Immunomodulatory effects of cyclosporin A on human peripheral blood dendritic cell subsets

Cyclosporin A (CsA) is a potent immuno-suppressant and is approved for the treatment of various disease conditions. The molecular biological mechanism of CsA has been investigated intensively in T cells and has been shown to involve the intracellular calcineurin pathway. Recently, it was reported that CsA has capacities to affect not only T cells but also antigen-presenting cells such as B cells and dendritic cells (DCs). DCs are a master regulator of immune responses that have an integral capacity to prime naive T cells. In the present study, we investigated the biological effects of CsA on human peripheral blood DC subsets: CD11c+ myeloid and CD11c- lymphoid subsets. CsA inhibited the up-regulation of co-stimulatory molecules induced with or without microbial stimuli and CD40L on both CD11c+ and CD11c- subsets. In addition, CsA negatively regulated the endocytic activity of CD11c+ DC during the immature state. CsA inhibited the interleukin-12 (IL-12) production, but augmented the IL-10 production from the LPS-stimulated CD11c+ subset, whereas CsA reduced the interferon-alpha (IFN-alpha) production from the CD11c- subset infected with Sendai virus (SV). Both the LPS-stimulated CD11c+ subset and SV-infected CD11c- subset preferentially induced the development of IFN-gamma-producing T helper-type 1 (Th1) cells. Pretreatment of these DC subsets with CsA inhibited the Th1 skewing. These findings suggested a DC-mediated mechanism of immunosuppression by CsA.     

Differential MHC class II expression on human peripheral blood monocytes and dendritic cells

Both monocytes (MO) and dendritic cells (DC) in human peripheral blood are of a plastic-adherent nature. The expression of the MHC class II sublocus products HLA-DP, -DQ and -DR on human peripheral blood transiently adherent cells (TA) was examined by an immunocytochemical staining technique. While most TA showed strong expression of molecules of the HLA-DR subtype, only a small proportion of cells (2-6%) showed strong HLA-DP or -DQ positivity. This strong expression of the HLA-DP and HLA-DQ sublocus products by a subset of TA was seen only after short-term culture; freshly isolated cells expressed comparatively low levels of these molecules. Enrichment for Fc receptor-negative or low-density cells from TA produced populations with strong HLA-DQ and -DP expression. Such co-enrichment of the strongly HLA-DQ+ and strongly HLA-DP+ cells suggests that the same cells express high levels of both types of MHC class II molecule. Immunocytochemical analysis of TA indicated that the strongly HLA-DQ+ cells, at least, were only weakly or non-reactive with the MO-specific monoclonal antibodies OKM1, UCHM1, MO2 and EB11. In addition, strongly HLA-DQ- or -DP-positive cells were poorly phagocytic in comparison with the majority of adherent cells. The apparent FcR-negative, low-density and weakly phagocytic nature of the strongly HLA-DQ/DP+ cells, combined with their lack of reactivity with several MO-specific antibodies, suggests that they may represent the DC component of TA. Such strong HLA-DQ/DP expression by DC may aid their positive identification in human peripheral blood and may be of relevance to DC function in antigen presentation.     

人外周血树突状细胞对LAK细胞杀伤活性的影响

从人外周血中分离出树突状细胞，体外观察了其对ＬＡＫ细胞活性的影响。发现：５×１０２～１×１０４／ｍｌ树突状细胞对ＬＡＫ细胞活性起增强作用，而５×１０４～１×１０５／ｍｌ树突状细胞却抑制ＬＡＫ杀伤活性。这说明人外周血树突状细胞对ＬＡＫ细胞活性起双相性调节作用。     

粒细胞集落刺激因子动员对供者外周血细胞树突状细胞亚群的影响

目的：研究粒细胞集落刺激因子(G-CSF)动员对外周血单个核细胞（PBMNC）中树突状细胞（DC）亚群及其功能的影响。方法：以CD34^-Lin^- HLA-DR⁺细胞群设门4色荧光分析方法检测25例无关供者G-CSF动员前、后外周血细胞DC亚群；ELISA检测其血清相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4活性，并分析DC₁/DC₂（CD11c⁺CD123^- /CD11c^-CD123⁺）比值与CD34⁺细胞含量的相关性。结果：G-CSF动员后, 供者PBMNC 的DC₁/DC₂比值显著低于动员前（P<0.05）；DC₁/DC₂与CD34⁺细胞含量呈负相关（r=-0.438, P<0.05），CD34⁺细胞/MNC≥0.4%时， DC₁/DC₂倒置；而血清相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4水平无显著改变（P>0.05）； DC₂ HLA-DR表达上调而CD83不表达。结论： G-CSF动员后，供者外周血CD34⁺细胞数量增加的同时，DC₂数量也增加，这些DC₂尽管HLA-DR表达上调，但仍处前体细胞状态，不直接分泌或调节Th2细胞分泌免疫抑制因子。     

