该项目是由美国国立卫生研究院NIAID的合同号HHSN2662000500021C支持。我们感谢他的技术援助名臣。

1。计划Amaxa 96穿梭Nucleofector <ol><li>打开一个新的参数文件。 </li><li>选择您将使用标准的转拖动光标在96孔板图，井的数量。使用至少3口井，池，每个实验样品。 </li><li>输入程序代码:在第一部分选择"FF"，并在第2部分选择&#39;168&#39;从下拉式菜单</li><li>从解决方案中，选择"单核细胞，人类" </li><li>在控制选项，选择"标准"。 </li><li>点击应用。 </li><li>包括无染控制，从图中选择进一步根据需要，然后选择"没有计划控制"控制选项，并单击Apply井。 </li><li>选择任何的板图上剩余的未使用的水井和点击取消定义。 </li></ol> 2。准备转区议会<ol><li>一切工作都要在细胞培养罩在可能的情况下无菌条件下进行。准备加入96 nucleofection解决方案以及补充96人单核细胞以及在450到2025年的比例Nucleofector解决方案。混合，让温暖室温。您需要的解决方案nucleofection20μL，每口井。作出超过10％，让移液错误。 </li><li>将nucleocuvette模块，你需要在正确的方向，即nucleocuvette板插入1和第2行第一个。 </li><li>确定为您的实验400克离心10min后，在50万个以及细胞和颗粒细胞计算所需的DC。小心去除上清。 </li><li>重悬浮在nucleofection溶液中轻轻吹打向上和向下几次的细胞。 </li><li>分成特定的治疗如GLO和RIG - I标记eppendorfs的悬浮细胞的正确的卷。 </li><li>添加吹打每50万细胞和混合0.25μg的siRNA。使用非针对你不转染对照样品的siRNA。 </li><li>移取20μL到nucleocuvette模块上述混合物中，根据实验布局，确保液体被传递到井底。 </li><li>盖上盖子nucleocuvette板和挖掘了几次，在坚硬的表面，以帮助确保去除气泡板。 </li></ol> 3。转染区议会<ol><li> Nucleofector 96穿梭托盘插入板，点击上传，然后开始。 </li><li>按照圈上方显示转染过程中的进展情况;一个绿色背景上的黑十字标志，以及在成功转染，而红色背景上的一个黑条意味着它是不成功的。 </li><li>在转染过程完成后，删除板和直流生长介质中添加每口井使用多道移液器80μl。 </li><li>盘10分钟在37 ° C和5％的CO 2。 </li><li>从nucleocuvettes转移到矩阵管的预热直流生长培养基中含有100μL100μL量，保持正确的方向。 </li><li>取下并丢弃那些管的地方转没有发生。 </li><li>在37 ° C和5％的CO 2为24小时或其他所需的时间间隔。 </li></ol> 4。感染新城疫病毒细胞<ol><li>每个实验样品取出矩阵管从孵化器到细胞培养罩和池管到eppendorfs </li><li>颗粒细胞轻轻在桌面离心纺丝5分钟，去除上清液。 </li><li>重悬在无血清的增长介质中含有新城疫病毒的细胞在MOI为1，并在37 ° C和5％ 的 CO 2为45分钟eppendorfs松散覆盖在无菌的方式。 </li><li>添加直流生长培养基900μl，和重新孵育8-10小时</li></ol> 5。收获细胞<ol><li>纺eppendorfs在桌面离心机，去除上清液，沉淀细胞。 </li><li>收获细胞RNA或蛋白质的提取，根据您的协议。 </li></ol> 6。代表性的成果: 使用我们的优化协议，我们转染的 DC瑞吉，针对24小时的siRNA，然后感染新城疫病毒（RIG - I检测到一个副粘病毒）的细胞刺激干扰素反应途径。 QRT - PCR分析表明敲的基因在转录水平的75％。我们还观察到一个类似IFNβ表达减少，在干扰素信号级联，这是一个RIG - I的下游效应。此外，我们观察到，在非疫区，控制转染的细胞表达IFNβ，没有检测到 ， 而 MXA，IFNβ下游反应基因，是最小的（图1A）。 一个区议会第二次转瑞吉靶向的siRNA进行足够的细胞包括免疫印迹分析。凡RT - PCR结果相似，我们看到以前（敲除RIG - I和IFNβ表达分别下降62％和66％）（图1B），免疫印迹检测RIG - I显示，该基因的表达已完全阻断（图1C）。 <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="图1A"/> 图1。 （一）MoDCs或者siRNA的转染靶向的RIG - I或GLO的siRNA和非特异性的RIG - I，IFNβ和QRT - PCR确定MXA的表达效果。转染协议，用于单核细胞特定的缓冲和nucleoporation方案FF168（龙沙Walkersville公司）以下潜伏期为24小时，转染的细胞被感染了新城疫病毒（+）或左未受感染者（ - ）。进一步潜伏期为10小时后，收获细胞和RNA提取的转录水平代表两个重复实验的结果。从鲍尔斯等 16。（二）从一个不同的棕黄色大衣图1A MoDCs再次转任的RIG - I靶向siRNA或使用相同的转染上述协议的非特异性GLO的siRNA 。一个额外的控制，使用未转染MoDCs纳入实验。所有细胞与鸡新城疫病毒感染，并为进一步的RNA和蛋白质的提取收获前10 h孵育后培养24 h。 RIG - I，IFNβ和MXA QRT - PCR确定的转录水平代表两个重复实验的结果。鲍尔斯等 16（C）从上面的图1B中描述的细胞裂解液，免疫印迹分析。泳道1和2，未转染细胞裂解液;泳道3和4，来自GLO siRNA转染细胞裂解液;泳道5和6，RIG -我靶向的siRNA转染细胞裂解液。样品探测RIG - I，也GAPDH的装载控制和重复运行。摘自鲍尔斯等 16。

