急性冠脉综合征患者CD4~+T淋巴细胞亚群的变化及临床意义

目的:研究急性冠脉综合征(ACS)患者CD4+T淋巴细胞亚群的变化及临床意义。方法:收集2013年3月-2014年3月入住我院首次行冠脉成形术(PCI)治疗的ACS患者88例以及造影结果正常患者28例。分别于冠脉造影时取冠脉血流式细胞技术检测CD4+T细胞亚群Th1/Th2、Th17/Treg比例表达变化。结果:与对照组相比,ACS组患者冠脉血中Th1、Th17细胞占CD4+T淋巴细胞的百分比显著升高,而Th1、Treg细胞占CD4+T淋巴细胞的百分比显著降低,差异具有统计学意义(P0.05)。结论:ACS患者体内免疫反应增强,CD4+T淋巴细胞不同功能亚群均参与了动脉粥样硬化(AS)的发生发展,Th1、Th17可促进AS的进展和不稳定性,而Th1、Treg作用相反。     

Pertussis toxin by inducing IL-6 promotes the generation of IL-17-producing CD4 cells

Compelling evidence has now demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to autoimmune pathogenesis, whereas CD4+CD25+ T regulatory cells (Treg) play a major role in suppression of autoimmunity. Differentiation of proinflammatory Th17 and immunosuppressive Treg from naive CD4 cells is reciprocally related and contingent upon the cytokine environment. We and others have reported that in vivo administration of pertussis toxin (PTx) reduces the number and function of mouse Treg. In this study, we have shown that supernatants from PTx-treated mouse splenic cells, which contained IL-6 and other proinflammatory cytokines, but not PTx itself, overcame the inhibition of proliferation seen in cocultures of Treg and CD4+CD25- T effector cells. This stimulatory effect could be mimicked by individual inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha. The combination of these cytokines synergistically stimulated the proliferation of CD4+CD25- T effector cells despite the presence of Treg with a concomitant reduction in the percentage of FoxP3+ cells and generation of IL-17-expressing cells. PTx generated Th17 cells, while inhibiting the differentiation of FoxP+ cells, from naive CD4 cells when cocultured with bone marrow-derived dendritic cells from wild-type mice, but not from IL-6-/- mice. In vivo treatment with PTx induced IL-17-secreting cells in wild-type mice, but not in IL-6-/- mice. Thus, in addition to inhibiting the development of Treg, the immunoadjuvant activity of PTx can be attributable to the generation of IL-6-dependent IL-17-producing CD4 cells.     

Systemic and local characterization of regulatory T cells in a chronic fungal infection in humans

The long-term persistence of pathogens in a host is a hallmark of certain infectious diseases, including schistosomiasis, leishmaniasis, and paracoccidioidomycosis (PCM). Natural regulatory T (Treg) cells are involved in control of the immune responses, including response to pathogens. Because CTLA-4 is constitutively expressed in Treg cells and it acts as a negative regulator of T cell activation in patients with PCM, here we investigated the involvement of Treg cells in the control of systemic and local immune response in patients with PCM. We found that the leukocyte subsets were similar in patients and controls, except for CD11c+CD1a+ cells. However, a higher frequency of CD4+CD25+ T cells expressing CTLA-4, glucorticoid-inducible TNFR, membrane-bound TGF-beta, and forkhead-box 3 were observed in PBMC of patients. In accordance, these cells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5% of inhibition of allogeneic T cell proliferation). In addition, the data showed that CD4+CD25+ T cells expressing CTLA-4+, glucocorticoid-inducible TNFR positive, CD103+, CD45RO+, membrane-bound TGF-beta, forkhead-box 3 positive, and the chemokines receptors CCR4 and CCR5 accumulate in the Paracoccidioides brasiliensis-induced lesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells to peripheral tissues, were also detected in the biopsies. Moreover, the CD4+CD25+ T cell derived from lesions, most of them TGF-beta+, also exhibited functional activity in vitro. Altogether, these data provide the first evidence that Treg cells play a role in controlling local and systemic immune response in patients with a fungal-induced granulomatous disease advancing our understanding about the immune regulation in human chronic diseases.     

Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H4(71-94), when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-beta. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H4(71-94) peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-beta, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H4(71-94) was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H4(71-94) peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.     

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.     

Enhanced regulatory T cell activity is an element of the host response to injury

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-beta1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

In vitro and in vivo down-regulation of regulatory T cell activity with a peptide inhibitor of TGF-beta1

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-beta1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-beta1 and TGF-beta2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-beta may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.     

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.     

Th1-Th17 Cells Contribute to the Development of Uropathogenic Escherichia coli-Induced Chronic Pelvic Pain

The etiology of chronic prostatitis/chronic pelvic pain syndrome in men is unknown but may involve microbes and autoimmune mechanisms. We developed an infection model of chronic pelvic pain in NOD/ShiLtJ (NOD) mice with a clinical Escherichia coli isolate (CP-1) from a patient with chronic pelvic pain. We investigated pain mechanisms in NOD mice and compared it to C57BL/6 (B6) mice, a strain resistant to CP-1-induced pain. Adoptive transfer of CD4+ T cells, but not serum, from CP-1-infected NOD mice was sufficient to induce chronic pelvic pain. CD4+ T cells in CP-1-infected NOD mice expressed IFN-γ and IL-17A but not IL-4, consistent with a Th1/Th17 immune signature. Adoptive transfer of ex-vivo expanded IFN-γ or IL-17A-expressing cells was sufficient to induce pelvic pain in naïve NOD recipients. Pelvic pain was not abolished in NOD-IFN-γ-KO mice but was associated with an enhanced IL-17A immune response to CP1 infection. These findings demonstrate a novel role for Th1 and Th17-mediated adaptive immune mechanisms in chronic pelvic pain.     

