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 functional insufficiency of human CD4+CD25 high T-regulatory cells in allergic asthma is subjected to TNF-alpha modulation

Background:  Natural CD4⁺CD25^highFoxp3⁺ regulatory T (nTreg) cells are important in maintaining immunologic tolerance, but their role in the pathogenesis of allergic asthma is unclear. We studied the function of nTreg cells in allergic asthmatic children and assessed the factors which may relate to the functional insufficiency of nTreg cells.
Methods:  The percentage of CD4⁺CD25^high Treg cells, the expression of Foxp3, and the cell-induced suppressive activity of nTreg cells isolated from nonatopic controls, allergic asthmatics, and allergen-specific immunotherapy (AIT)-treated asthmatic patients were studied.
Results:  Although the percentage of nTreg in peripheral blood mononuclear cells was increased, the expression of Foxp3 and its cell-induced suppressive activity were significantly lower in Dermatophagoides pteronyssinus (Der p)-sensitive asthmatic children when compared to nonatopic controls. In contrast, the expression of Foxp3 and the functional activity of nTreg cells were reversed in allergic asthmatics who received AIT. The addition of recombinant tumor necrosis factor (TNF)-α directly downregulated Foxp3 expression and abrogated the cell-induced suppressive function of Treg cells. The anti-TNF-α reagent, etanercept, restored the functional activity and Foxp3 expression of CD4⁺CD25^high Treg derived from allergic asthmatics.
Conclusions:  The functional insufficiency of nTreg cells in patients with allergic asthma may be related to the enhanced production of TNF-α and its effect on the Foxp3 expression. These results may explain, in part, the effectiveness of anti-TNF-α therapy in the treatment of allergic asthma.     

人外周血CD4+CD25+调节性T细胞的分离、鉴定和功能特征

目的： 分离人外周血CD4＋ CD25＋ Treg细胞, 并检测其功能.方法： RT-PCR技术检测CD4＋ CD25＋ Treg细胞中Foxp3的mRNA表达;与CD8＋ T细胞和CD4＋ CD25- T细胞共同培养, 或加入外源性IL-2及IL- 4, 检测其抑制功能;流式细胞术检测IFN-γ、 IL- 4和IL-10.结果： CD4＋ CD25＋ Treg细胞高表达Foxp3, 主要分泌IL-10, 能够抑制CD8＋ T细胞和CD4＋ CD25- T细胞的增殖, 高浓度IL-2能够阻断CD4＋ CD25＋ Treg细胞的抑制功能.结论： CD4＋ CD25＋ Treg细胞是一群具有免疫抑制功能的调节性T细胞, 这种抑制作用能够被高浓度IL-2阻断.     

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.     

Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells

CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3(-) T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3(-) T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy.     

Monocyte-derived dendritic cells from breast cancer patients are biased to induce CD4+CD25+Foxp3+ regulatory T cells

DCs orchestrate immune responses contributing to the pattern of response developed. In cancer, DCs may play a dysfunctional role in the induction of CD4(+)CD25(+)Foxp3(+) Tregs, contributing to immune evasion. We show here that Mo-DCs from breast cancer patients show an altered phenotype and induce preferentially Tregs, a phenomenon that occurred regardless of DC maturation stimulus (sCD40L, cytokine cocktail, TNF-α, and LPS). The Mo-DCs of patients induced low proliferation of allogeneic CD3(+)CD25(neg)Foxp3(neg) cells, which after becoming CD25(+), suppressed mitogen-stimulated T cells. Contrastingly, Mo-DCs from healthy donors induced a stronger proliferative response, a low frequency of CD4(+)CD25(+)Foxp3(+) with no suppressive activity. Furthermore, healthy Mo-DCs induced higher levels of IFN-γ, whereas the Mo-DCs of patients induced higher levels of bioactive TGF-β1 and IL-10 in cocultures with allogeneic T cells. Interestingly, TGF-β1 blocking with mAb in cocultures was not enough to completely revert the Mo-DCs of patients' bias toward Treg induction. Altogether, these findings should be considered in immunotherapeutic approaches for cancer based on Mo-DCs.     

