Mechanisms regulating the development and function of natural regulatory T cells

The key of the immune system is to protect the host from foreign threat posed by pathogens and from the internal threat posed by self-attacking lymphocytes. The ability to discriminate self versus non-self ensures that only “non-self” pathogens, but not the self antigens, are attacked. Such tolerance to “self” arises from the central tolerance mechanisms that include the deletion of thymocytes with high reactivity to self antigens and also the induction of unresponsiveness of autoreactive T cells in the periphery. Natural regulatory T cells (nTregs) directly inhibit effector T cells, and keep their proliferation in control. Apart from preventing autoimmune reactions, Tregs also contribute to peripheral immune homeostasis as evidenced by the excessive lymphocyte accumulation in peripheral lymphoid organs and intestinal inflammation in the absence of nTregs. Here we discuss the molecular aspects of the development and suppressive function of naturally occurring Tregs. Accumulating evidence shows the importance of these Tregs in autoimmunity, tumor immunity, organ transplantation, allergy, and microbial immunity.     

Differential requirement of PKC-theta in the development and function of natural regulatory T cells

CD4+CD25+ natural Treg cells, which are developed in the thymus, migrate to the periphery to actively maintain self-tolerance. Similar to conventional T cells, TCR signals are critical for the development and activation of Treg cell inhibitory function. While PKC-theta-mediated TCR signals are required for the activation of peripheral na?ve T cells, they are dispensable for their thymic development. Here, we show that mice deficient in PKC-theta had a greatly reduced number of CD4+Foxp3+ Treg cells, which was independent of PKC-theta-regulated survival, as transgenic Bcl-x(L) could not restore the Treg cell population in PKC-theta(-/-) mice. Active and WT PKC-theta markedly stimulated, whereas inactive PKC-theta and dominant negative NFAT inhibited Foxp3 promoter activity. In addition, mice-deficient in calcineurin Abeta had a decreased Treg cell population, similar to that observed in PKC-theta deficient mice. It is likely that PKC-theta promoted the development of Treg cells by enhancing Foxp3 expression via activation of the calcineurin/NFAT pathway. Finally, Treg cells deficient in PKC-theta were as potent as WT Treg cells in inhibiting T cell activation, indicating that PKC-theta was not required for Treg cell-mediated inhibitory function. Our data highlight the contrasting roles PKC-theta plays in conventional T cell and natural Treg cell function.     

PD‐1 is not required for natural or peripherally induced regulatory T cells: Severe autoimmunity despite normal production of regulatory T cells

The expression of the coinhibitor PD‐1 on T cells is important for the establishment of immune homeostasis. We previously found that PD‐1 is particularly critical for the control of self‐tolerance during lymphopenia‐induced proliferation of recent thymic emigrants (RTEs). Previous studies suggested that PD‐1 modulates the generation of Treg cells, particularly peripherally induced Treg (pTreg) cells, and controls Th17 cells. However, these conclusions were derived indirectly from studies on the ligand PD‐L1, and not PD‐1 itself. Herein we directly tested whether T‐cell PD‐1 expression was needed for Treg cell generation and examined if a paucity of Treg cells or enhanced Th17 cells could explain the severe lymphopenia‐potentiated autoimmunity caused by PD‐1 KO RTEs. Employing the murine FoxP3^EGFP reporter system to simultaneously monitor conversion of WT and PD‐1 KO T cells to pTreg cells in the same animal, we found that PD‐1 deficiency did not inhibit pTreg cell generation or lead to Th17‐cell‐mediated autoimmunity. Surprisingly, pTreg cell numbers were increased in PD‐1 KO versus WT cell populations. Furthermore, we noted an increased conversion to pTreg cells by RTEs. Our data suggest that the primary role for PD‐1 is to restrain T‐cell activation/proliferation to self‐Ags rather than promote generation of Treg cells.     