The glucocorticoid dexamethasone programs human dendritic cells for enhanced phagocytosis of apoptotic neutrophils and inflammatory response

GCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte-derived human DCs develop their capacity to engulf apoptotic cells by up-regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow-derived DCs of ADORA3 KO mice were treated with Dex. The GC-treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN-γ, the inflammatory cytokine TNF-α. Furthermore, the Dex-treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.     

Differential uptake and cross‐presentation of soluble and necrotic cell antigen by human DC subsets

Cross‐presentation is the mechanism by which exogenous Ag is processed for recognition by CD8⁺ T cells. Murine CD8α⁺ DCs are specialized at cross‐presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross‐presentation of soluble and cellular Ag by human blood CD141⁺ DCs, the human equivalent of mouse CD8α⁺ DCs, and compared them with human monocyte‐derived DCs (MoDCs) and blood CD1c⁺ DC subsets. MoDCs were superior in their capacity to internalize and cross‐present soluble protein whereas CD141⁺ DCs were more efficient at ingesting and cross‐presenting cellular Ag. Whilst cross‐presentation by CD1c⁺ DCs and CD141⁺ DCs was dependent on the proteasome, and hence cytosolic translocation, cross‐presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross‐presentation by CD1c⁺ DCs and MoDCs but not by CD141⁺ DCs. These data demonstrate that CD1c⁺ DCs, CD141⁺ DCs, and MoDCs are capable of cross‐presentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141⁺ DCs, like their murine CD8α⁺ DC counterparts, are specialized at cross‐presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.     

Enrichment of dendritic cells from human peripheral blood

A simplified method is described for purification of dendritic cells from human peripheral blood. The method is based on depletion of phagocytes with carbonyl iron and magnet, followed by centrifugation of nonphagocytic cells on Percoll and elimination of contaminating T lymphocytes, B lymphocytes, natural killer cells, and monocytes from the low-density cell fraction by treatment with monoclonal antibodies and complement. The purity of enriched dendritic cells was about 80% and these cells represented 0.2% of the starting mononuclear cell population. Dendritic cells were potent autologous and allogeneic stimulators in mixed leukocyte cultures.     

Harnessing human dendritic cell subsets for medicine

Summary: Immunity results from a complex interplay between the antigen-non-specific innate immune system and the antigen-specific adaptive immune system. The cells and molecules of the innate system employ non-clonal recognition receptors including lectins, Toll-like receptors, NOD-like receptors, and helicases. B and T lymphocytes of the adaptive immune system employ clonal receptors recognizing antigens or their derived peptides in a highly specific manner. An essential link between innate and adaptive immunity is provided by dendritic cells (DCs). DCs can induce such contrasting states as immunity and tolerance. The recent years have brought a wealth of information on the biology of DCs revealing the complexity of this cell system. Indeed, DC plasticity and subsets are prominent determinants of the type and quality of elicited immune responses. In this article, we summarize our recent studies aimed at a better understanding of the DC system to unravel the pathophysiology of human diseases and design novel human vaccines.     

Differential production of inflammatory chemokines by murine dendritic cell subsets

Dendritic cells (DC) are efficient antigen presenting cells with the ability to activate na?ve T cells. Murine DC represent a heterogeneous population that can be subdivided into distinct subsets, including the conventional DC (cDC) which are either CD4(-)CD8(-) (DN), CD4(+)CD8(-) (CD4+) or CD4(-)CD8(+) (CD8+) subsets and the plasmacytoid DC (pDC), which have different immune regulatory functions. In this study, we investigated the differential expression of genes encoding the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes, and the secretion of these chemokines, among splenic DC subsets. These chemokine genes were expressed at higher levels by the splenic CD4+ and DN cDC subsets compared with the CD8+ cDC, in both the resting and activated states in vivo. Both the pDC and cDC subsets displayed increases in chemokine secretion in response to a range of toll-like receptor (TLR) stimuli in vitro. Whilst the pDC were the highest producers of Mip-1alpha and Mip-1beta in response to some TLR stimuli, the DN and CD4+ cDC subsets were the superior producers of Rantes. Overall, of the cDC, the CD4+ cDC produced all chemokines most efficiently, both at a basal level, and in response to most TLR stimuli. Thus, we report a new functional difference between the murine splenic cDC subsets, with the CD4+ cDC demonstrating the most efficient production of the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes.     

