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.     

Natural and inducible Tregs in swine: Helios expression and functional properties

Within the population of regulatory T cells (Tregs) natural Tregs (nTregs) and inducible Tregs (iTregs) can be distinguished. Although information about Tregs in swine exists, porcine iTregs were not under investigation yet. In this study, Foxp3⁺ iTregs were generated from CD4⁺Foxp3⁻ T cells by in vitro stimulation in the presence of IL-2 and TGF-β. In comparison to ex vivo Tregs these iTregs had a similar suppressive capacity on the proliferation of CD3-stimulated PBMC, caused higher levels of IL-10 in PBMC/Treg co-cultures, but did not suppress IFN-γ levels. The Ikaros family member Helios is currently discussed to distinguish iTregs and nTregs or to serve as an activation marker of Tregs. In this study, we demonstrate the cross-reactivity of an anti-mouse/human Helios mAb with porcine Helios. Flow cytometric analyses with this antibody showed that porcine iTregs do not express Helios after in vitro iTreg induction. Nevertheless, thymic Foxp3⁺ T cells, which arise at the CD4/CD8α single-positive stage of T-cell development and are defined as nTregs, entirely expressed Helios. Although this might suggest the suitability of Helios as an nTreg–iTreg differentiation marker we also found that Helios⁻ Tregs displayed a phenotype of naive CD4⁺ T cells in vivo. Since iTregs are by definition activated/differentiated Tregs, this finding precludes that all Helios⁻ Tregs are iTregs and thus also the use of Helios as a selection marker for porcine nTregs. Furthermore, Helios⁺ Tregs displayed a more differentiated phenotype indicating that Helios might rather serve as a Treg activation/differentiation marker.     

Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other

Foxp3(+) CD4(+) CD25(+) regulatory cell (Treg) subsets that maintain immunologic homeostasis have been considered to be a homogeneous population of naturally occurring, thymus-derived CD4(+)CD25(+) cells (nTregs). However, similar Foxp3+ Tregs can be induced from CD25(-) precursors in vivo, and ex vivo with interleukin 2 (IL-2) and transforming growth factor beta (TGF-beta) (iTregs). These two subsets differ in their principal antigen specificities and in the T-cell receptor signal strength and co-stimulatory requirements needed for their generation. However, whether iTregs have any unique functions in vivo has been unclear. Although IL-6 can convert nTregs to Th17 cells, iTregs induced by IL-2 and TGF-beta are resistant to this cytokine and thereby might retain suppressive function at inflammatory sites. Thus, nTregs and iTregs may have different roles in the adaptive immune response.     

Diabetes in non-obese diabetic mice is not associated with quantitative changes in CD4+ CD25+ Foxp3+ regulatory T cells

The role of regulatory T cells (Tregs) in maintaining self tolerance has been intensively researched and there is a growing consensus that a decline in Treg function is an important step towards the development of autoimmune diseases, including diabetes. Although we show here that CD25+ cells delay diabetes onset in non-obese diabetic (NOD) mice, we found, in contrast to previous reports, neither an age-related decline nor a decline following onset of diabetes in the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. Furthermore, we demonstrate that CD4+ CD25+ cells from both the spleen and pancreatic draining lymph nodes of diabetic and non-diabetic NOD mice are able to suppress the proliferation of CD4+ CD25- cells to a similar extent in vitro. We also found that pretreatment of NOD mice with anti-CD25 antibody allowed T cells with a known reactivity to islet antigen to proliferate more in the pancreatic draining lymph nodes of NOD mice, regardless of age. In addition, we demonstrated that onset of diabetes in NOD.scid mice is faster when recipients are co-administered splenocytes from diabetic NOD donors and anti-CD25. Finally, we found that although diabetic CD4+ CD25+ T cells are not as suppressive in cotransfers with effectors into NOD.scid recipients, this may not indicate a decline in Treg function in diabetic mice because over 10% of CD4+ CD25+ T cells are non-Foxp3 and the phenotype of the CD25- contaminating population significantly differs in non-diabetic and diabetic mice. This work questions whether onset of diabetes in NOD mice is associated with a decline in Treg function.     

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.     

In vitro-expanded CD4CD25Foxp3 regulatory T cells controls corneal allograft rejection

Aims

Natural CD4⁺CD25⁺ regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model.

Methods

Natural CD4⁺CD25⁺ T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4⁺CD25⁻ T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling.

Results

Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4⁺CD25⁻ T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression.

Conclusion

Therapeutic use of in vitro-expanded CD4⁺CD25⁺ T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.     

