Flow cytometric analysis of intracellular IFN-gamma, IL-4 and IL-10 in CD3(+)4(+) T-cells from rat spleen

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma, IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.     

IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells

Interleukin-2 (IL-2) treatment is currently used to enhance T cell-mediated immune responses against tumors or in viral infections. At the same time, IL-2 is essential for the peripheral homeostasis of CD4(+)CD25(+)Foxp3(+ )regulatory T cells (Treg). In our study, we show that IL-2 is also an important activator of Treg suppressive activity in vivo. IL-2 treatment induces Treg expansion as well as IL-10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL-2 via gene-gun vaccination using IL-2 encoding DNA plasmids (pIL-2) inhibited naive antigen-specific T cell proliferation as well as a Th1-induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL-2-treated mice and the suppression depends on the synergistic action of IL-10 and TGF-beta. These data highlight that during therapeutic treatment with IL-2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.     

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.     

Immunoregulation of encephalitogenic MBP-NAc1-11-reactive T cells by CD4+ TCR-specific T cells involves IL-4, IL-10 and IFN-gamma

The generation of TCR transgenic (Tg) mice expressing a BV8S2 (Vbeta8 subfamily 2) chain specific for the encephalitogenic NAc1-11 region of MBP provides a unique system for evaluating the mechanisms involved in anti-TCR immunoregulation of EAE. In a previous study, we showed that vaccination with BV8S2 protein induced specific T cells that inhibited proliferation responses and encephalitogenic activity of MBP-reactive T cells in vitro, and resulted in a skewed production of Th2 cytokines by the MBP-reactive T cells. These data suggested that regulation of the encephalitogenic T cells was mediated by inhibitory cytokines rather than through a deletional mechanism. In the current study, we have employed the BV8S2 Tg mouse model to address the issue of which cytokines produced by anti-TCR-reactive T cells can regulate the function of encephalitogenic Th1 cells. Utilizing neutralizing anti-cytokine antibodies to reverse inhibitory effects of supernatants from BV8S2-specific T cells, we found that IL-4, IL-10, and to a lesser extent, IFN-gamma and TGF-beta, were the major regulatory cytokines responsible for inhibiting encephalitogenic activity, proliferation, and IFN-gamma secretion of MBP-NAc1-11-reactive Th1 cells. These results indicate that cytokine regulation is the major mechanism through which TCR specific CD4+ T cells regulate encephalitogenic and potentially other bystander Th1 cells.     

Induction of IL-10 producing CD4+ T cells with regulatory activities by stimulation with IL-10 gene-modified bone marrow derived dendritic cells

Dendritic cells (DCs) can induce both tolergenic as well as effective immune responses in the lung. Pulmonary DCs producing interleukin (IL)-10 mediated tolerance induced by respiratory exposure to antigen. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DC. To assess whether IL-10 producing DCs can exert the tolergenic effect through the differentiation of regulatory T cells, bone marrow derived DCs were genetically modified by IL-10 expressing adenovirus. IL-10 gene modified DCs (Ad-IL-10-DC) displayed a characteristic phenotype of immature DCs. Here we showed that in vitro repetitive stimulation of naïve DO11·10 CD4⁺ T cells with Ad-IL-10-DCs resulted in a development of IL-10 producing T-cell regulatory cells. These T cells could not proliferate well but also lost their ability to produce interferon-γ upon restimulation with irradiated splenocytes and ovalbumin peptide. Furthermore, in co-culture experiments these T cells inhibited the antigen-driven proliferation of naïve CD4+ T cells in a dose-dependent manner. Our findings demonstrated that IL-10 producing DCs had the potential to induce the differentiation of Tr1-like cells and suggested their therapeutic use.     

Chronic exposure to superantigen induces regulatory CD4(+) T cells with IL-10-mediated suppressive activity

