我想感谢Sreemanti巴蜀图1提供的数据。这项工作是支持部分由美国国立卫生研究院授予AI069358和BloodCenter研究基金会。

A.兔补<ol><li>股票兔补充应在室温下解冻，然后分装成0.5和/或1毫升卷，并冻结在-20-80 ° C下长期贮存。 </li><li>每批补应滴定，以确定最佳的工作浓度。通常情况下，稀释的1:10-1:20是最佳选择。 </li><li>补兔补充并不总是presterilized在出售前，根据细胞的最终用途，应过滤消毒，分装时冻结或之前，除了细胞。 </li></ol> B.抗体选择<ol><li>并非所有的抗体是有效激活补体的抗体，从而必须先测试活动。 </li><li>小鼠和大鼠单抗是有效的补体活化。 </li><li> IgM抗体通常是最有效的使用耳机，在补体诱导的细胞裂解。最好的补体激活的IgG亚型不同物种之间。 </li><li>每个抗体，应在10 7细胞/ ml的细胞密度的最佳使用滴定。 </li></ol> C.基本协议<ol><li>细胞群开始调整至5 × 10 7细胞/ ml的培养基中含有热灭活的FCS。最终的细胞量将两倍量。记住要保存一些细胞，可以量化的枯竭，使。 </li><li>每个抗体被添加到预定在适当的浓度或稀释的细胞悬液。混合反演细胞。 </li><li>计算所需的补充量。在室温下解冻的补充库存。如果是没有出息的补充，消毒，使用低蛋白结合注射器喷嘴过滤器。注射器，加几毫升的中型的补充除了之前和过滤直接进入管细胞和抗体。 </li><li>调整在管的最终体积，使细胞在1 × 10 7细胞/ ml。 </li><li>在37 ° C培养细胞一小时，每15分钟混合。 </li><li>离心机的细胞和上清液。 </li><li>中等洗两次。细胞碎片，将坚持以聚苯乙烯管方。使用细胞过滤器或密度梯度离心法，死细胞和碎片可以消除。 </li><li>检查细胞纯度比较耗尽之前和之后的细胞群体的利益。流式细胞仪和免疫组化确定细胞纯度的两个好方法。 </li></ol>代表性的成果当使用一个回合后激活补体，细胞耗竭，是非常高效的单克隆抗体是大于95％。这是显示在图1中，我们开始与32.2％αβT细胞组成的小鼠脾细胞所表达的TCRβ流式细胞仪（eBioscience公司，加利福尼亚州圣迭戈）发现。我们耗尽使用mAb特异性为Thy1，所有的T细胞所表达的一种蛋白质，但没有其他白细胞T细胞。我们使用的抗体，Y - 19大鼠IgG2c 2。下面的过程描述，TCRβ人口减少到1.56％，大于95％减少。 <img src="/files/ftp_upload/1487/1487fig1.jpg" alt="图1" /> 图1。 thy1耗尽+补体活化脾T细胞的小鼠脾细胞总数分别获得健脾和溶解红细胞的同质化。反thy1和婴儿在预定的浓度兔补充被添加到一个单细胞悬液，最终细胞浓度为1 × 10 7细胞/ ml，脾等。潜伏期为1小时后在37 ° C时，细胞​​洗两次和细胞活力的台盼蓝排斥评估。枯竭的效率评估TCRβ+细胞的百分比流量比较之前耗尽（左图）和（右图）耗尽后流式细胞仪。单杠表示阳性，上述酒吧是积极的％。

<ol>
	<li>Janeway, C., Travers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Immune+System+in+Health+and+Disease.">The Immune System in Health and Disease.</a> Immunobiology. , (1994).</li><li>Jones, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+activities+of+antibodies+against+brain-associated+T+cell+antigens+II.+Stimulation+of+T+cell-induced+B+cell+proliferation.">Functional activities of antibodies against brain-associated T cell antigens II. Stimulation of T cell-induced B cell proliferation.</a> Eur J Immunol. 12, 30-37 (1982).</li></ol>

