交叉呈递—树突状细胞行使功能的重要途径

树突状细胞（DC）是天然免疫和获得性免疫的重要调节剂。DC的一重要特征是通过交叉呈递使外源性抗原进入MHC I类途径，从而将外源性的蛋白质抗原呈递给CD8^ T细胞，以诱导机体产生抗原特异性CTL。本文从主要的交叉呈递细胞DC入手，阐述了影响交叉呈递的因素；交叉呈递中细胞之间的相互作用及交叉呈递的最终结果交叉激活和交叉耐受。对宿主抵抗病原微生物感染、诱导抗肿瘤免疫反应、疫苗研制和维持外周耐受有重要的指导意义。     

交叉呈递-树突状细胞行使功能的重要途径

树突状细胞 (DC)是天然免疫和获得性免疫的重要调节剂。DC的一重要特征是通过交叉呈递使外源性抗原进入MHCⅠ类途径 ,从而将外源性的蛋白质抗原呈递给CD8+ T细胞 ,以诱导机体产生抗原特异性CTL。本文从主要的交叉呈递细胞DC入手 ,阐述了影响交叉呈递的因素 ;交叉呈递中细胞之间的相互作用及交叉呈递的最终结果交叉激活和交叉耐受。对宿主抵抗病原微生物感染、诱导抗肿瘤免疫反应、疫苗研制和维持外周耐受有重要的指导意义。     

023 交叉呈递-树突状细胞行使功能的重要途径

树突状细胞(DC)是天然免疫和获得性免疫的重要调节剂。DC的一重要特征是通过交叉呈递使外源性抗原进入MHCⅠ类途径，从而将外源性的蛋白质抗原呈递给CD8+T细胞，以诱导机体产生抗原特异性CTL。本文从主要的交叉呈递细胞DC入手，阐述了影响交叉呈递的因素；交叉呈递中细胞之间的相互作用及交叉呈递的最终结果交叉激活和交叉耐受。对宿主抵抗病原微生物感染、诱导抗肿瘤免疫反应、疫苗研制和维持外周耐受有重要的指导意义。     

少量人外周血体外培养鉴定单核细胞来源树突状细胞方法初探

目的:在少量人外周血条件下体外培养并鉴定单核细胞来源树突状细胞(Monocyte-derived dendritic cells,Mo DC)。方法:取健康成人少量新鲜外周血经改良密度梯度离心法分离获得单核细胞,加入重组人粒-巨噬细胞集落刺激因子(rh GM-CSF)、重组人白细胞介素4(rh IL-4)诱导Mo DC生长,并用肿瘤坏死因子α(TNF-α)刺激成熟,倒置显微镜及扫描电镜观察细胞形态;分别于培养第4天和第7天用流式进行表型鉴定、CCK-8法检测同种异体混合淋巴细胞反应鉴定抗原递呈能力。结果:Mo DC呈类圆形,聚集成团,悬浮生长,扫描电镜观察其表面有典型毛刺状突起;流式检测Mo DC高表达CD11c、CD1a,经TNF-α刺激后的Mo DC表面MHCⅡ、CD80、CD83、CD86表达均较培养4 d的Mo DC明显升高,具有统计学意义(P0.05);经TNF-α刺激后的Mo DC刺激同种异体淋巴细胞增殖能力较培养4 d的Mo DC明显升高,具有统计学意义(P0.05)。结论:用本实验方法可从少量人外周血中获得成熟Mo DC,为其在多种变态反应疾病、自身免疫性疾病及肿瘤疫苗等领域的研究提供基础保障。     

人骨髓来源的树突状细胞的诱导扩增及鉴定

目的:以人骨髓细胞为来源,建立体外诱导扩增树突状细胞(DC)的方法并进行形态学和免疫表型鉴定。方法:取正常人骨髓,以淋巴细胞分离液分离骨髓单核细胞后,用rhGM-CSF和rhIL-4诱导DC产生,再用rhTNF促进其成熟。收集细胞,用扫描电镜观察细胞的形态特征,用流式细胞术分析细胞的表型。结果:人骨髓细胞经rhGM-CSF、rhIL-4和rhTNF诱导可得到大量成熟的DC,电镜观察具有典型的DC形态。流式细胞术分析细胞的表型表明,诱导后第5天,CD1a 细胞的百分率为70%~75%,CD83 细胞为3%~3.2%;诱导后第7天,CD1a 细胞为84%~86%,CD83 细胞为30%~32%。结论:由人骨髓细胞可成功地诱导出具有典型形态特征的DC,为DC的深入研究和临床应用提供又一细胞来源。     