The repeated injection of bacterial superantigens (SAg), such as staphylococcus enterotoxin (SE) A or B, has been shown in mice to induce a state of unresponsiveness characterized by the lack of secretion of Th1 lymphokines, such as IL-2 and IFN-gamma, following subsequent SAg challenge. We made the observation, in vivo as well as in vitro, that unresponsiveness to SAg could be transferred from SEA- to SEB-reactive T cells (and reversibly from SEB- to SEA-specific T cells) in C57BL/6 mice but not in BALB/c mice. Since C57BL/6 mice, unlike BALB/c mice, possess TCR V(beta)3+ and V(beta)11+ T cells able to react with both SEA and SEB, we hypothesized that SAg-unresponsive V(beta)3(+) and V(beta)11+ T cells could mediate linked suppression of other SAg-reactive T cells. To analyze further this possibility, spleen cells from BALB/c mice made unresponsive to SEB were tested for their capacity to suppress the response of normal BALB/c cells to SEB. The production of both IFN-gamma and IL-2 following SEB stimulation was greatly impaired in co-cultures containing CD4(+) T cells, but not CD8(+) T cells, isolated from unresponsive animals. In vivo, the production of both IFN-gamma and IL-2 responses to SEB was dramatically reduced in animals adoptively transferred with unresponsive spleen cells. This suppression was abrogated in recipients injected with neutralizing anti-IL-10 antibodies. Moreover, in animals made unresponsive to SEB, SAg-reactive CD4(+) T cells were found to express high levels of CTLA-4, a molecule recently described to play an essential role in the suppressive function of regulatory T cells. Taken together these results demonstrate that the repetitive injection of SAg induces the differentiation of regulatory CD4(+) T cells capable of suppressing SAg-reactive naive T cells.     

Intracellular IL-4, IL-10, and IFN-gamma levels of leukemic cells and bone marrow T cells in acute leukemia

The quantitative levels of intracellular cytokines IL-4, IL-10, and IFN-gamma (ie, the number of bound PE-conjugated antibody molecules/cell) of leukemic cells and bone marrow T cells (bmT cells) of acute leukemia patients were analyzed by flow cytometry. One hundred, thirty-one (95 AML, 25 ALL, 11 ABL) patients were studied. The leukemic cell IL-4 level was highest in the monocytic AML group (1735 +/- 1056) and lowest in the dysplastic AML group (960 +/- 545). The IFN-gamma level was highest in the acute promyelocytic leukemia (APL) group (495 +/- 159), and lowest in the ALL group (252 +/- 119). The IL-10 level was not significantly different among the diagnosis groups. In bmT cells, the IL-10 level was highest in the dysplastic AML group (972 +/- 1049) and lowest in the APL group (397 +/- 352). The leukemic cell cytokine levels were lowest and bmT cell cytokine levels were highest in the dysplastic AML group. There were no significant correlations of these cytokine levels with 2-yr survival rate, complete remission (CR) rate, or relapse rate. The cytokine levels of bmT cells at the time of CR became normal and were not different among the diagnosis groups. In summary, leukemic cell and bmT cell cytoplasmic expression profiles of IL-4, IL-10, and IFN-gamma are characteristic for each diagnostic group of acute leukemia patients and the profiles of bmT cells are normal at the time of CR.     

Total glucosides of paeony induces regulatory CD4(+)CD25(+) T cells by increasing Foxp3 demethylation in lupus CD4(+) T cells

Total glucosides of paeony (TGP), an active compound extracted from Paeony root, has been used in therapy for autoimmune diseases. However the molecular mechanism of TGP in the prevention of autoimmune response remains unclear. In this study, we found that TGP treatment significantly increased the percentage and number of Treg cells in lupus CD4(+) T cells. Further investigation revealed that treatment with TGP increased the expression of Foxp3 in lupus CD4(+) T cells by down-regulating Foxp3 promoter methylation levels. However, we couldn't observe similar results in healthy control CD4(+) T cells treated by TGP. Moreover, our results also showed that IFN-γ and IL-2 expression was enhanced in TGP-treated lupus CD4(+) T cells. These findings indicate that TGP inhibits autoimmunity in SLE patients possibly by inducing Treg cell differentiation, which may in turn be due to its ability to regulate the methylation status of the Foxp3 promoter and activate IFN-γ and IL-2 signaling.     

Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells

We have shown previously that the generation of tumor-reactive CD8(+) cytotoxic T lymphocytes require qualitatively different signals from CD4(+) and CD8(+) T cells that most likely are provided to dendritic cells (DCs). This raises the question of whether the two T cell subsets are equally able to deliver the initial activation signal to DCs. Using ovalbumin as a model antigen we show that naive CD4(+) T cells cannot activate immature DCs and do not become activated, even though they recognize antigen on immature DCs. In contrast, naive CD8(+) T cells rapidly activate DCs and subsequently start to proliferate. This suggests that CD8(+) T cells contribute to DC activation prior to CD4(+) T cells and implies that CD8(+) T cells can provide help to CD4(+) T cells.     

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.     

