Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice

A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4(+)CD25(+) Treg cells in Balb/c mice, which were older than 20 months. Significantly enhanced percentages of CD4(+)CD25(+) Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vbeta family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4(+)CD25(+) Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (IL-2 and IFN-gamma) but not cell proliferation of effector T cells, as compared with CD4(+)CD25(+) Treg cells of young mice. In addition, the percentages of CD4(+)CD8(-)CD25(+) Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4(+)CD25(+) Treg cells were altered significantly with aging in mice. The functional defects of CD4(+)CD25(+) Treg cells may shed light on the role of CD4(+)CD25(+) Treg cells in the increased sensitivity to autoimmune diseases of aged populations.     

血红素加氧酶-1上调CD4~+ CD25~+ Treg foxp3表达以增强Treg的免疫抑制功能

本研究探讨血红素加氧酶-1(heme oxygenase-1,HO-1)诱导CD4+CD25+调节性T细胞(regulatory T cells,Treg)foxp3表达,增加IL-10分泌,提高CD4+CD25+Treg的免疫抑制功能。选用磁珠分离正常BALB/c小鼠脾脏CD4+CD25+Treg,转染含HO-1质粒pcDNA3HO-1,或用血红素(hemin)、锡-原卟啉(Sn-protoporphyrin,SnPP)干预,培养48 h。用卵清蛋白致敏、激发BALB/c小鼠建立哮喘模型,并在致敏、激发阶段分别经血红素和SnPP干预。用Real-Time PCR和Western blot方法分别测定培养细胞内HO-1、foxp3 mRNA及蛋白量;ELISA方法分别测定细胞上清液和动物血清中IL-10、TGF-β水平;用磁珠分离哮喘动物脾脏CD4+CD25+Treg进行功能抑制试验。结果显示:经pcDNA3HO-1和血红素上调CD4+CD25+Treg HO-1表达,foxp3表达及蛋白水平相应增加,上清液IL-10水平明显升高。而OVA致敏、激发的哮喘小鼠模型,经血红素干预后,血清IL-10分泌亦增多,CD4+CD25+Treg功能抑制作用显著增强。该结果表明HO-1诱导CD4+CD25+Treg特异性转录因子foxp3表达,促进IL-10分泌,增强CD4+CD25+Treg的调节功能,具有显著的免疫抑制作用。     

CD4+CD25+ naturally occurring regulatory T cells and not lymphopenia play a role in the pathogenesis of iodide-induced autoimmune thyroiditis in NOD-H2h4 mice

NOD-H2(h4) mice, which express I-A(k) on the NOD background, spontaneously develop autoimmune thyroiditis, a model of Hashimoto thyroiditis in humans, by adding iodide in the drinking water. Parental NOD mice have previously been shown to have intrinsic numerical abnormalities in peripheral lymphocytes and lymphocyte subpopulations such as CD4(+)CD25(+) naturally occurring regulatory T cells (Treg). Therefore we first investigated whether the similar abnormalities exist in NOD-H2(h4) mice. We observed that, compared with other non-autoimmune disease prone BALB/c and C57BL/6 mice, NOD-H2(h4) mice have lower numbers of splenocytes, CD3(+)T, CD4(+)T and CD8(+)T cells but the ratios of Treg to CD4(+)T cells were comparable. Increasing the numbers of peripheral lymphocytes by Complete Freund's Adjuvant immunization or splenocyte transfer did not affect development of thyroiditis, indicating that lymphopenia does not play a critical role in the pathogenesis of thyroiditis. We next examined the significance of Treg by depleting this lymphocyte subset with anti-CD25 antibody. Treg depletion, performed 4days before the administration of NaI water for 8 weeks, significantly exacerbated thyroiditis (p<0.01). Anti-thyroglobulin antibody titers also increased by Treg depletion (p<0.01) without changing the IgG2b to IgG1 ratios. In addition, expression levels of mRNA for IFN-gamma and IL-4 were enhanced in parallel. However, T(4) levels were similar between antibody-treated and untreated groups. Additional anti-CD25 administration at 3 weekly intervals did not influence these results. These data, together with previous studies on other mouse models of inducible thyroiditis and Graves' disease, indicate the role played by Treg in keeping anti-thyroid autoimmune reaction in check in experimental autoimmune thyroid diseases.     

CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4^＋CD25^＋T细胞凋亡的影响。结果MACS成功分离CD4^＋CD25^＋T细胞，纯度可达98％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。     

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.     

Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4+ effector cells that are resistant to regulatory T cell activity

In Fas/FasL-deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3(+) Treg cells is elevated in BALB/c-lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220(-) CD4(+) T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-gamma differentiation of Th cells from BALB/c or young BALB-lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c-lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.     

A method of generating antibodies against exogenously administered self-antigen by manipulating CD4+CD25+ regulatory T cells

The generation of antibodies against self-antigens or antigens having a high degree of structural homology with self-antigens is a difficult task because of immunological tolerance. CD4(+)CD25(+) regulatory T cells play an important role in maintaining peripheral tolerance. Sakaguchi et al. previously reported that the transfusion of CD25(+) cell-depleted mouse splenocytes into syngeneic nude mice results in a breaking of peripheral tolerance that leads to the development of autoimmunity. In this study, we attempted to apply this mouse model to the generation of antibodies against self-antigens. We depleted CD25(+) cells from BALB/c mouse splenocytes and transferred the rest of the cells into syngeneic nude mice. The animals were immunized with mouse thyroglobulin. We observed a significant increase of the anti-mouse thyroglobulin antibody titer in the group of mice immunized twice within 10 days after the cell transfer (P<0.05). From these mice, we established hybridoma cell lines producing anti-mouse thyroglobulin monoclonal antibodies, including a clone with a dissociation constant of 10(-8)M. Control nude mice which received CD25(+) cell-containing BALB/c splenocytes did not produce anti-mouse thyroglobulin antibodies. When the CD25(-)cell-transferred nude mice were immunized with mouse Gα12, another self-antigen, anti-Gα12 antibodies were produced in the sera. This method should prove highly useful in the generation of antibodies against self-antigens or antigens for which the structure is highly conserved across species.     

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.     

Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells

Tolerance to self-antigens expressed in peripheral organs is maintained by CD4(+) CD25(+) Foxp3(+) Treg cells, which are generated as a result of thymic selection or peripheral induction. Here, we demonstrate that steady-state migratory DCs from the skin mediated Treg conversion in draining lymph nodes of mice. These DCs displayed a partially mature MHC II(int) CD86(int) CD40(hi) CCR7(+) phenotype, used endogenous TGF-β for conversion and showed nuclear RelB translocation. Deficiency of the alternative NF-κB signaling pathway (RelB/p52) reduced steady-state migration of DCs. These DCs transported and directly presented soluble OVA provided by s.c. implanted osmotic minipumps, as well as cell-associated epidermal OVA in transgenic K5-mOVA mice to CD4(+) OVA-specific TCR-transgenic OT-II T cells. The langerin(+) dermal DC subset, but not epidermal Langerhans cells, mediated conversion of naive OT-II×RAG-1(-/-) T cells into proliferating CD4(+) CD25(+) Foxp3(+) Tregs. Thus, our data suggest that steady-state migratory RelB(+) TGF-β(+) langerin(+) dermal DCs mediate peripheral Treg conversion in response to epidermal antigen in skin-draining lymph nodes.     

The different effects of indirubin on effector and CD4+CD25+ regulatory T cells in mice: potential implication for the treatment of autoimmune diseases

CD4(+)CD25(+) regulatory T (Treg) cells play an essential role in the induction and maintenance of peripheral self-tolerance. Indirubin, a traditional Chinese medicine, was clinically used in the treatment of chronic myelocytic leukemia as well as some autoimmune diseases, including Alzheimer's disease, diabetes, and so on. The effects of indirubin on CD4(+)CD25(+)Treg cells, which play a critical role in controlling autoimmunity, have not been addressed. In the present study, we observed the cell levels, phenotypes, and immunoregulatory function of CD4(+)CD25(+)Treg cells in indirubin-treated mice. Treatment with indirubin significantly enhanced the ratios of CD4(+)CD25(+)Treg cells or CD4(+)CD25(+)Foxp3(+)Treg cells to CD4(+)T cells in peripheral blood, lymph nodes, and spleens (P < 0.01 compared with control mice). CD4(+)CD25(+)Foxp3(+)Treg cells to CD4 single positive cells in the thymi of indirubin-treated mice were significantly higher than those in control mice. Furthermore, splenic CD4(+)CD25(+)Treg cells in indirubin-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4(+)CD25(+)Treg cells in vitro. The present studies indicate that CD4(+)CD25(+)Treg cells are more resistant to indirubin than effector T cells in vivo. The selectively enhanced CD4(+)CD25(+)Treg cell levels by indirubin made host to be more favorable for immune tolerance induction, which opened one possibility for indirubin to treat autoimmune diseases.     

Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes

Although regulatory T cells (Tregs) are well described, identifying autoaggressive effector T cells has proven more difficult. However, we identified CD4loCD40+ (Th40) cells as being necessary and sufficient for diabetes in the NOD mouse model. Importantly, these cells are present in pancreata of prediabetic and diabetic NOD mice, and Th40 cells but not CD4+CD40(-) T cells transfer progressive insulitis and diabetes to NOD.scid recipients. Nonobese-resistant (NOR) mice have the identical T cell developmental background as NOD mice, yet they are diabetes-resistant. The seminal issue is how NOR mice remain tolerant to diabetogenic self-antigens. We show here that autoaggressive T cells develop in NOR mice and are confined to the Th40 subset. However, NOR mice maintain Treg numbers equivalent to their Th40 numbers. NOD mice have statistically equal numbers of CD4+CD25+forkhead box P3+intrinsic Tregs compared with NOR or nonautoimmune BALB/c mice, and NOD Tregs are equally as suppressive as NOR Tregs. A critical difference is that NOD mice develop expanded numbers of Th40 cells. We suggest that a determinant factor for autoimmunity includes the Th40:Treg ratio. Mechanistically, NOD Th40 cells have low susceptibility to Fas-induced cell death and unlike cells from NOR and BALB/c mice, have predominantly low Fas expression. CD40 engagement of Th40 cells induces Fas expression but further confers resistance to Fas-mediated cell death in NOD mice. A second fundamental difference is that NOD Th40 cells undergo much more rapid homeostatic expansion than Th40 cells from NOR mice.     

Complementary role of CD4+CD25+ regulatory T cells and TGF-beta in oral tolerance

CD4(+)CD25(+) regulatory T cells are thought to be generated in the periphery as well as in the thymus. We sought to determine the roles played by CD4(+)CD25(+) T cells and transforming growth factor-beta (TGF-beta) in the induction and maintenance of tolerance generated by oral antigens in BALB/c mice. We found that oral administration of a high dose of ovalbumin (OVA) suppressed OVA-specific proliferation and antibody production in BALB/c mice depleted of CD25(+) cells. In contrast, the unresponsiveness induced by lower doses of OVA was only partially blocked by CD25 depletion prior to feeding. Depletion of CD4(+)CD25(+) cells after mice were orally tolerized did not reverse the tolerant status, indicating that these cells were not required to maintain the established tolerance. Furthermore, the induction of oral tolerance was not hampered by the administration of TGF-beta-neutralizing antibodies. However, in mice depleted of CD25(+) cells, anti-TGF-beta-neutralizing antibodies blocked the induction of tolerance, regardless of whether the mice followed the high- or low-dose regimens of oral OVA. CD25 depletion together with TGF-beta neutralization led the expansion of OVA-specific CD4 T cells against the subsequent antigen challenge, and each treatment alone did not. Our findings indicate that CD4(+)CD25(+) T cells and TGF-beta play a complementary role in the induction of oral tolerance, at least in part, by regulating the expansion of antigen-specific CD4 T cells.     

Pertussis toxin as an adjuvant suppresses the number and function of CD4+CD25+ T regulatory cells

We observed a remarkable reduction in the frequency and immunosuppressive activity of splenic CD4+CD25+ T cells in C57BL/6 mice with MOG33-55-induced experimental autoimmune encephalomyelitis (EAE). Our study revealed that pertussis toxin (PTx), one component of the immunogen used to induce murine EAE, was responsible for down-regulating splenic CD4+CD25+ cells. Treatment of normal BALB/c mice with PTx in vivo reduced the frequency, suppressive activity and FoxP3 expression by splenic CD4+CD25+ T cells. However, PTx treatment did not alter the expression of characteristic phenotypic markers (CD45RB, CD103, GITR and CTLA-4) and did not increase the expression of CD44 and CD69 by the residual splenic and lymph node CD4+CD25+ T cells. This property of PTx was attributable to its ADP-ribosyltransferase activity. PTx did not inhibit suppressive activity of purified CD4+CD25+ T regulatory (Treg) cells in vitro, but did so in vivo, presumably due to an indirect effect. Although the exact molecular target of PTx that reduces Treg activity remains to be defined, our data suggests that alteration of both distribution and function of splenic immunocytes should play a role. This study concludes that an underlying cause for the immunological adjuvanticity of PTx is down-regulation of Treg cell number and function.     