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

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

2-Gy whole-body irradiation significantly alters the balance of CD4+CD25? T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice

CD4⁺CD25⁺ T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4⁺CD25⁺Foxp3⁺ Treg cells has not been determined. The proportion, phenotypes and function of CD4⁺CD25⁺ Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁺Foxp3⁺ Treg cells to CD4⁺ T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4⁺CD25⁺ Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4⁺CD25⁻ T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4⁺CD25⁺Foxp3⁺ Treg cells than in CD4⁺CD25⁻ T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.     

NOD小鼠自然状态下CD4~+CD25~+T细胞的动态变化及意义

研究自发的1型糖尿病雌鼠模型(NOD)在自然状态下发生1型糖尿病过程中CD4+CD25+T细胞的动态变化,旨在初步探讨调节性T细胞参与1型糖尿病发病的可能机制。采用雌性NOD小鼠作动物模型,每2周尾静脉采血1次,采用三色流式细胞术测定NOD小鼠外周血中CD4+CD25+T细胞(CD3+CD4+CD25+)的百分率。在32周时,对比发生糖尿病和未发生糖尿病NOD小鼠不同脏器中的CD4+CD25+T细胞阳性率。HE法检测胰岛炎。结果显示:(1)自第6周起NOD小鼠CD4+CD25+T细胞百分率逐渐降低。发生糖尿病NOD小鼠CD4+CD25+T细胞比率低于未发病NOD小鼠对照组(外周血分别为0.94%±0.21%、1.62%±0.23%,P=0.01;脾脏2.09%±0.14%、2.77%±0.36%,P=0.019),提示糖尿病NOD小鼠外周血中存在异常比例的CD4+CD25+T细胞;(2)32周龄糖尿病NOD小鼠与未发病NOD小鼠的CD4+CD25+T细胞抑制功能减低,与阳性对照组有显著性差异;(3)HE染色结果示糖尿病NOD小鼠胰岛结构完全破坏,胰岛炎程度较未发病NOD小鼠严重。该结果提示NOD小鼠发生糖尿病时免疫功能紊乱与CD4+CD25+T细胞参与调节及T细胞亚群变化相关,糖尿病的发生受致病性T细胞和调节性T细胞的调节。     

Enhancement of antitumor immunity by low-dose total body irradiation is associated with selectively decreasing the proportion and number of T regulatory cells

Low-dose total body irradiation (LTBI) is used in the treatment of somecancers mainly for immune enhancement rather than cell killing. However, themechanism underlying LTBI remains unknown. In this study, by analyzing theimmune patterns of lymphocytes, we found that the percentage and absolutenumber of CD4⁺CD25⁺Foxp3⁺regulatory T cells are markedly decreased in naive mice following treatmentwith LTBI. On the contrary, the CD4⁺CD44⁺/CD8⁺CD44⁺effector-memory T cells are greatly increased. Importantly, naive mice treatedwith dendritic cell-gp100 tumor vaccines under LTBI induced an enhancementof antigen-specific proliferation and cytotoxicity as well as interferon-γ(IFN-γ) secretion against F10 melanoma tumor challenge, compared to treatmentwith either the tumor vaccine or LTBI alone. Consequently, the treatment resultedin a reduced tumor burden and prolonged mouse survival. Our data demonstratethat LTBI''s enhancement of antitumor immunity was mainly associatedwith selectively decreasing the proportion and number of T regulatory cells,implying the potential application of the combination of LTBI and a tumorvaccine in antitumor therapy.     

CD4~+CD25~+Foxp3~+调节性T细胞与LSCC发病的相关研究

为研究调节性T细胞在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)、发展中的变化及其参与疾病进展的作用机制,收集2010～2011年上海市五官科医院收治的50例LSCC患者的肿瘤组织和外周血,应用流式细胞术检测CD4+CD25+Foxp3+Treg细胞及趋化因子受体CCR6的表达变化,Real-time PCR法检测转录因子Foxp3以及细胞因子mRNA的表达量。结果发现:LSCC患者外周血中CD4+CD25+Foxp3+Treg的百分比较正常人显著增加,并与临床分期相关;CD4+CD25+CCR6+Treg Foxp3的表达,以及肿瘤组织Foxp3mRNA的表达皆明显高于对照组,且与临床分期、淋巴结转移相关。同时发现,LSCC患者外周血中TGF-β和IL-10mRNA的检出水平分别高于对照组,但IFN-γ、IL-2、IL-12mRNA的水平低于对照组。提示此类Foxp3+Treg属于一类诱导性T抑制细胞(Foxp3+iTreg),可通过产生IL-10和TGF-β抑制LSCC患者的细胞免疫功能。Foxp3的检测可能对判断LSCC的预后有一定价值。     