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幼稚的初级树突状细胞的高效转染重要的是，在这关键的细胞介导的​​先天适应性免疫过渡的细胞炎症通路的高通量分析和逆向工程。然而，大多数研究人员发现，这些细胞是难以转染效率和转染过程诱导细胞成熟时，使用标准的转染技术。我们研究了克服这些限制是否可以由高吞吐量协议优化使用，同时，独立96以及商业核转染系统（龙沙）。 利用荧光激活细胞分选（FACS），我们测得的绿色荧光蛋白表达质粒转染效率和标志物CD86和MHCII免疫细胞成熟的读数。一系列实验评估缓冲器和电的方案，最终确定的条件显示&gt; 50％的转染和成熟的标志物增加的情况下。 为了进一步探讨的nucleofection过程中可能激活区议会，我们分析，在mRNA水平的IFNβ的表达水平和其下游反应基因 MXA。MXA表达是极其敏感IFNβ生产的，可用于生物活性检测到非常低的水平在cytokine18。通过定量RT - PCR分析，我们看到没有检测 IFNβ表达，但确实看到一些MXA从而表明上述基准IFNβ诱导上调。然而，这在非疫区，控制转染细胞的MXA最小上调相比是微不足道的，从病毒介导的刺激IFNβ信号级联反应（图1A），并确认不激活转染区议会，我们可以成功地使用我们的nucleoporation协议任何重大的水平。 Western blot分析证实了我们的优化协议的效用。针对RIG - I造成完全丧失了检测的RIG - I蛋白（图1C）。siRNA的转染区议会我们应该注意到，其他基因的成功扰动，用量的siRNA转染时间长短可能要优化。 据我们所知，这项工作使首次设计的高通量功能丧失，在人类的主要区议会的研究，从而解决了一个困难的障碍，在这一领域的研究。这应提供新的机遇和直流电信号的研究可能有助于在开发基于DC免疫治疗的进步。