SHARPIN controls the development of regulatory T cells

SHARPIN is an essential component of the linear ubiquitin chain assembly complex (LUBAC) complex that controls signalling pathways of various receptors, including the tumour necrosis factor receptor (TNFR), Toll‐like receptor (TLR) and antigen receptor, in part by synthesis of linear, non‐degrading ubiquitin chains. Consistent with SHARPIN's function in different receptor pathways, the phenotype of SHARPIN‐deficient mice is complex, including the development of inflammatory systemic and skin diseases, the latter of which depend on TNFR signal transduction. Given the established function of SHARPIN in primary and malignant B cells, we hypothesized that SHARPIN might also regulate T‐cell receptor (TCR) signalling and thereby control T‐cell biology. Here, we focus primarily on the role of SHARPIN in T cells, specifically regulatory T (Treg) cells. We found that SHARPIN‐deficient (Sharpin^cpdm/cpdm) mice have significantly reduced numbers of FOXP3⁺ Treg cells in lymphoid organs and the peripheral blood. Competitive reconstitution of irradiated mice with mixed bone marrow from wild‐type and SHARPIN‐deficient mice revealed an overall reduced thymus population with SHARPIN‐deficient cells with almost complete loss of thymic Treg development. Consistent with this cell‐intrinsic function of SHARPIN in Treg development, TCR stimulation of SHARPIN‐deficient thymocytes revealed reduced activation of nuclear factor‐κB and c‐Jun N‐terminal kinase, establishing a function of SHARPIN in TCR signalling, which may explain the defective Treg development. In turn, in vitro generation and suppressive activity of mature SHARPIN‐deficient Treg cells were comparable to wild‐type cells, suggesting that maturation, but not function, of SHARPIN‐deficient Treg cells is impaired. Taken together, these findings show that SHARPIN controls TCR signalling and is required for efficient generation of Treg cells in vivo, whereas the inhibitory function of mature Treg cells appears to be independent of SHARPIN.     

Interaction between natural killer cells and regulatory T cells: perspectives for immunotherapy

Regulatory T (Treg) cells and natural killer (NK) cells are key players in the immune system. The interaction between these two cell types has been reported to be beneficial in healthy conditions such as pregnancy. However, in the case of certain pathologies such as autoimmune diseases and cancer this interaction can become detrimental, as Treg cells have been described to suppress NK cells and in particular to impair NK cell effector functions. This review aims to discuss the recent information on the interaction between Treg cells and NK cells under healthy and pathologic conditions, to describe the specific conditions in which this interaction takes place, the effect of Treg cells on hematopoietic stem cell differentiation and the consequences of this interaction on the optimization of immunotherapeutic protocols.     

调节性T细胞介导的肿瘤免疫耐受

恶性肿瘤能够有效的逃避免疫系统的监视源于免疫反应系统功能的障碍,多种因素都可导致抗肿瘤免疫的失效.调节性T细胞(Tr)在肿瘤浸润的淋巴细胞及肿瘤引流的淋巴结中高频出现,意味着其能促进肿瘤生长.Tr在肿瘤免疫病理以及调节免疫治疗的效果中起重要作用.因此,肿瘤患者的Tr可能成为重要的治疗靶细胞,并将会为抗肿瘤免疫治疗提供新策略.     

Isolation and expansion of human natural T regulatory cells for cellular therapy

Natural T regulatory cells (nTregs) play a key role in inducing and maintaining immunological tolerance. Cell-based therapy using purified nTregs is under consideration for several conditions, but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. We have established a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants. The Robosep instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures. The resulting cell population exhibited a stable CD4⁺CD25^++brightFoxp3⁺ phenotype, had potent functional ability to suppress CD4⁺CD25^negative T cells without evidence of conversion to effector T cells including TH17 cells, and manifested little to no production of pro-inflammatory cytokines upon in vitro stimulation. Boolean gating analysis of cytokine-expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat, approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers are adequate for infusion of approximately one million cells kg⁻¹ for cell therapy in clinical trials.     

The role of natural regulatory T cells in infection

Naturally occurring regulatory T cells (T(Reg)) suppress multiple cell types of the immune system to maintain dominant tolerance to protect from autoimmunity, down-modulate anti-tumor immunity and restrain allergic diseases. In addition to these functions, T(Reg) can alter effector responses to invading pathogens, leading to a variety of outcomes affecting both the host and infecting microorganisms. Here, we review how T(Reg) can influence the immune responses to chronic infections where pathogen-specific T(Reg) can contribute to pathogen persistence and, in some cases, concomitant immunity, as well as control immunopathology associated with robust immune responses. We also review the data on T(Reg) during acute infection, focusing on the questions these studies raise regarding the most appropriate model(s) to examine T(Reg) during infection. Finally, we discuss the ways in which the T(Reg) function can be altered by invading pathogens and how these can be exploited to develop methods therapeutically to influence disease and vaccine outcomes.     