Hepatitis C virus core protein triggers expansion and activation of CD4+CD25+ regulatory T cells in chronic hepatitis C patients

CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) are increased in patients with chronic hepatitis C, which may contribute to the sustained suppression of hepatitis C virus (HCV)-specific T-cell responses and viral persistence in HCV-infected individuals. We postulated that HCV core protein (HCVc) directly contributes to the expansion of Tregs in HCV-infected patients, and we provide evidence to support this hypothesis in the report. Peripheral blood mononuclear cells (PBMCs) and sera were collected from 87 treatment-naïve chronic HCV-infected patients, CD4⁺CD25⁺ Tregs were measured by flow cytometry, and HCV RNA and HCVc levels were detected using qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. CD4⁺, CD8⁺, CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were purified from healthy donors and cultured with recombinant HCVc and Toll-like receptor (TLR) ligands. Flow cytometry was used to analyze cell proliferation, and ELISA was performed to measure cytokine production. In the 87 chronic HCV-infected patients, HCVc showed a significant correlation with HCV RNA and CD4⁺CD25⁺ Tregs. Mechanistic studies showed that HCVc, together with anti-CD3 antibody, augmented CD4⁺CD25⁺ Treg proliferation, but inhibited CD4⁺CD25⁻ T-cell proliferation and IFN-γ production, in a dose-dependent and Treg-dependent manner. Moreover, unlike the TLR3 ligand (poly I:C) and the TLR4 ligand (lipopolysaccharide, LPS), the TLR2 ligand (lipoteichoic acid, LTA) and HCVc both inhibited TCR-induced CD4⁺ T-cell proliferation and IFN-γ secretion in a Treg-dependent manner. These data indicate that HCVc, like other TLR2 ligands, triggers CD4⁺CD25⁺ Treg activation and expansion to inhibit host immune responses, which may play a critical role in viral persistence in HCV-infected patients.     

A Role for Trichosanthin in the Expansion of CD4+CD25+ Regulatory T Cells

CD4⁺CD25⁺ regulatory T cells (Tregs) are critical for the peripheral immune tolerance. Understanding the signals for the generation of Tregs is important for the clinical immunotherapy, but only limited progress has been made on obtaining enough peripheral Tregs. The aim of this study was to evaluate the role of trichosanthin (Tk) extracted from Chinese medicinal herb Trichosanthes kirilowi on the function of Tregs in vitro and in vivo. We reported here that Tk is needed for the expansion of freshly isolated CD4⁺CD25⁺Tregs (nTregs) into Tk‐expanded CD4⁺CD25⁺Tregs (Tk‐Tregs) through up‐regulating CD25 and Foxp3 expression. The dose–response analyses indicated that 100 ng/ml Tk was the most appropriate dose. The result of real‐time PCR showed that Tk‐Tregs expressed 1.5‐fold higher levels of Foxp3 than those observed in nTregs. Tk ‐ Tregs markedly suppressed activation of effector T cells at a suppressor/responder ratio of 1:1, 1:2, 1:4, 1:8 or 1:16, and their effect was dose dependent. Moreover, Tk‐Tregs secreted more immunosuppressive cytokines interleukin (IL)‐10 and transforming growth factor (TGF)‐β1 after stimulating with antigen and antigen‐presenting cells (APC). Transwell experiments showed that not only cell‐to‐cell contact but also soluble cytokines were involved in suppressive mechanism of Tk‐Tregs. And Tk‐Tregs were more efficient in suppressing CD25^?T cell response to specific antigen than to irrelative antigen. Most importantly, it was revealed for the first time that Tk‐Tregs could prolong the survival duration of mice with acute graft‐versus‐host disease (aGVHD). In conclusion, the study suggests a possible therapeutic potential of Tk‐Tregs for clinical treatment on aGVHD.     