CD4(+) T cells stimulate memory CD8(+) T cell expansion via acquired pMHC I complexes and costimulatory molecules, and IL-2 secretion

The rapid and efficient expansion of CD8(+) memory T cells after the second encounter with a pathogen constitutes a hallmark trait of adaptive immunity. Yet, the contribution of CD4(+) T cells to the expansion of memory CD8(+) T cells remains the subject of controversy. Here, we show that, antigen-specific CD4(+) T cells, once activated by dendritic cells (DC) in vitro, have the capacity to stimulate expansion of memory CD8(+) T cells in vivo. The memory CD8(+) T cell expansion triggered by active CD4(+) T cells are mediated through DC-derived MHC I/peptide complexes and CD80 molecules displayed on the active CD4(+) T cells, with the involvement of IL-2 secreted by the active CD4(+) T cells. These results highlight a previously undescribed role of active CD4(+) T cells in triggering expansion of memory CD8(+) T cells.     

Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.     

IL-12 increases CD80 expression and the stimulatory capacity of bone marrow-derived dendritic cells

Dendritic cells (DC) are potent antigen-presenting cells derived from CD34 bone marrow stem cells. They undergo a series of maturational steps that allow them to stimulate primary T cell responses. Several cytokines are known to contribute to this process. In this study murine DC maturing from bone marrow progenitors under the influence of granulocyte macrophage colony stimulating factor and tumour necrosis factor-alpha were found to produce IL-12 as measured by ELISA and by flow cytometry to detect intracellular cytokine. Administration of additional IL-12 from day 3 to 7 of culture altered the function and phenotype of DC; enhanced stimulation of T cell proliferation by DC in allogeneic mixed leukocyte reactions was associated with an increase in the surface expression of CD80 on DC. These effects were dose dependent, and were consistently seen with IL-12 at 25 ng/ml and were less marked with IL-12 at 50 ng/ml. These results show that IL-12 is both produced by DC and can increase their stimulatory capacity. The findings suggest that there may be an autocrine effect of IL-12 on DC maturation and function.      

Identification of a rat bone marrow-derived dendritic cell population which secretes both IL-10 and IL-12: evidence against a reciprocal relationship between IL-10 and IL-12 secretion

The qualitative nature of immune responses induced by dendritic cells (DCs) is influenced by the balance of pro-inflammatory (e.g. IL-12) and anti-inflammatory (e.g. IL-10) cytokines that they secrete. Evidence to date suggests that IL-12 and IL-10 secretion is reciprocally regulated and that IL-10 inhibits IL-12 secretion. This study identifies a population of resting, immature rat bone marrow-derived DCs (BMDCs) which secretes IL-10, the IL-12(p70) heterodimer and the free IL-12(p40) subunit, the latter in vast excess of IL-12(p70). Counter-intuitively, activation with LPS induces the secretion of high and equivalent levels of IL-10 and IL-12(p40), but only quantitatively small increases in IL-12(p70). Neutralization of IL-10 increased the secretion of IL-12(p40) by resting BMDCs, but decreased IL-12(p40) secretion by LPS-activated BMDCs. Pre-incubation of resting BMDCs for 24h with neutralizing antibody to IL-10 reduced the subsequent secretion of IL-10 in allogeneic cultures of Lewis CD3(+) T cells with resting and LPS-activated Wistar BMDCs, and enhanced IL-12(p40) secretion in allogeneic cultures with LPS-activated BMDCs. IL-10 neutralization had no effect on the levels of IL-12(p70), IFN-gamma or IL-4 in allogeneic cultures. In summary, this study has identified a population of rat BMDCs that secretes low levels of bioactive IL-12(p70), but high levels of IL-10 and IL-12(p40). These findings argue against the concept that there is a reciprocal relationship between IL-10 and IL-12 secretion. They might also have implications for understanding the role of DCs in post-activation qualitative skewing of immune responses.     

Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18

The role of CD8+ T cells in adaptive immunity is well documented and involves numerous effector mechanisms including direct cytolysis of targets and secretion of cytokines. The role of CD8+ T cells in innate immunity has not been previously appreciated. Using J774 macrophages infected in vitro with the intracellular bacterium, Listeria monocytogenes (LM), we show that CD8+ T cells isolated from na?ve C57BL/6 (B6) mice respond rapidly by secreting IFN-gamma. CD8+ T cells secreting IFN-gamma can also be found in na?ve B6 mice 16 h after infection with LM. This rapid IFN-gamma response is TCR-independent and mediated through the actions of IL-12 and IL-18. Cell surface staining and cell sorting experiments indicate that these novel CD8+ T cells express memory markers. In vitro CFSE-labeling experiments show that IFN-gamma-secreting CD8+ T cells proliferate rapidly after 2 days in culture and after 4 days constitute the majority of the CD8+ T cell population. Together, these data suggest an important role for IFN-gamma-secreting CD8+ T cells in the innate response to bacterial pathogens.     