CTLA4-Ig modifies dendritic cells from mice with collagen-induced arthritis to increase the CD4+CD25+Foxp3+ regulatory T cell population

Cytotoxic T lymphocyte antigen-4 (CTLA4) and IgG fusion protein, CTLA4-Ig, is a therapeutic agent used for rheumatoid arthritis. It binds B7 molecules on dendritic cells (DCs) and thereby blocks B7/CD28 costimulatory interaction and inhibits effective T cell proliferation. However, the effect of CTLA4-Ig on the regulatory T cell (Treg) is still not known. In this study, we investigated the influence of CTLA4-Ig on the CD4+CD25+Foxp3+ Treg population in collagen-induced arthritis (CIA) mouse model. CTLA4-Ig suppressed CIA and increased the CD4+CD25+Foxp3+ Treg population in joint and spleen. When CD11c + DCs and CD4+T cells from CIA mice were cultured with anti-CD3, CTLA4-Ig increased the CD4+CD25 + Foxp3+ Treg population in a TGF-β-dependent manner. When CD11c + DCs from CIA mice were treated with CTLA4-Ig and adoptively transferred into CIA-induced mice, arthritis did not develop in association with the increase in CD4+CD25+Foxp3+ Treg population. However, in CTLA4-Ig-untreated DC-transferred CIA mice, arthritis developed and then rapidly progressed. Our study demonstrated that CTLA4-Ig suppressed CIA by modifying DCs from CIA mice into tolerogenic DCs to increase the CD4+CD25+Foxp3+ Treg population and this seems to be the new immune regulatory mechanism of CTLA4-Ig.     

Regulatory function of CD4+CD25+ T cells from Class II MHC-deficient mice in contact hypersensitivity responses

Contact hypersensitivity (CHS) is a CD8+ T cell-mediated, inflammatory response to hapten sensitization and challenge of the skin. During sensitization, the magnitude and duration of hapten-specific CD8+ T cell expansion in the skin-draining lymph nodes (LN) are restricted by CD4+CD25+ T regulatory cells (Treg). The regulation of hapten-specific CD8+ T cell priming in Class II MHC-deficient (MHC-/-) mice was investigated. Although hapten-specific CD8+ T cell priming and CHS responses were elevated in Class II MHC-/- versus wild-type mice, presensitization depletion of CD4+ or CD25+ cells in Class II MHC-/- mice further increased CD8+ T cell priming and the elicited CHS response. Flow cytometry analyses of LN cells from Class II MHC-/- mice revealed a population of CD4+ T cells with a majority expressing CD25. Forkhead box p3 mRNA was expressed in LN cells from Class II MHC-/- and was reduced to background levels by depletion of CD4+ or CD25+ cells. Isolated CD4+CD25+ T cells from wild-type and Class II MHC-/- mice limited in vitro proliferation of alloantigen- and hapten-specific T cells to antigen-presenting stimulator cells. These results identify functional CD4+CD25+ Treg in Class II MHC-/- mice, which restrict hapten-specific CD8+ T cell priming and the magnitude of CHS responses.     

CD4+ CD25+ [corrected] regulatory T cells render naive CD4+ CD25- T cells anergic and suppressive

CD4(+) CD25(+) Foxp3(+) naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4(+) CD25(-) T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG(-/-)) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4(+) CD25(-) T cells and CD8(+) T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-beta partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4(+) CD25(-) responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.     

Suppression by CD4+CD25+ regulatory T cells is dependent on expression of heme oxygenase-1 in antigen-presenting cells

Heme oxygenase-1 (HO-1) has been viewed as a cytoprotective protein, ameliorating the effects of inflammatory cellular damage, and as beneficial in allograft protection from acute and chronic rejection, suggesting important functions in both innate and adaptive immune responses. Mice deficient in HO-1 exhibit defective immune regulation characterized by a proinflammatory phenotype. We examined if impaired regulatory T cell (Treg) function contributes to the immunoregulatory defects observed in HO-1(-/-) mice. HO-1(-/-) mice exhibited a significantly higher proportion of Foxp3-expressing cells among total CD4(+) and CD4(+)CD25(+) cells in comparison to HO-1(+/+) mice, and HO-1(-/-) Treg cells were at least as effective as HO-1(+/+) Treg cells in suppressing proliferation of effector T cells in vitro from either HO-1(+/+) or HO-1(-/-) mice. However, the absence of HO-1 in antigen-presenting cells abolished the suppressive activity of Treg cells on effector T cells. These findings demonstrate that HO-1 activity in antigen-presenting cells is important for Treg-mediated suppression, providing an explanation for the apparent defect in immune regulation in HO-1(-/-) mice.     

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

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