Human Th17 cells share major trafficking receptors with both polarized effector T cells and FOXP3+ regulatory T cells

It is a question of interest whether Th17 cells express trafficking receptors unique to this Th cell lineage and migrate specifically to certain tissue sites. We found several Th17 cell subsets at different developing stages in a human secondary lymphoid organ (tonsils) and adult, but not in neonatal, blood. These Th17 cell subsets include a novel in vivo-stimulated tonsil IL17+ T cell subset detected without any artificial stimulation in vitro. We investigated in depth the trafficking receptor phenotype of the Th17 cell subsets in tonsils and adult blood. The developing Th17 cells in tonsils highly expressed both Th1- (CCR2, CXCR3, CCR5, and CXCR6) and Th2-associated (CCR4) trafficking receptors. Moreover, Th17 cells share major non-lymphoid tissue trafficking receptors, such as CCR4, CCR5, CCR6, CXCR3, and CXCR6, with FOXP3+ T regulatory cells. In addition, many Th17 cells express homeostatic chemokine receptors (CD62L, CCR6, CCR7, CXCR4, and CXCR5) implicated in T cell migration to and within lymphoid tissues. Expression of CCR6 and CCR4 by some Th17 cells is not a feature unique to Th17 cells but shared with FOXP3+ T cells. Interestingly, the IL17+IFN-gamma+ Th17 cells have the features of both IL17-IFN-gamma+ Th1 and IL17+IFN-gamma- Th17 cells in expression of trafficking receptors. Taken together, our results revealed that Th17 cells are highly heterogeneous, in terms of trafficking receptors, and programmed to share major trafficking receptors with other T cell lineages. These findings have important implications in their distribution in the human body in relation to other regulatory T cell subsets.     

良性前列腺增生患者外周血Th17和Treg细胞比率的变化

目的:探讨良性前列腺增生患者外周血Th17和Treg细胞比率的变化。方法:选择33例良性前列腺增生患者及19例正常对照者为研究对象,采用流式细胞术检测和比较其外周血中T淋巴细胞亚群及Th17和Treg细胞占CD4~+T细胞的比率。结果:良性前列腺增生患者外周血Th17和Treg细胞占CD4~+T细胞的比率分别为1.58±0.71和1.76±0.83,Th17/Treg的比率为0.89±0.42。正常健康对照者外周血Th17和Treg细胞占CD4~+T细胞的比率分别为0.75±0.46和1.83±0.75,Th17/Treg的比率为0.41±0.32。良性前列腺增生患者外周血Th17占CD4~+T细胞的比率和Th17/Treg的比率明显高于正常健康对照者(P0.05)。结论:良性前列腺增生患者体内Th17细胞比率升高,Th17/Treg比率失衡,可能与良性前列腺增生的发生、发展有关。     

Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets

We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 mug nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H4(71-94) was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+ CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN-gamma responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90-100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+ CD25+ and CD8+ Treg cells produced and required TGF-beta1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+ CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF-beta-producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

The Circulating Treg/Th17 Cell Ratio Is Correlated with Relapse and Treatment Response in Pulmonary Sarcoidosis Patients after Corticosteroid Withdrawal

Objectives

Pulmonary sarcoidosis is an immune-mediated disease, and some patients can be effectively treated with corticosteroids. However, nearly half of all sarcoidosis patients relapse after corticosteroid withdrawal. Different subsets of CD4+ helper T cells participate in the immunopathogenesis of sarcoidosis. Thus, the aims of our study were to investigate whether the circulating subsets of CD4+ helper T cells were associated with sarcoidosis relapse and with its remission after retreatment. Additionally, we identified a useful biomarker for predicting the relapse and remission of sarcoidosis patients.

Methods

Forty-two patients were enrolled in the present study who had previously been diagnosed with pulmonary sarcoidosis and treated with corticosteroids. The patients were allocated into either a stable group if they exhibited sustained remission (n = 22) or a relapse group if they experienced clinical or radiological recurrence after treatment withdrawal (n = 20). Peripheral blood cells were collected from these patients and analyzed to determine the frequencies of subsets of circulating CD4+ helper T cells by flow cytometry. The patients in the relapse group were retreated with corticosteroids and immunosuppressive agents and were then reevaluated to determine the frequencies of dynamic subsets of circulating CD4+ helper T cells after remission.

Results

The frequencies of circulating Tregs were significantly increased concomitant with a decrease in the circulating Th17 cell frequency in the relapsed patients compared with the stable patients. The Treg/Th17 ratio was negatively correlated with sarcoidosis activity and was sensitive to retreatment. In addition, the percentage of isolated CD45RO+Ki67+ Tregs was higher in the patients who were stable and in those who recovered after retreatment than in those who relapsed.

Conclusions

An imbalance between Tregs and Th17 cells is associated with pulmonary sarcoidosis relapse after corticosteroid withdrawal. The circulating Treg/Th17 ratio could serve as an alternative marker for monitoring pulmonary sarcoidosis relapse after the end of corticosteroid treatment and for rapidly predicting the response to retreatment.