新生儿脐带血CD4+CD25high调节性T细胞Foxp3的表达

目的:检测新生儿脐带血CD4 CD25high调节性T细胞(Treg)数量及胞内转录因子Foxp3的表达,探讨Treg细胞在新生儿期的表达特点.方法:采集新生儿脐带血(n=15)和成人外周血(n=12),密度梯度离心法获取单个核细胞用荧光标记单克隆抗体(mAb)作表面和胞内染色后,在流式细胞仪上检测CD4 CD25highTreg细胞的数量及其胞内转录因子Foxp3的表达.结果:脐带血Treg细胞占CD3 CD4 T细胞的比例(3.86%±1.63%)明显高于成人外周血(0.87%±0.74%,P<0.01);而脐带血Treg细胞中表达Foxp3的比例明显低于外周血Treg细胞(23.21%±8.9%vs71.3%±11.6%,P<0.01).结论:虽然新生儿脐带血CD4 CD25highTreg细胞数量明显高于成人外周血,但Foxp3 细胞数量明显低于成年人,提示在功能上可能尚未成熟.     

Der p2重组BCG诱导适应性CD4+CD25+ Treg产生并下调小鼠变应性气道炎症反应

目的： 卡介苗（BCG）是广泛应用的Th1应答诱导疫苗。近年来的几项研究认为分支杆菌疫苗可作为免疫佐剂诱导生成调节性T细胞并抑制哮喘气道炎症。我们前期构建了胞壁表达屋尘螨抗原Der p2的重组BCG疫苗（Der p2 rBCG）。本研究的目的是阐明Der p2 rBCG的免疫调节机制。方法：小鼠分别给予生理盐水、BCG、Der p2 rBCG免疫后,观察脾细胞中相关调节性T细胞亚群的相对比例和绝对数量。体外及在体观察Der p2 rBCG诱导产生的CD4+CD25+ Treg的抑制功能。结果：(1)Der p2 rBCG可以诱导CD4+CD25+ Treg的产生;(2)Der p2 rBCG诱导产生的CD4+CD25+ T细胞可以在体外以变应原特异的方式抑制效应CD4+T细胞的增殖;(3)Der p2 rBCG诱导产生的CD4+CD25+ T细胞可以在体内下调Der p2诱导的小鼠变应性气道炎症。结论：Der p2 rBCG可以诱导适应性CD4+CD25+ Treg的产生,并通过其介导的免疫抑制作用下调小鼠变应性气道炎症反应。     

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.     

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.     

Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells

Studies in both animal models and humans have shown a subset of B cells behaving as immuno-regulatory cells, being a source of inhibitory cytokines such as IL-10 and TGF-β. Our aims were to establish the presence of human B regulatory (Breg) cells and to assess their ability to suppress proliferation of CD4(+) T cells and to mediate T regulatory (Treg) cells' properties. For this purpose, human Breg, CD4(+) T and Treg cells were purified using magnetic microbeads. CFSE-labeled CD4(+) T cells were stimulated and cultured alone or with Breg cells. Their proliferative response was determined 72 hours later based on the CFSE staining. In parallel, Treg cells were cultured alone or with Breg cells in different conditions for 24 hours, and then stained and analyzed for Foxp3 and CTLA-4 expression. We found that, the co-culture of Breg cells (defined as CD25(high) CD27(high) CD86(high) CD1d(high) IL-10(high) TGF-β(high)) with autologous stimulated CD4(+) T cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4(+) T cells. Furthermore, Foxp3 and CTLA-4 expression in Treg cells were enhanced by non-stimulated and further by ODN-CD40L stimulated Breg cells. The regulatory function of Breg cells on Treg cells was mainly dependent on a direct contact between Breg and Treg cells, but was also TGF-β but not IL-10 dependent. In conclusion, human Breg cells decrease the proliferation of CD4(+) T cells and also enhance the expression of Foxp3 and CTLA-4 in Treg cells by cell-to-cell contact.     

Aurintricarboxylic acid promotes the conversion of naive CD4+CD25- T cells into Foxp3-expressing regulatory T cells

Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-β revealed that ATA requires the presence of TGF-β to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-β had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.     