Targeting human dendritic cell subsets for improved vaccines

Dendritic cells (DCs) were discovered in 1973 by Ralph Steinman as a previously undefined cell type in the mouse spleen and are now recognized as a group of related cell populations that induce and regulate adaptive immune responses. Studies of the past decade show that, both in mice and humans, DCs are composed of subsets that differ in their localization, phenotype, and functions. These progresses in our understanding of DC biology provide a new framework for improving human health. In this review, we discuss human DC subsets in the context of their medical applications, with a particular focus on DC targeting.     

G-CSF对外周血树突状细胞亚群比例的影响

目的 :了解G CSF对小鼠外周血树突状细胞 (DC)亚群比例的影响。方法 :以不同剂量 (0、5 .0、10 .0或 15 .0 μg)的G CSF ,分别皮下注射于BALB/c小鼠 ,连续 6d ,每天 1次。第 6天分离外周血DC ,用流式细胞仪检测DC1(CD11c+ CD8a-)和DC2 (CD11c+ CD8a+ )的比例并计算其绝对值。结果 :经不同剂量的G CSF刺激后 ,外周血DC1的绝对值无显著变化 ,而DC2的绝对值增加 5倍 (9.6× 10 6/Lvs 5 5 .1× 10 6/L ,P <0 .0 1) ,DC1/DC2比值降低 (4.2vs 0 .7,P <0 .0 1)。结论 :G CSF在体内可提高外周血DC2的绝对数 ,对DC1的数无明显影响 ,从而使DC1/DC2的比例倒置     

结核病患者外周血树突状细胞及其亚群的变化

目的:分析不同结核病患者外周血树突状细胞(DCs)及其亚群的变化,并进行免疫机制探讨。方法:就诊于我院2008-11/2009-08的结核病患者32例(实验组),同期在我院进行健康体检者11例(对照组),采用流式细胞术(FCM)检测了结核病患者(包括19例初治患者和13例复治患者,以及痰涂片结果不同的19例肺结核患者)和对照组中外周血中DCs及其亚群的变化。结果:实验组DC1亚群的比率和DCs的总数分别为(0.28±0.13)%和(0.42±0.19)%,明显低于对照组的DC1亚群的比率和DCs的总数(0.47±0.23)%和(0.65±0.22)%(P0.01)。痰涂片阳性患者外周血中DC1亚群的比率和DCs的总数分别为(0.16±0.04)%和(0.24±0.06)%,明显低于痰涂片阴性患者DC1亚群的比率和DCs的总数(0.28±0.14)%和(0.43±0.12)%(P0.05)。初治患者与复治患者外周血中DCs的总数及其亚群的比率无统计学意义(P0.05)。结论:DCs可作为结核病传染源初筛和抗结核疗效观察的参考指标,反映不同结核病患者的免疫状态。     

From skin dendritic cells to a simplified classification of human and mouse dendritic cell subsets

Recent studies have identified several DC subsets within the mouse skin and showed that functional specialization exists among them. This Viewpoint summarizes recent data on functional specialization of skin DC subsets and integrates this knowledge into a unifying DC classification that emphasizes the similarities between the DC subsets found in both lymphoid and nonlymphoid tissues of several mammalian species.     