纯化的细胞群，是研究其职能所需的一个共同的过程。在这里，我们描述了一个快速和有效的方法消耗使用抗原特异性抗体和补体消耗的细胞群。的补充，激活抗体，如果是一个宗族的特定标记，可以实现任何细胞人口枯竭。在这里，我们展示了Thy1使用一个mAb特异性T细胞耗竭，基本上所有的T细胞群所表达的蛋白质。我们耗尽αβT细胞超过95％，余下的脾细胞从68％提高到98％。这种细胞纯度水平是相似的其它净化策略，包括流式细胞仪和磁珠分离。补枯竭是一种快速的技术，可在1.5小时内进行一次起始细胞群。补充枯竭的另一个优点是，任何实验室都可以执行的技术，因为它不需要昂贵的设备和试剂。唯一的主要件装备需要水浴和离心机。兔补充，可从不同厂商购买，以合理的成本（见试剂表）和许多单克隆抗体可以购买从美洲热带金枪鱼委员会和当地种植，以控制成本。相比之下，流式细胞仪需要与流式细胞仪检测细胞的分拣能力，将成本的六个数字和昂贵的荧光标记单克隆抗体。磁珠分离是价格适中，但需要购买一块磁铁和磁珠，这可能是昂贵的的。此外，一些磁珠分离战略，也需要购买的列。 补体细胞耗竭使用的主要绊脚石是所需的抗原特异性的单克隆抗体激活补体的可用性。虽然人类，大鼠和小鼠抗体所有有能力修正补充，一些亚型比别人做得更好。虽然从所有三个品种的IgM是高度有效地固定补充的IgG亚型有所不同。因此，每一个单克隆抗体，必须先在试点研究测试，以确定其有效性。对于不高效的抗体，，可以进行两轮补充枯竭，而不影响其余的细胞群的生存能力。由于节约了成本，抗体滴定为达到最佳效果。此外，使用的抗体并不需要得到净化。杂交瘤细胞培养上清和腹水的工作一样有效，这是特别方便的IgM抗体，这是很难净化。与抗体，它必须在一项试验研究滴定的补充。的浓度过高会导致非目标细胞群的生存能力损失。同样重要的是，孵化时间不超过60分钟。 在我们的协议，我们共同孵育抗体和补在一起。然而，细胞可以标记抗体为15-30分钟，冲前补此外。当使用此策略，确保执行冰，以防止损失的细胞在细胞表面的抗原抗体孵化，与某些蛋白质发生修补和上限或受体的内化。虽然高效，与其他方法相比，以补充枯竭的主要缺点是，它不能为正选择使用。因此，它是很难获得的细胞亚群。例如，将难以净化边缘区B细胞滤泡B细胞，最好是用流式细胞仪做。在这种情况下，激活补体可用于消耗性T细胞，以减少细胞被排序。该战略将减少排序的时间，这将节省的钱，将在不太苛刻的细胞。 由于特定的细胞群的净化，是一种常见的要求，已开发的战略。补充枯竭战略的主要优点是其经济承受能力，技术简单和节省时间。

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<td>3-4 week baby rabbit complement</td>
<td>&nbsp;</td>
<td>Pel-Freez Biologicals</td>
<td>31061-3</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>&nbsp;</td>
<td>various</td>
<td>various </td>
<td>An incubator set at 37 &deg;C can also be used.</td>
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depletion of specific cell populations by complement depletion

免疫细胞群的净化，往往需要以研究其独特的功能。特别是，如实时PCR和基因芯片分析的分子的方法，需要高纯度的细胞群的隔离。常用的净化策略包括荧光激活细胞分选（FACS），磁珠分离和补充枯竭。补充枯竭的三个战略，提供了快速，价格低廉，对细胞和细胞产量高温和的优势。补体系统是由大量的血浆蛋白被激活时启动一种蛋白水解级联反应，最终在一个膜攻击复合物的形成，形成了一个在细胞表面，从而导致细胞死亡的孔隙<sup&gt; 1</sup&gt;。 IgM和IgG抗体激活经典途径和杀死细菌的机制。产生的单克隆抗体（单抗），补体级联可用于裂解抗原特异性的方式在任何细胞群。补体级联反应的细胞耗竭是通过增加补充固定抗原特异性抗体和兔补充的起始细胞群。这些细胞在37℃，裂解细胞，其后两轮洗涤除去了一个小时。单克隆抗体与补体结合的高效率通常消耗95-100％的有针对性的细胞群。根据上有针对性的细胞群的净化战略，可用于补充枯竭细胞纯化或浓缩的细胞，然后随后的方法可以进一步纯化人口。

Watch this Scientific Journal Video about Depletion of Specific Cell Populations by Complement Depletion at JoVE.com

Depletion of Specific Cell Populations by Complement Depletion

为了有效地研究其净化往往需要的人群的免疫细胞的功能。补枯竭是一个快速和廉价的技术，高纯度的免疫细胞群的隔离。

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

depletion-of-specific-cell-populations-by-complement-depletion

Research

JoVE Journal

Immunology and Infection

22.0K 次观看.  Blood Research Institute. 为了有效地研究其净化往往需要的人群的免疫细胞的功能。补枯竭是一个快速和廉价的技术，高纯度的免疫细胞群的隔离。

视频: Depletion of Specific Cell Populations by Complement Depletion

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

科研

免疫与感染

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

测量50％的溶血性补充（CH 50）活性的血清

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

免疫突触由双色时间选通受激发射损耗（STED）纳米显微可视化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

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 ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

利用受刺激的排放损耗 (STED) 显微镜可视化肌动蛋白和微管骨架在 B 细胞免疫突触中的应用

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...