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.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

肺癌患者CD4+CD25high Foxp3+调节性T细胞的格局变化及意义

目的：研究肺癌患者外周血（PBMC）及肿瘤浸润淋巴细胞（TIL）中CD4^＋ CD25^high Foxp3^＋调节性T细胞（Treg）的比例改变，探讨其在抗肿瘤免疫中的调节作用。方法：分离肺癌患者PBMC及TIL，FACS分析CD4^＋／CD8^＋T细胞的比值及CD4^＋ CD25^highT细胞占CD4^＋T细胞的比例。Real-time PCR检测Treg特异性转录因子Foxp3基因在PBMC及TIL中的表达。结果：肺癌患者PBMC及TIL中CD4^＋／CD8^＋比值降低；而CD4^＋ CD25^high T细胞在CD4^＋T细胞中所占比例升高；Foxp3基因仅在TIL中高表达，而在PBMC中低或不表达，表明肿瘤局部的CD4^＋ CD25^high T细胞主要是CD4^＋ CD25^high Foxp3^＋ Treg。结合临床资料分析显示Treg在肺腺癌比例较高。结论：CD4^＋ CD25^high Foxp3^＋ Treg在肺癌患者肿瘤浸润淋巴细胞中明显升高，可能与其通过细胞与细胞间接触抑制CD8^＋T细胞的杀伤效应，最终发挥免疫抑制效应相关。     

Foxp3(high) and Foxp3(low) Treg cells differentially correlate with T helper 1 and natural killer cells in peripheral blood

Regulatory T (Treg) cells interact with B, natural killer (NK), and dendritic cells in addition to other T cells. In this study, we aimed at determining whether Foxp3(+) T cells and subpopulations have any correlation with other lymphocyte subsets and their functions in a systemic immune environment. Peripheral blood was drawn from 22 nonpregnant healthy women. T, B, and NK cell subpopulations were measured by immunophenotype analysis. Intracellular Foxp3, cytokine expression (tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], and interleukin-10 [IL]-10), and NK-cell cytotoxicity were analyzed by flow cytometric analysis. Correlations between Foxp3(+) T cells and other immune variables were analyzed under control of age and menstrual phases. Foxp3(+), Foxp3(low), and CD4(+)Foxp3(+) cells significantly correlated with CD4(+)CD25(+), CD4(+)CD25(dim), and CD4(+)CD25(bright) cells. Foxp3(+), Foxp3(low), and CD4(+)Foxp3(+) cells positively correlated with CD3(+) and CD3(+)CD4(+) T cells, but negatively correlated with CD3(-)CD56(+) and CD3(-)CD56(dim) NK cells. CD4(+)Foxp3(high) Treg cells were positively correlated with CD3(+)CD4(+)TNF-α(+) (p = 0.014) and negatively correlated with CD3(+)CD8(+)IL-10(+) T cells (p = 0.001). The ratio of type 1/2 cytokine-producing CD3(+)CD8(+) cells demonstrated a positive correlation with CD4(+)Foxp3(high) cells (p ≤ 0.01). CD8(+)Foxp3(+) cells were positively correlated with CD3(+)CD4(+)IL-10(+) cells (p = 0.007) and negatively correlated with CD3(+)CD8(+)TNF-α(+) cells (p = 0.008). In conclusion, each Foxp3(+) Treg cell subpopulation has unique immune interaction, which controls particular subsets of lymphocytes.     

Ethylenecarbodiimide-coupled allogeneic antigen presenting cells induce human CD4+ regulatory T cells

Adoptive transfer of naturally occurring CD4(+)CD25(+) regulatory T cells can tolerize transplantation alloresponses in animal models. However isolation of these cells in sufficient numbers from humans is cumbersome and prone to contamination with alloreactive CD25(+) T cells. Incubation of ethylenecarbodiimide-coupled antigen presenting cells (APC) with na?ve T cells and antigen has been shown to induce tolerance in various experimental models. We therefore investigated whether ECDI-coupled allogeneic APC were able to induce an expandable human CD4(+) Treg population. CD4(+) and CD4(+) CD25(-) cells cultured for 5 days with ECDI-treated human PBMC exhibited potent suppressive capacity in a mixed lymphocyte reaction. Induction of these ECDI-Tregs was associated with up-regulation of Foxp3 mRNA and protein expression and they maintained high expression of CD62L and CD27 as well as low CD127 expression. ECDI-treated APC displayed reduced expression of the co-stimulatory signaling molecules CD40 and CD80, and failed to stimulate proliferation and cytokine secretion in co-cultured CD4(+) T cells. Restimulation in the presence of rapamycin and hrIL-2 led to expansion of ECDI-Tregs with increasing Foxp3 levels and suppressive activity significantly higher than expanded naturally occurring CD4(+)CD25(+) Tregs. In summary these findings support the hypothesis that ECDI-coupled APC can convert na?ve CD4(+) T cells into functional Tregs with different phenotypic characteristics than naturally occurring CD4(+)CD25(+) Tregs. These inducible Tregs could provide a novel approach that might facilitate the translation of ex vivo generated and expanded Tregs into clinical settings.