<table border="1"><tbody><tr><th>设备/试剂</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td> Amaxa Nucleofector 96以及班车</td><td>龙沙</td><td> 108S0109 </td><td>序列号</td></tr><tr><td> Amaxa人类单核细胞96 Nucleofector套件</td><td>龙沙</td><td> VHPA - 2007 </td><td>包含96 Nucleofector解决方案的人单核细胞，96的补充和Nucleocuvettes和板</td></tr><tr><td> RIG -我的siRNA </td><td> Dharmacon </td><td> L - 012511 - 00 </td><td></td></tr><tr><td> GLO的siRNA </td><td> Dharmacon </td><td> D - 001600 - 01 - 20 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司</td><td> 11875 </td><td>辅以10％的FCS，2毫米L -谷氨酰胺，100 U / ml青霉素和100微克/ ml链霉素，使直流生长介质</td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11965 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> Invitrogen公司</td><td> 25030081 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15070063 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> 3070.03 </td><td></td></tr><tr><td>树突状细胞</td><td>纽约血液中心</td><td></td><td>区议会是从Buffy大衣使用的标准程序纯化</td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

树突状细胞可以被认为是其中发挥关键作用，在其发起和响应感染 1的免疫系统的哨兵。天真区议会致病抗原的检测是通过模式识别受体（PRRS）是能够识别特定病原体相关分子模式（PAMPS）的保守结构。区议会PAMPS检测触发细胞内的信号级联，导致他们的活化和转化为成熟DC。这个过程通常是通过对1型干扰素的生产特点以及与其他炎性细胞因子，如MHCII和CD86和迁移的淋巴结，与T细胞相互作用启动适应性免疫反应的成熟DC的细胞表面标志的上调， 3。因此，区议会的联系先天免疫和适应性免疫系统。 解剖的基本直流响应，以各种病原体的分子网络的能力是至关重要的，以更好地了解了这些信号通路的调控和诱导基因。它也应当有助于促进DC为基础的疫苗针对传染病和肿瘤的发展。然而，此行的研究已经严重阻碍，难以转染的主要区议会 4 。 病毒转导的方法，如慢病毒系统，通常使用的，但携带很多限制，如复杂性和有害生物风险（ 相关费用）5,6,7,8。此外，该病毒基因产物的交付增加转区议会 9,10,11,12的免疫原性。电穿孔技术已用于13,14,15结果好坏参半，但我们是第一个报告使用一种高通量的转染协议和确凿证明其效用。 在这份报告中，我们总结了一个优化的商业协议，高通量转染人类的主要区议会，与有限的细胞毒性和一个 DC成熟16的情况下。转染效率（GFP质粒）和细胞活力分别超过50％和70％。流式细胞仪分析建立的成熟标志物CD86和MHCII转染细胞中表达增加的情况下，而定量RT - PCR结果显示没有IFNβ上调。使用此电协议，我们提供证据议会成功转染siRNA和有效的敲有针对性的基因RIG - I，16,17一个关键的病毒识别受体的mRNA和蛋白水平，。

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

我们目前我们优化的高通量nucleofection协议，要么质粒DNA或不会造成细胞的成熟的siRNA转染初级人类单核细胞衍生的树突状细胞的有效方法。我们进一步提供证据证明为成功的siRNA沉默靶基因的mRNA和蛋白水平的RIG - I。

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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Immunology and Infection

20.3K 次观看.  Mount Sinai School of Medicine. 我们目前我们优化的高通量nucleofection协议，要么质粒DNA或不会造成细胞的成熟的siRNA转染初级人类单核细胞衍生的树突状细胞的有效方法。我们进一步提供证据证明为成功的siRNA沉默靶基因的mRNA和蛋白水平的RIG - I。

视频: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

科研

免疫与感染

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

制备肿瘤抗原负载成熟树突状细胞免疫治疗

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...