尼古丁对哮喘大鼠树突状细胞髓样分化因子88及白介素-10、白介素-12表达的影响

目的通过研究尼古丁对哮喘大鼠模型树突状细胞(DCs)Toll受体转导途径中重要接头蛋白髓样分化因子88(MyD88)表达影响及其与白介素-10(IL-10)、白介素-12(IL-12)的关系,从细胞水平探讨尼古丁加重哮喘的免疫学机制。方法制备哮喘大鼠模型,培养骨髓来源的DCs及脾脏来源的淋巴细胞,实验随机分为4组:对照组、哮喘组、尼古丁组和哮喘加尼古丁组,其中对照组和哮喘组大鼠DCs在培养5 d后在培养液中加入一定体积的PBS继续培养72 h,尼古丁组和哮喘加尼古丁组大鼠DCs在培养5 d后加入质量浓度为400μg/ml尼古丁作用72 h。收集各组细胞及细胞上清,采用酶联免疫吸附双抗体夹心法(ELISA)测定各组IL-10和IL-12的浓度。采用免疫印迹(Western blot)法测定细胞内MyD88的含量。采用MTT法测定DCs刺激同种异体淋巴细胞增殖的能力。结果①哮喘加尼古丁组和哮喘组大鼠DCs IL-10的表达均高于对照组,IL-12的表达均低于对照组,差异均具有统计学意义(P<0.01)。哮喘加尼古丁组大鼠DCs IL-10和IL-12的表达均低于哮喘组大鼠,差异均具有统计学意义(P<0.05)。②尼古丁组、哮喘组和哮喘加尼古丁组大鼠DCs MyD88的表达低于对照组,差异均具有统计学意义(P<0.05)。哮喘加尼古丁组大鼠DCs MyD88的表达低于哮喘组,差异均具有统计学意义(P<0.01)。③哮喘组大鼠DCs刺激同种异体淋巴细胞增殖的能力与对照组相比明显增强,差异具有统计学意义(P<0.01)。哮喘加尼古丁组大鼠DCs刺激同种异体淋巴细胞增殖的能力与哮喘组和对照组相比明显减弱,差异均具有统计学意义(P<0.05)。④DCs MyD88的表达与IL-12呈显著的正相关(r=0.644,P<0.01),与IL-10的表达无明显相关性。IL-12与IL-10的表达呈显著的负相关(r=-0.388,P<0.05)。DCs中IL-10的表达与淋巴细胞增殖活性呈显著的正相关(r=0.524,P<0.01);IL-12的表达与淋巴细胞增殖活性无明显相关性。结论尼古丁可以抑制哮喘大鼠DCs MyD88以及IL-10、IL-12的表达,并降低DCs刺激淋巴细胞的增殖能力。这可能是吸烟加重哮喘的免疫学机制之一。其中MyD88依赖的Toll受体转导途径可能在尼古丁下调IL-12的表达中发挥了一定作用。     

Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.     

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon (IFN) in response to virus. Although type I IFN is a natural killer (NK) cell modulator, a direct role for pDC in coordinating NK cell functions has not yet been elucidated in detail, especially in humans. Here we report that human pDC, following engagement of Toll-like receptor (TLR) 9, not only activate autologous NK cells, as indicated by the induction of CD69 expression, but also enhance their effector functions, especially cytotoxicity. Moreover, they can induce selective proliferation of CD56bright CD16- NK cells. This activity can be strongly augmented by the addition of autologous CD4+ CD25- T helper cells in an IL-2-dependent fashion. Strikingly, CD4+ CD25hi T regulatory (Treg) cells completely abrogate this IL-2-dependent proliferation of NK cells, while they are not able to influence NK cell activation or proliferation solely induced by pDC. Our data show that TLR9-engaged pDC represent a critical stimulus for human NK cells and that CD4+ Th cells and CD4+ CD25hi Treg cells play an important role in modulating human NK cell responses.     

Heterogeneity of CD4(+) and CD8(+) T cells

There is extensive plasticity in the T-cell response to antigen. Helper CD4(+) T cells, cytotoxic CD8(+) T cells, the progression from na?ve to effector and memory T cells, and differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. More recently it has become apparent that T-cell populations display additional diversity in terms of phenotype, anatomical distribution and effector function.     

Role of CD4(+)CD25(+) T regulatory cells in IL-2-induced vascular leak

T regulatory cells (CD4(+)CD25(+)) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent T(h)1/T(h)2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (T(h)1-biased) and BALB/c (T(h)2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4(+)CD25(+) T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4(+)CD25(+) T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4(+)CD25(+) T regulatory cells play an important role in the regulation of IL-2-induced EC injury.