SLE patients when compared with normal cells, although the difference was significant only in the CD8⁺ T-cell subset ( P  = 0.05). Cellular activation increased CD69 expression. When stimulated with anti-CD2/CD2R or phytohaemagglutinin (PHA), the percentage and absolute numbers of CD69⁺ cells were lower in patients than in controls. Addition of anti-interleukin (IL)-10 monoclonal antibody (MoAb) increased the percentage of in-vitro CD69 expression in SLE cells. These results suggest that the peripheral blood lymphocytes from patients with SLE have an intrinsic defect that alters their activation process, including the expression of CD69, and might explain some of the T immunoregulatory abnormalities observed in these patients.     

Variation in Gut-Homing CD27-Negative Lymphocytes in Inflammatory Colon Disease

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin-4 (IL-4). Co-expression of α4β7 identifies those cells with gut-homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non-inflammatory bowel disease (non-IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, α4β7 and intracellular cytokines was performed. Circulating CD4+CD27– populations were increased in patients with CD (8.8 ± 0.8%, P  < 0.001), UC (12.2 ± 1.9%, P  < 0.001) and non-IBD colitis (10.5 ± 1.3%, P  < 0.01) as compared with controls (6.1 ± 0.5%). CD4⁺CD27⁻α4β7⁺ cells were increased in CD ( P  < 0.01). Lamina propria CD4⁺CD27⁻ populations were depressed significantly in CD ( P  < 0.05), UC ( P  < 0.02) and non-IBD colitis ( P  < 0.03). Mucosal CD4⁺CD27⁻ cells synthesized IL-4 in preference to interferon-γ. Thus, colonic inflammation is associated with alterations in gut-tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL-4-synthesizing cells.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Interleukin-6 is essential for Staphylococcal exotoxin B-induced T regulatory cell insufficiency in nasal polyps

Background The pathogenesis of nasal polyps is still unclear. There is increasing evidence indicating that Staphylococcal aureus (S. aureus) is associated with the formation of nasal polyps, but the mechanism has not been well documented to date.
Methods We stimulated cultured nasal polyps and turbinate tissues with Staphylococcal exotoxin B (SEB), detected the expression of pro-inflammatory cytokines (IL-2, IL-6, and IL-8) and T cell cytokines (IFN-γ, IL-4, IL-5, IL-10, and IL-17) in the supernatants, and evaluated mRNA expression (T-bet, GATA-3, Foxp3, and RORγt) and frequencies of CD4⁺CD25⁺ T regulatory cells (Tregs) in nasal tissues. We also evaluated the effects of blocking IL-6 with monoclonal antibodies to T cell profiles in cultured nasal tissues stimulated by SEB.
Results Levels of IL-6, IFN-γ and IL-4 increased significantly in SEB-stimulated nasal polyps. Meanwhile, mRNA expressions of T-bet and GATA-3 were significantly up-regulated, while Foxp3 was inhibited and the frequencies of CD4⁺CD25⁺ Tregs were decreased after SEB stimulation. After blocking IL-6, the levels of IL-10 and Foxp3 mRNA, as well as the frequencies of CD4⁺CD25⁺ Tregs, were significantly increased, while IFN-γ and IL-4 production and the mRNA expression of T-bet and GATA-3 were significantly inhibited.
Conclusions SEB is able to modulate pro-inflammatory factors, T-helper type 1/Th2 profiles and suppress Treg activity in cultured nasal polyps, which were rescued by blocking IL-6 activity. Therefore, IL-6 is essential for SEB-induced Treg insufficiency in nasal polyps.     