骨髓CD34+细胞体外扩增诱导树突状细胞实验研究

目的探讨应用不同细胞因子组合方案从骨髓CD34+细胞体外扩增诱导树突状细胞(DC)的可行性及评价不同诱导方案诱导DC的效果.方法免疫磁珠法纯化骨髓CD34+细胞.在有血清条件下应用两步法SCF+FL+TPO+IL-3扩增2周,然后以GM-CSF+IL-4+TNF-α(GI方案)或GM-CSF+TNF-α(GT方案)诱导DC;或者一步法SCF+FL+TPO+IL-3+GM-CSF+TNF-α直接作用2周扩增诱导DC.通过相差显微镜、电子显微镜、流式细胞仪分析、异硫氰酸荧光素标记的葡聚糖(FITC-DX)内吞实验检测DC的生物学特性.结果诱导后细胞较0 d或诱导前细胞高表达DC相关标记(CD1a、CD80、CD86、CD40、CD54、HLA-DR).两步法GI方案诱导10 d,总细胞扩增倍数、CDla+DC扩增倍数分别为(198±178)倍和(122±129)倍,与GT方案比较无统计学意义,但诱导细胞CD1a、CD80、CD86的表达明显高于后者.一步法扩增诱导2周时总细胞数扩增(43±16)倍,CD1a+DC数是0 d接种细胞的(4±2)倍.结论两步法能从正常CD34+细胞诱导产生大量DC,GI方案优于GT方案.两者扩增效率均优于一步法.     

树突状细胞影响肝移植

据Supter TL[Hepatology，2007，46（6）：2021-2031]报道，肝脏树突状细胞对肝移植有重要影响。 树突状细胞（dendritic cells，DC）是近年来倍受人们关注的专职抗原呈递细胞（antigen presenting cells，APC），能摄取、加工及呈递抗原，启动T淋巴细胞介导的免疫反应。     

Lewis大鼠骨髓来源成熟和未成熟树突状细胞的提取与鉴别

背景：树突状细胞在未成熟阶段表现出极强的抗原吞噬功能,它可以在免疫耐受、癌症的免疫治疗等方面都表现出极大的优越性。但由于未成熟树突状细胞在生物体内含量极微,这就严重限制了它在临床、科研方面的应用。目的：提取鉴别Lewis大鼠骨髓来源成熟和未成熟树突状细胞。方法：从Lewis大鼠骨髓中分离骨髓前体细胞,应用20 ng/m L粒细胞集落刺激因子、10 ng/m L白细胞介素4培养7 d诱导为未成熟树突状细胞,然后在未成熟树突状细胞中加入1μg/m L脂多糖继续培养2 d诱导为成熟树突状细胞。采用荧光倒置显微镜观察树突状细胞形态,流式细胞仪鉴定成熟和未成熟树突状细胞表面特异性分子,ELISA检测成熟和未成熟树突状细胞培养上清白细胞介素10、白细胞介素12和白细胞介素17A因子的分泌水平,混合淋巴细胞反应检测成熟和未成熟树突状细胞对T淋巴细胞的刺激反应。结果与结论：(1)普通荧光倒置显微镜下观察树突状细胞具有明显的突起结构;(2)流式细胞仪可见未成熟树突状细胞低表达CD40、CD86等共刺激分子;相反,成熟树突状细胞高表达上述共刺激分子;(3)未成熟树突状细胞的白细胞介素10、白细胞介素17A...     