CD4+CD25+Treg在急性淋巴细胞白血病患者外周血中的表达及其体外实验研究

目的初步探讨CD4 CD25 调节性T细胞(CD4 CD25 regulatory T cells,CD4 CD25 Treg)在急性淋巴细胞白血病(acute lymphocytic leukemia,ALL)患者化疗前及化疗缓解后外周血中的表达水平,并研究患者血清能否诱导外周血CD4 CD25-T细胞转化为CD4 CD25 Treg。方法①采用流式细胞术分别检测ALL初诊组、化疗完全缓解或部分缓解组及正常对照组外周血中CD4 CD25 T细胞所占比例,然后通过荧光定量RT-PCR检测各组外周血中转录因子Foxp3mRNA的表达水平,并逐层分析比较。②采集正常人外周血单个核细胞后,对照组用正常人血清,实验组用患者血清并分别设浓度梯度进行培养,72h后采用流式细胞术、荧光定量RT-PCR分别检测CD4 CD25 T细胞和Foxp3mRNA表达。结果ALL化疗缓解组CD4 CD25 T细胞及Foxp3mRNA表达水平均明显高于ALL初诊组和正常对照组(P<0.05),后两者之间CD4 CD25 T细胞水平无统计学差异(P>0.05),但ALL初诊组Foxp3mRNA含量较正常对照组明显升高(P<0.01),差异具有统计学意义;并且血清培养对照组CD4 CD25 T细胞水平及Foxp3mRNA含量均明显低于实验组(P<0.05),且其表达并不随血清浓度的增加而升高。结论CD4 CD25 Foxp3 Treg在ALL初诊组及化疗缓解组患者外周血中比例明显升高,且初步表明患者血清中的可溶性物质可诱导外周血CD4 CD25 T细胞转化为CD4 CD25 Treg,提示CD4 CD25 Treg可能是ALL免疫抑制的一个重要原因。     

Recipient-Derived Allo-iTregs Induced by Donor DCs Effectively Inhibit the Proliferation of Donor T Cells and Reduce GVHD

To compare the potency of recipient-derived, antigen-specific regulatory T cells induced by different dendritic cells (DCs; iTregs) and freshly isolated natural regulatory T cells (nTregs) in preventing mouse graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). CD4⁺ T cells from recipient BALB/c mice were stimulated with DCs from recipient BALB/c (syn-DCs), donor B6 (allo-DCs), and third-party C3H (third-party-DCs) mice to induce different iTregs. In parallel, nTregs were isolated from spleen cells of recipient BALB/c (syn-nTregs) and donor B6 (allo-nTregs) mice using magnetic-activated cell sorting. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the suppressive ability of these various regulatory T cells (Tregs). Both the iTregs and nTregs were transfused to GVHD mice on Days 0, 1, 3, and 5. Body weight, GVHD score, and survival time were monitored. Peripheral Tregs were subsequently examined on Days 7, 14, 21, and 28 after BMT, while chimerism was evaluated on Days 14 and 60. Histopathology of colon, liver, and spleen were also performed. DCs markedly induced CD25⁺ and Foxp3⁺ expression on CD4⁺ T cells. The allo-DC-induced Tregs (allo-iTregs) suppressed the proliferation of alloreactive T cells better than the other iTregs/nTregs in MLR assays (P < 0.05). Meanwhile, transfusion of the allo-iTregs reduced the severity of GVHD (P < 0.05), increased survival time compared with the GVHD group (P < 0.05), and enhanced the chimerism proportion. On Day 28 after BMT, the allo-iTregs group had the highest frequency of peripheral Tregs (P < 0.05). Recipient-derived allo-iTregs induced by donor DCs included predominant clones that specifically recognized donor antigens. These allo-iTregs not only prevented GVHD by suppressing the proliferation of donor-alloreactive T cells, but also promoted engraftment, and prolonged the survival of GVHD mice. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:825–836, 2019. © 2018 Wiley Periodicals, Inc.     

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.     

Essential roles of TGF-beta in anti-CD3 antibody therapy: reversal of diabetes in nonobese diabetic mice independent of Foxp3+CD4+ regulatory T cells

Anti-CD3 mAb have potentials to treat overt autoimmunity as reported recently. However, the underlying mechanisms remain unclear. In this report, using an animal model of type 1 diabetes, we found that TGF-beta1, an important immunoregulatory cytokine, plays a critical role in anti-CD3-mediated diabetes reversion and immune tolerance. Anti-CD3 treatment increased the TGF-beta1 production, lasting for a long period of time, which contributed to maintaining peripheral tolerance by controlling pathogenic cells. Furthermore, we found that anti-CD3 treatment did not increase the forkhead box p3+ (Foxp3+)CD4+ regulatory T cells (Tregs). When fractionated from anti-CD3-treated, remitting mice and cotransferred with splenic cells from diabetic NOD mice, these Tregs failed to inhibit diabetes development in NOD.scid mice. Moreover, we found that the depletion of these Tregs did not affect an anti-CD3-mediated, therapeutic effect and the level of TGF-beta1 production, which suggested that an increased level of TGF-beta1 may not derive from these Tregs. Thus, our data showed a dispensable role of Foxp3+CD4+ Tregs in anti-CD3 antibody-reversed diabetes in NOD mice. These findings may have an important implication for understanding the involved mechanisms responsible for immunomodulatory function of anti-CD3 antibody on autoimmune diseases.     