CD 127- and FoxP3+ expression on CD25+CD4+ T regulatory cells upon specific diabetogeneic stimulation in high-risk relatives of type 1 diabetes mellitus patients

Abnormalities in CD4⁺CD25⁺ regulatory T cells (Treg) may contribute to type 1 diabetes (T1D) development. First-degree relatives of T1D patients are at increased risk especially when they carry certain HLA II haplotypes. Using two novel markers of CD4⁺CD25⁺ Treg (CD127⁻ and FoxP3⁺ respectively), we evaluated number and function of Treg after specific stimulation with diabetogeneic autoantigens in 11 high-risk (according to HLA-linked risk) relatives of T1D patients and 14 age-matched healthy controls using a cytokine secretion assay based on interferon- γ (IFN- γ ) production. High-risk relatives of T1D patients had significantly lower pre- and post-stimulatory number of CD127⁻ Treg than that of healthy controls ( P  < 0.05). Labelling Treg with FoxP3⁺ demonstrated similar trend but did not reach statistical significance. Although the stimulation with diabetogenic autoantigens did not lead to a significant change in number of Treg in both groups, the defective function of Treg was performed by significantly higher activation of diabetogeneic T cells in high-risk relatives of T1D patients compared to healthy controls ( P  ≤ 0.02). Individuals at increased HLA-associated genetic risk for T1D showed defects in Treg.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

T-Lymphocyte Functions in Acute Leukaemia Patients with Severe Chemotherapy-Induced Cytopenia: Characterization of Clonogenic T-Cell Proliferation

Intensive chemotherapy for acute leukaemia is followed by a period of severe chemotherapy-induced leukopenia. We used a limiting dilution assay to investigate whether remaining CD4⁺ and CD8⁺ T lymphocytes derived from such leukopenic patients could be activated and undergo clonogenic proliferation. The activation signal in our model was accessory cells (irradiated normal peripheral blood mononuclear cells) + phytohaemagglutinin (PHA) + interleukin-2 (IL-2). During severe leukopenia a majority of circulating lymphocytes were CD4⁺ T cells. Clonogenic proliferating T lymphocytes were detected for all patients. Higher frequencies of clonogenic cells were detected in the CD8⁺ subset as compared to the CD4⁺ subset. However, for both subsets frequencies of proliferating cells were decreased compared with healthy individuals. The CD4⁺ and CD8⁺ lymphocytes were also capable of proliferation in response to alloactivation, and accessory cells mainly containing acute myelogenous leukaemia blast were efficient as accessory cells for activation. For the CD4⁺ cells, increased proliferation was detected in the presence of acute myelogenous leukaemia (AML) blasts compared with normal accessory cells. Based on our results we conclude that: (1) although acute leukaemia patients with therapy-induced leukopenia have both a quantitative and a qualitative T-cell defect, (2) the remaining T-cell population includes a subset capable of clonogenic proliferation. However, (3) proliferation of the clonogenic CD4⁺ cells can be modulated by AML blasts.     

The Allogeneic but not Syngeneic Dendritic Cells Effectively Generated Regulatory T Cells from Total Cd4+ Population without Exogenous Cytokines

Dendritic cells (DC) play a critical role in both the expansion of natural regulatory T cells (nTreg) and conversion of induced Treg (iTreg) from their precursors. In the present study, we evaluated the potential of DC to generate Treg from total CD4⁺ population which contains both nTreg and the precursors, and found that allogeneic (allo-DC) but not syngeneic DC (syn-DC) could effectively generated Foxp3⁺ Treg from total CD4⁺ population in the absence of exogenous cytokines. Compared with freshly purified CD4⁺ T cells, allo-DC-stimulated CD4⁺ T cells showed increased percentage of CD4⁺CD25⁺Foxp3⁺ Treg by 5–7-folds while syn-DC-stimulated CD4⁺ T cells did not. Furthermore, we demonstrated that the significant amounts of endogenous IL-2 and TGF-β, at least partially, contributed to the expansion of nTreg and conversion of iTreg in this cocultural system, respectively. Importantly, similar to nTreg, these allo-DC-generated Treg were capable of suppressing T cell response in vitro . Thus, our research provides a novel and efficient strategy for generation of Treg from total CD4⁺ population.