乙型肝炎病毒感染者树突状细胞(DC)亚群的变化及意义

目的　分析乙型肝炎病毒 (HBV)感染引起的慢性肝炎和肝炎肝硬变患者体内树突状细胞 (DC)亚群 (包括MDC和PDC)的数量和功能变化。方法　采用流式细胞分析技术检测 12例急性乙型肝炎、4 3例慢性乙型肝炎、15例肝炎肝硬变患者及 2 2例健康人体内MDC和PDC细胞的比例和数量 ;体外培养患者及健康人外周血DC1并检测其表型和刺激混合淋巴细胞反应 (MLR)的能力 ,评价MDC的功能 ;用 1型单纯疱疹病毒 (HSV 1)刺激外周血单个核细胞 (PBMCs)并与PBMCs共培养 ,通过检测PBMCs产生的α 干扰素水平评估PDC的功能。结果　慢性乙型肝炎患者的DC1表面CD80、CD86的表达水平低于健康人 ;健康人外周血MDC的比例为 0 .4 5 %± 0 .14 % ,绝对数为 (11.3± 6 .3)× 10 6个 L ,两者在肝炎肝硬变患者下降 ;而健康人外周血PDC的比例为 0 .36 %± 0 .15 % ,绝对数为 (8.9±4 .2 )× 10 6 个 L ,两者在慢性肝炎和肝炎肝硬变患者均下降 ;慢性肝炎和肝硬变患者PDC分泌的α 干扰素量也低于健康人。结论　乙肝病毒感染慢性化后 ,患者体内两种DC均出现数量和功能异常 ,这种异常可能是导致HBV感染的慢性化和疾病进程的原因之一     

人外周血和扁桃体中Tfh细胞与其他Th细胞亚群之间的关系

目的比较观察人外周血和扁桃体Tfh细胞的表型以及与Th1、Th17、Th22细胞亚群之间的关系。方法分离正常人PBMC及扁桃体单个核细胞,利用anti-CD3+anti-CD28或PMA+ionomycin刺激后,采用ELISA和流式细胞术(FCM)检测其细胞因子的产生,分析Tfh与Th1、Th17、Th22细胞亚群之间的关系。结果与PBMC中CD4+T细胞不同,扁桃体CD4+T细胞高表达CXCR5和CD45RO,低表达CCR7和CD62L;与PBMC中CD4+T细胞相比,扁桃体CD4+T细胞IL-21和IL-17产生水平较高,IFN-γ产生水平较低,IL-22水平无显著差异;外周血和扁桃体CD4+T细胞中均存在一定比例的IL-21+IL-17+双阳性、IL-21+IL-22+双阳性、IL-21+IFN-γ+双阳性细胞,IL-21单阳性细胞在扁桃体CD4+T细胞中所占比例明显高于外周血;外周血和扁桃体CD4+CXCR5+细胞除表达IL-21外,还表达IL-17、IL-22和IFN-γ。结论扁桃体中存在较多数量的Tfh细胞,大多数Tfh细胞是不同于Th1、Th17和Th22的细胞亚群。     

Differential effects of necrotic or apoptotic cell uptake on antigen presentation by macrophages.

The induction of pathogenic immune responses may be dependent on the immune system receiving 'danger' signals resulting from tissue damage, rather than tolerogenic stimuli associated with normal cell turnover. The aim was to test this hypothesis by comparing the effects of the uptake of necrotic and apoptotic cells on the ability of antigen-presenting cells (APC) to stimulate immune responses in vitro. The experiments focused on presentation by the macrophage, which is the main cell type adapted for clearing cellular debris in vivo. Murine bone marrow-derived macrophages were pulsed with neutrophils that had been rendered apoptotic or necrotic, and tested for the ability to induce T cell responses. The macrophages that had taken up necrotic, but not apoptotic, cells were able to stimulate recall proliferation by ovalbumin-specific T cells. Furthermore, the response to the mitogen concanavalin A (Con A) was more than 6 times higher when macrophages had been pulsed with necrotic, in comparison with apoptotic, cells. In control experiments, macrophages that had not been exposed to dying neutrophils stimulated weak responses to ovalbumin and Con A. To determine why the uptake of apoptotic and necrotic cells exert opposing effects on the ability of macrophages to stimulate T cells, the expression of costimulatory molecules by treated macrophages, and their production of potentially immunomodulatory cytokines were measured. Flow cytometry revealed that macrophages that had taken up necrotic, but not apoptotic, neutrophils expressed increased levels of CD40 compared to untreated controls within 4 h. Macrophages pulsed with apoptotic cells secreted higher levels of transforming growth factor-beta1 than those ingesting necrotic cells or untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic cell debris present antigens to T lymphocytes with greater efficiency due to transient CD40 upregulation, whereas those that have ingested apoptotic cells are ineffective APC since they secrete inhibitory cytokine.     