Thymic development and peripheral homeostasis of regulatory T cells

The development and maintenance of regulatory T (T-reg) cells is crucial for determining the level of reactivity in the immune system. Until recently, however, surprisingly little was known about the factors involved in the development of these cells in the thymus or the mechanisms that maintain them in the periphery. Studies have now demonstrated that thymic development of T-reg cells is facilitated by TCRs with increased affinity for self-peptide-MHC complexes. Increased TCR affinity alone, however, is not sufficient to support the development of T-reg cells, and external factors such as CD80 and CD86, ligands for co-stimulatory receptor CD28, and interleukin 2 are required. These factors are also needed to maintain the T-reg cell subset in the periphery.     

胎盘蛋白14增强调节性T细胞诱导滤泡调节性T细胞比例增加的作用及机制研究

目的：研究胎盘蛋白14（PP14）增强调节性T细胞（Treg细胞）诱导滤泡调节性T细胞（Tfr细胞）增殖的作用及机制。方法：分离健康志愿者外周血中的淋巴细胞;磁珠分选淋巴细胞中的Treg细胞;流式检测CD4⁺CD25⁺Foxp3⁺Treg细胞比例及其表面CTLA-4和HLA-G的表达;ELISA检测上清中IL-10和TGF-β的浓度;采用Transwell小室进行Treg细胞和淋巴细胞的共培养实验;流式检测淋巴细胞中CD4⁺CXCR5⁺Foxp3⁺Tfr细胞的比例。结果：相对于对照组,PP14组Treg细胞的比例增加了1.26倍（t=4.748,P=0.009 0）,而其表面CTLA-4和HLA-G的表达与对照组没有显著的统计学差异（t=0.108,P=0.918 6;t=0.439,P=0.682 8）;ELISA的结果显示相对于对照组,PP14组Treg细胞培养上清中IL-10和TGF-β的浓度分别上升了3.66倍和3.86倍（t=6.817,P=0.002 4;t=7.537,P=0.001 7）;共培养实验显示PP14能够增强Treg细胞诱导Tfr细胞比例的增加,相对于共培养组,PP14组Tfr细胞比例增加了2.71倍（t=4.815,P=0.008 6）,IL-10和TGF-β的阻断均能部分阻断这种增强作用（t=4.868,P=0.008 2;t=3.670,P=0.021 4）。结论：PP14能够通过分泌IL-10和TGF-β,显著增强Treg细胞诱导Tfr细胞比例的增加,这为不明原因复发性流产的治疗提供了一个新的靶点。     

A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection that is responsible for a significant number of deaths in children and nonimmune adults. A failure to control blood parasitemia and subsequent sequestration of parasites to brain microvasculature are thought to be key events in many CM cases. Here, we show for the first time, to our knowledge, that CD4(+)CD25(+)Foxp3(+) natural regulatory T (Treg) cells contribute to pathogenesis by modulating immune responses in P. berghei ANKA (PbA)-infected mice. Depletion of Treg cells with anti-CD25 monoclonal antibody protected mice from experimental CM. The accumulation of parasites in the vasculature and brain was reduced in these animals, resulting in significantly lower parasite burdens compared with control animals. Mice lacking Treg cells had increased numbers of activated CD4(+) and CD8(+) T cells in the spleen and lymph nodes, but CD8(+) T-cell recruitment to the brain was selectively reduced in these mice. Importantly, a non-Treg-cell source of interleukin-10 was critical in preventing experimental CM. Finally, we show that therapeutic administration of anti-CD25 monoclonal antibody, even when blood parasitemia is established, can prevent disease, confirming a critical and paradoxical role for Treg cells in experimental CM pathogenesis.     

Emerging possibilities in the development and function of regulatory T cells

CD25+CD4+ Regulatory T cells (Treg) represent a unique population of lymphocytes capable of powerfully suppressing immune responses. A large body of experimental data have now confirmed the essential role played by these cells in a host of clinically relevant areas such as self-tolerance, transplantation, allergy and tumor/microbial immunity. Despite this mass of knowledge, significant gaps in our understanding of fundamental Treg biology remain, particularly regarding their development and mechanisms of suppression. In this review we attempt to highlight the current controversies and directions in which this exciting field is moving.     

TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells

Transforming growth factor (TGF)-beta-activating kinase 1 (TAK1) is critical for Toll-like receptor- and tumor necrosis factor-mediated cellular responses. In B cells, TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs), but not nuclear factor-kappaB (NF-kappaB), in antigen receptor signaling. In this study, we generate T cell-specific TAK1-deficient (Lck(Cre/(+))Tak1(flox/flox)) mice and show that TAK1 is indispensable for the maintenance of peripheral CD4 and CD8 T cells. In thymocytes, TAK1 is essential for TCR-mediated activation of both NF-kappaB and MAPKs. Additionally, Lck(Cre/(+))Tak1(flox/flox) mice developed colitis as they aged. In these mice, accumulations of activated/memory T cells as well as B cells were observed. Development of regulatory T (Treg) cells in thymus was abrogated in Lck(Cre/(+))Tak1(flox/flox) mice, suggesting that the loss of Treg cells is the cause of the disease. Together, the results show that TAK1, by controlling the generation of central Treg cells, is important for preventing spontaneously developing colitis.     

Cellular and molecular basis of natural killer and natural killer-like activity

The capability of killing various tumors or cells infected by certain viruses is a property shared by natural killer (NK) cells and by a subset of cytolytic T lymphocytes (CTLs) termed NK-CTL. Recent analysis of the molecular basis in these phenomena, however, revealed rather different molecular mechanisms. Thus, while NK cell cytotoxicity is regulated by a complex balance between activating signals (delivered by non HLA-class I-specific triggering receptors) and inhibitory signals (delivered by HLA-class I-specific receptors) the effector function of NK-CTL reflects the TCR-mediated recognition of the poorly polymorphic HLA-E.     

Contribution of neuraminidase 3 to the differentiation of induced regulatory T cells

Neuraminidase family enzymes that hydrolyze the terminal sialic acid linkage in biomolecules are involved in various immune responses. We previously showed that Th1 and Th2 cells differentially express several neuraminidases. Herein, the expression of neuraminidases in induced regulatory T (iTreg) cells was investigated in comparison with that in other T‐cell subsets. Contrary to the tendency toward higher neuraminidase 1 mRNA expression in in vitro‐differentiated Th2 cells, compared to Th1, Th17 and iTreg cells, we observed significantly higher expression of neuraminidase 3 (Neu3) in iTreg cells. Furthermore, the expression of Neu3 in FoxP3⁺CD62L^? spleen cells was higher than that in FoxP3⁺CD62L⁺ and FoxP3^? cells. Lentiviral expression of Neu3 in naïve CD4⁺ T cells during the stimulation culture led to upregulation of FoxP3 expression. On the basis of these findings, we conclude that Neu3 contributes to the differentiation of iTreg cells by upregulation of FoxP3.     

Quantification and molecular characterization of regulatory T cells in connective tissue diseases

The aim of our study was to investigate and characterize regulatory T cells (Treg) in peripheral blood of patients with connective tissue diseases (Systemic lupus erythematosus, systemic sclerosis, Sj?gren's syndrome, poly- and dermatomyositis) as compared with blood from healthy controls. Treg cells were quantified and phenotypically characterized by flow cytometry while the expression level of Foxp3 mRNA was evaluated by real time PCR. A reduced percentage of peripheral blood Treg cells was found in patients than in controls, irrespective of the type of connective tissue disease. Treg cells, especially those expressing one of the phenotypical markers, seemed to differ not only between patients and healthy controls but also among types of diseases. Additionally, the presence of autoantibodies as well as disease activity appeared to be correlated with particular Treg cell populations, especially those expressing one of the examined phenotypical markers. Correlations with therapy suggested that glucocorticoids plus antimalarial or other immunosuppressor drugs diminished the percentage of Treg cells, especially of those with memory phenotype. These findings indicated dysregulations at the level of Treg cells and suggested an involvement of these cells in the pathology of connective tissue diseases. Moreover, our data are in agreement with the suggestion that Treg cells could be therapeutic targets for some autoimmune diseases.     

BTLA对调节性T细胞发育及功能的影响

目的 研究B和T淋巴细胞弱化因子(B and T lymphocyte attenuator,BTLA)对调节性T细胞(regulatory T cell,Treg)发育和功能的影响.方法 构建在Treg中特异性敲除BTLA基因的小鼠模型,使用流式细胞术检测该模型小鼠中枢及外周各淋巴器官中T细胞外周环境稳态、T细胞的活...