Tat-Rac1 C-末端肽促进树突状细胞的交叉递呈

目的 研究Rac1、Rac2、Rac3、RhoA以及Cdc42的C-末端肽对树突状细胞(DC)交叉递呈的影响.方法 合成Rac1、Rac2、Rac3、RhoA及Cdc42 C末端区与人工改造的穿膜肽Tat47-57的融合肽,负载DC2.4细胞后,采用荧光显微镜及流式细胞术检测DC2.4细胞吞噬能力的变化,采用B3Z细胞检测DC2.4细胞抗原交叉递呈能力的变化.结果 负载了Tat-Rac1 C-末端肽的DC2.4细胞吞噬能力增强,并显著促进DC2.4细胞经MHC I类分子途径递呈外源性抗原的能力.结论 Tat-Rac1 C-末端肽能够有效促进DC的交叉递呈,为病毒感染和肿瘤等疾病的治疗奠定了基础.     

树突状细胞的表面标志及其免疫学意义

树突状细胞（ＤＣ）是免疫应答中重要的免疫细胞，在体内有移行成熟的特点，成熟及非成熟ＤＣ在免疫答答中有着不同的作用。本文综述ＤＣ的表面标志，共刺激分子的表达及其免疫学意义和体外培养学方面的最新进展，有助于对ＤＣ进一步了解。     

Anatomic location defines antigen presentation by dendritic cells to T cells in response to intravenous soluble antigens

In the spleen, exogenous antigen is preferentially presented by CD8alpha+CD11b- DC to CD8 T cells and by CD8alpha-CD11b+ DC to CD4 T cells. However, it is not yet clear whether the same rule applies to other secondary lymphoid organs. To address this issue, we first classified secondary lymphoid tissues into three categories based on the expression pattern of CD8alpha and CD11b in C57BL/6 and BALB/c mice: (a) spleen, (b) mesenteric lymph node (MLN) and (c) other peripheral lymph nodes (PLN). We then analyzed the OVA-specific T cell-stimulating capacity of each DC subset after intravenous injection with soluble OVA. Our results show that, regardless of tissue origin, CD8alpha-CD11b+ DC generally present OVA to CD4 T cells, a finding that held true as well for CD8alpha+CD11b+ DC in PLN. In striking contrast, CD8alpha+CD11b- DC in spleen, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN mainly cross-present OVA to CD8 T cells in their respective tissues. Of note, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN present OVA to both CD4 T and CD8 T cells. Therefore, the antigen-presenting capacity of each distinct DC subset is determined by its anatomic environment in combination with its surface phenotype.     

Rab4蛋白参与DC细胞内源性抗原递呈

目的 建立稳定表达卵白蛋白的DC细胞株DC-OVA,研究Rab蛋白对DC内源抗原递呈的影响.方法 首先构建含OVA蛋白基因慢病毒表达质粒pLentimycOVA;以DNA-磷酸钙共沉淀法转染293FT细胞制备慢病毒,用病毒感染DC2.4细胞及杀稻瘟毒素(Blasticidin)筛选方法建立稳定表达OVA的细胞株,Western blot鉴定DC-OVA细胞中OVA蛋白表达;然后用识别MHC Ⅰ类分子-OVA肽复合物的T细胞杂交瘤B3Z细胞以及针对该复合物的单抗25D1.16检测DC-OVA细胞表面MHC-OVA肽复合物的形成情况,建立内源性抗原呈递的检测方法;最后采用脂质体法转染化学合成的Rab4、Rab5A、Rab7、Rab11的siRNA于DC-OVA中,B3Z检测DC内源抗原递呈的变化.结果 酶切和测序分析证实转移质粒克隆成功,Western blot可在DC-OVA细胞中检测到OVA蛋白,B3Z细胞和25D1.16单抗可检测到DC-OVA细胞表面存在MHCⅠ类分子-OVA表位多肽复合物,表明内源性抗原呈递系统成功建立.在此基础上,发现下调DC细胞中Rab4蛋白的表达,DC-OVA细胞刺激B3Z生成IL-2(白细胞介素2)的量明显下降.结论 成功构建了OVA蛋白的慢病毒表达载体,获得了表达内源OVA蛋白的DC细胞株,建立了内源性抗原呈递系统,初步证实下调Rab4蛋白可抑制DC细胞的内源性抗原递呈,为病毒感染和肿瘤等疾病的治疗奠定了基础.     

Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: Definition of a maturative step

Human CD1⁺ CD14^- dendritic cells (DC) can be derived from CD14⁺ monocytes using granulocyte/monocyte colony-stimulating factor and interleukin (IL)-4. We have previously shown that IL-10 pre-treatment of such DC significantly inhibited their antigen-presenting capacity to CD4⁺ T cell clones. In this study, we further analyze how IL-10 influences antigen presentation. We first investigated whether IL-10 could alter the early stage of antigen presentation, the capture of antigen. This can be mediated by mannose receptor (MR)-mediated endocytosis and by fluid-phase uptake through macropinocytosis. IL-10-treated DC showed an enhancement of both mechanisms of antigen capture, as indicated by the increase of fluorescein isothiocyanate-dextran uptake through MR and lucifer yellow uptake. However, IL-10-treated DC, irradiated or glutaraldehyde-fixed, were less efficient than untreated DC in stimulating mixed leukocyte reaction as well as in inducing the activation of peptide-specific T cell clones, indicating that IL-10 achieves its effects mainly by modifying the cell surface phenotype of DC. HLA class I and II, as well as intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen-3, B7-1, B7-2 and ICAM-3 expression were either significantly increased or essentially unchanged, and the ability to bind the epitope recognized by the T cell clones was also unaffected regardless of IL-10 treatment. Our study also indicates that as-yet unidentified accessory molecules may play an essential role in T cell activation. Thus, the IL-10-treated DC possess an increased capacity to capture antigen, with a concomitant decreased stimulatory activity. Our study suggests that IL-10-treated DC have the characteristics of highly immature DC (high capture ability, low stimulatory potency) and may represent an early maturative step of human DC of monocytic origin.     

Classical MHC class I peptide presentation of a bacterial fusion protein by bone marrow-derived dendritic cells

Dendritic cells (DC) expanded in the presence of GM-CSF from the bone marrow of C57BL/6 mice process Gram-negative bacteria expressing the model antigen Crl-OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257 – 264) processed by DC co-incubated with E. coli expressing Crl-OVA, which contains the K^b-binding OVA(257 – 264) epitope, occurs by a cytosolic MHC-I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1^−/− mice co-incubated with E. coli expressing Crl-OVA did not result in K^b presentation of OVA(257 – 264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257 – 264) on K^b when C57BL/6 DC phagocytosed and processed E. coli expressing Crl-OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257 – 264) processed from bacteria expressing Crl-OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257 – 264) on neighboring bystander K^b-expressing antigen-presenting cells after BALB/c (H-2^d) DC phagocytosed E. coli expressing Crl-OVA could not be detected. Together, these data support a cytosolic MHC-I presentation pathway for OVA(257 – 264) processed from E. coli expressing Crl-OVA by bone marrow-derived DC.     

Targeting antigen to bone marrow stromal cell‐2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation

Bone marrow stromal cell‐2 (BST‐2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST‐2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST‐2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST‐2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8⁺ and CD8^? DCs was determined. T‐cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST‐2 was compared with that via receptors DEC205 or Siglec‐H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST‐2, BST‐2‐targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST‐2 was inferior in its capacity to deliver antigen to the MHCI cross‐presentation pathway. In contrast, BST‐2 was superior to Siglec‐H at initiating either MHCI or MHCII antigen presentation. In summary, BST‐2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.     

Cross presentation of antigen by dendritic cells: mechanisms and implications for immunotherapy

Dendritic cells (DCs) possess the specialized potential to present exogenously derived antigen to cytotoxic T lymphocytes to elicit an immune response. This process, termed cross presentation, is crucial in the generation of immune response to viruses, tumors and in autoimmune disease. The ability of DCs to cross-present exogenous antigen to cytotoxic T lymphocytes makes them an attractive target for exploitation in immunotherapy. In recent years, significant advances have been made in understanding the mechanism of cross-presentation and the DC subsets involved. The recent discovery of the human cross presenting DC has given this field a new lease of life. In this report, the authors provide an overview of cross-presentation of antigen by DCs, focusing on the current understanding of the molecular mechanisms of the process. The authors also discuss the DC subsets involved in cross presentation and its role in health and disease.     