Regulatory T cells in many flavors control asthma

That regulatory T cells (Tregs) have a crucial role in controlling allergic diseases such as asthma is now undisputed. The cytokines most commonly implicated in Treg-mediated suppression of allergic asthma are transforming growth factor-β (TGF-β) and interleukin (IL)-10). In addition to naturally occurring Tregs, adaptive Tregs, induced in response to foreign antigens, have been shown in recent studies. The concept of inducible/adaptive Tregs (iTregs) has considerable significance in preventing asthma if generated early enough in life. This is because cytokines such as IL-4 and IL-6 inhibit Foxp3 induction in naive CD4+ T cells and therefore de novo generation of Tregs can be expected to be less efficient when it is concomitant with effector cell development in response to an allergen. However, if iTregs can be induced, the process of infectious tolerance would facilitate expansion of the iTreg pool as suggested in the recent literature. It is tempting to speculate that there is a window of opportunity in early life in the context of a relatively immature immune system that is permissive for the generation of iTregs specific to a spectrum of allergens that would regulate asthma for lifelong. The focus of this review is the relevance of nTregs and iTregs in controlling asthma from early life into adulthood, the mechanisms underlying Treg function, and the prospects for using our current concepts to harness the full potential of Tregs to limit disease development and progression.     

Decreased 4-1BB expression on CD4+CD25 high regulatory T cells in peripheral blood of patients with multiple sclerosis

As a tumour necrosis factor receptor superfamily member, 4-1BB (CD137) is preferentially expressed in CD4+CD25+ regulatory T cells (Tregs) and has been suggested to play an important role in regulating the generation or function of Tregs. Recent studies of human Tregs have shown that blood CD4+CD25(high) T cells were much closer to Tregs in terms of their functionality. Furthermore, CD4+CD25(high) Tregs have been found to have a decreased effector function in patients with multiple sclerosis (MS). In this study, we examined the expression of 4-1BB and soluble 4-1BB (s4-1BB) protein levels in the peripheral blood of MS patients. Compared with healthy controls, MS patients had decreased 4-1BB expression in their CD4+C25(high) Tregs and increased plasma s4-1BB protein levels. Moreover, the plasma s4-1BB levels of MS patients were shown to be inversely correlated with the 4-1BB surface expression of CD4+CD25(high) Tregs. The down-regulated 4-1BB expression on CD4+CD25(high) Tregs of MS patients may be involved in the impaired immunoactivity of these Tregs. The elevated s4-1BB levels may, at least in part, function as a self-regulatory attempt to inhibit antigen-driven proliferation of Tregs or their immunosuppressive activity.     

CD5 plays an inhibitory role in the suppressive function of murine CD4(+) CD25(+) T(reg) cells

A subset of CD4(+) T cells, the CD4(+) CD25(+) regulatory T (T(reg)) cells in the lymphoid organs and peripheral blood are known to possess suppressive function. Previous in vitro and in vivo studies have indicated that T cell receptor (TCR) signal is required for development of such 'natural regulatory (T(reg)) cells' and for activation of the effector function of CD4(+) CD25(+) regulatory T cells. CD5 is a cell surface molecule present on all T cells and a subtype of B lymphocytes, the B-1 cells, primarily localized to coelomic cavities, Peyer's patches, tonsils and spleen. CD5 acts as a negative regulator of T cell and B cell signaling via recruitment of SHP-1. Here, we demonstrate that T(reg) cells obtained from CD5(-/-) mice are more potent than those from wild type mice in suppressing the in vitro cell proliferation of anti-CD3 stimulated CD4(+) CD25(-) responder T cells. This phenomenon was cell contact and GITR dependent. Lack of CD5 expression on T(reg) cells (from spleen, lymph node and thymus) did not affect the intracellular levels of Foxp3. However, CD5(-/-) T(reg) thymocytes were able to elicit a higher Ca(2+) response to TCR + co-stimulatory signals than the wild type cells. CD5(-/-) mice expressed more Foxp3 mRNA in the colon than wild type mice, and additionally, the severity of the dextran sulfate sodium (DSS)-induced colitis in CD5(-/-) mice was less than the wild type strain. We suggest that manipulation of CD5 expression or the downstream signaling components of CD4(+) CD25(+) T(reg) cells as a potential strategy for therapeutic intervention in cases of auto-immune disorders.