Differential CD4(+) T-cell memory responses induced by two subsets of human monocyte-derived dendritic cells

Dendritic cells (DC) are powerful inducers of primary T-cell responses, but their role in secondary responses has not been extensively analysed. Here, we address the role of two DC subsets derived from human CD16(+) (16(+) mDC) or CD16(-) (16(-) mDC) monocytes on the reactivation of memory responses. CD4(+) CD45RA(-) memory T cells were obtained from adult blood donors, and central (T(CM)) and effector (T(EM)) memory T cells were isolated by fluorescence-activated cell sorting with anti-CCR7 antibodies. The 16(+) mDC and 16(-) mDC were cocultured with autologous lymphocytes, either unpulsed or loaded with purified protein derivatives of Mycobacterium tuberculosis (PPD) or tetanus toxoid (TT), and were analysed for up to 8 days. Over a range of doses, 16(+) mDC drove stronger T-cell proliferative responses against both antigens. Overall, antigen-specific memory cells tended to acquire a phenotype of T(EM) at later time-points in the culture, whereas cells that had completed fewer cycles of division were similar to T(CM). The 16(+) mDC induced higher rates of proliferation on both T(CM) and T(EM) lymphocytes than 16(-) mDC. This phenomenon was not related to the ability of both DC to induce CD25 expression on T cells, to lower secretion of interleukin-2, or to raise production of interleukin-10 during T-cell/16(-) mDC cocultures. The induction of T(CM) effector capacity in terms of interferon-gamma production was faster and more pronounced with 16(+) mDC, whereas both DC had similar abilities with T(EM). In conclusion, these data might reveal new potentials in vaccination protocols with 16(+) mDC aimed at inducing strong responses on central memory T cells.     

Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets

The induction of antigen-specific tolerance is critical for maintaining immune homeostasis and preventing autoimmunity. Because the central tolerance that eliminates potentially harmful autoreactive T cells is incomplete, peripheral mechanisms for suppressing self-reactive T cells play an important role. Dendritic cells (DCs) are professional antigen-presenting cells, which have an extraordinary capacity to stimulate naïve T cells and initiate primary immune responses. Recent accumulating evidence indicates that several subsets of human DCs also play a critical role in the induction of peripheral tolerance by anergizing effector CD4⁺ and CD8⁺ T cells or by inducing the differentiation of naïve T cells into T-regulatory cells, which produce interleukin (IL)-10. Human DC subsets with the property of suppressing an antigen-specific T-cell response include plasmacytoid DCs, which are either in an immature state or in a mature state induced by CD40 ligand stimulation, and monocyte-derived DCs, which are either in an immature state or have had their state modulated by treatment with IL-10 or CD8⁺CD28⁻ T cells. These “tolerogenic” DCs may be relevant to therapeutic applications for autoimmune and allergic diseases as well as organ transplant rejection.     

子痫前期患者外周血树突状细胞与T细胞亚群的变化

目的 观察子痫前期患者外周血中的树突状细胞亚群与T细胞亚群相关细胞因子的变化.方法 实验组为子痫前期患者32例,对照组为未孕妇女20例,正常妊娠妇女20例.采集研究对象外周血细胞,流式细胞术检测全血细胞中髓系树突状细胞(mDC)和浆细胞样树突状细胞(pDC);分离外周血单个核细胞,经胞内细胞染色检测Th1、Th2、Th17细胞数量及Th1/Th2比值.结果 子痫前期组mDC百分比(0.33±0.12)％和mDC/pDC比值(2.96±1.65)均高于正常妊娠组,二组数据有明显差异(P＜0.05);pDC百分比(0.16±0.13)％较正常妊娠组(0.21 ±0.12)％有所下降,二组差异有统计学意义(P＜0.05).子痫前期组IFN-γ、IL-4和IL-17的百分比分别为(18.67 ±1.96)％、(1.88±0.51)％和(1.36±0.59)％,与正常妊娠组相比均有显著性差异(P＜0.01).子痫前期组mDC/pDC比率和Th1/Th2之间呈显著正相关(r=0.637,P＜0.01);Th17表达率与pDC表达率之间呈负相关(r=-0.670,P＜0.05),与mDC/pDC比率之间呈显著正相关(r=0.772,P＜0.01).结论 子痫前期患者外周血中树突状细胞亚群和Th1、Th2、Th17型细胞因子异常表达,可能是患者发生免疫失衡的重要原因.