Circulating specific antibodies enhance systemic cross-priming by delivery of complexed antigen to dendritic cells in vivo

Increasing evidence suggests that antibodies can have stimulatory effects on T‐cell immunity. However, the contribution of circulating antigen‐specific antibodies on MHC class I cross‐priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross‐presentation of antigen to CD8⁺ T cells. Mice with hapten‐specific circulating antibodies, but na?ve for the T‐cell antigen, were infused with haptenated antigen and CD8⁺ T‐cell induction was measured. Mice with circulating hapten‐specific antibodies showed significantly enhanced cross‐presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross‐presentation in mice with circulating antigen‐specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c⁺ APCs were responsible for the enhanced and sustained cross‐presentation, although CD11c^? APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen‐antibody immune complexes improves MHC class I cross‐presentation, and CD8⁺ T‐cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor‐associated antigens intensively used in the clinic nowadays.     

树突状细胞摄取和提呈颗粒化抗原的能力与颗粒载体的大小相关

目的：研究体外小鼠骨髓树突状细胞对2种不同大小bead-OVA复合物（0.04 μm bead和1.0μm bead）的摄取及class I途径抗原提呈能力。方法：以2h骨髓粘附细胞为前体细胞，用GM－CSF（1000U／ml）和IL－3（10ng/ml）培养5d，观察细胞对FITC标记的2种bead-OVA复合物的摄取，PMA、amiloride、cytochalasin D对摄取的抑制，以及细胞摄取后表达MHC分子和共刺激分子的情况，同时用OVA表位特异性T细胞杂交检测细胞摄取后通过class I途径活化CTL应答的能力。结果：树突状细胞对1．0μm bead-OVA的摄取明显高于对0．04μm bead-OVA，前者被上述3种抑制剂显著抑制，后者仅对amiloride和PMA抑制作用敏感，CCD无明显抑制作用。与摄取结果相反，0．04μm bead－OVA较1．0μm bead－OVA诱导更强的CD8细胞免疫应答，表型分析显示，细胞摄取0．04μm bead后，MHC分子和共刺激分子表达显著高于1．0μm的bead。结论：树突状细胞对2种bead的摄取能力和摄取机制不一样，0．04μm bead尽管摄取效率不如1．0μm bead，但通过class I途径提呈抗原的效率显著高于后者。     

Pathways for antigen cross presentation

Dendritic cells (DCs) have the unique ability to capture cellular tissue antigens, and to present them on MHC class I molecules to antigen-specific CD8⁺ T lymphocytes after migration to the draining lymph nodes. This process, called cross presentation can lead either to the tolerization or activation of antigen-specific CD8⁺ T cells. Antigen capture is believed to occur by phagocytosis of antigen-bearing dead cells. Recent studies suggest that the antigen transferred from the phagocytosed cell to the DC during cross presentation is a proteasome substrate, rather than a proteasomal degradation product. In most cases, the formation of the peptide-MHC class I complexes in DCs requires the export of protein antigens from phagosomes to the cytosol, where they undergo proteasomal degradation. The resulting peptides are then translocated by TAP to the lumen of a cross presentation-loading compartment, for association to MHC class I under the control of chaperones and oxido-reductases. This loading compartment may be either the endoplasmic reticulum (ER) or a mix phagosome-ER compartment. MHC class I egress from the loading compartment to cell surface remains to be analyzed.     

Carbon monoxide impairs mitochondria‐dependent endosomal maturation and antigen presentation in dendritic cells

Heme‐oxygenase 1 (HO‐1) prevents T cell‐mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m‐chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome‐to‐lysosome fusion, and antigen processing, dampening the production of peptide‐MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4⁺ T‐cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8⁺ T cell‐dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T‐cell priming by blocking an unknown mitochondria‐dependent antigen‐processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen‐containing endosomes. In conclusion, CO produced by HO‐1 impairs antigen‐dependent inflammation by regulating DC immunogenicity by a mitochondria‐dependent mechanism.