Name: an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection
Uploaded: 2018-03-30T13:00:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection at JoVE.com

这项工作得到了中国科学院重点项目 (KFZD-SW-205)、中国科学院战略生物资源技术支持系统 (CZBZX-1 和 ZSSB-004) 的支持, 并获得中国国家科学基金会资助 (81401640) 和上海自然科学基金 (14ZR1434800)。

这项研究获得中国科学院遗传学与发展生物学研究所伦理委员会的批准, 该议定书遵循人类福利的机构指南。
注意: 有关工作流的示意图, 请参见图 1 。
1. CAEBV 患者 PBMC 的分离
<ol><li>根据制造商的指示, 通过梯度 (例如, Ficoll 菌斑) 离心法从 CAEBV 患者的2毫升全血中纯化原 PBMC。或者, 用 RPMI 1640 培养基从液氮中解冻冷冻 PBMC。</li>
	<li>增加2µL 的台盼蓝 (0.4%) 到18µL 细胞悬浮, 等待3分钟染色细胞, 转移悬浮到细胞计数器板, 并测量细胞的浓度和活力与自动细胞计数器。</li>
	<li>准备细胞悬浮在密度 2-3×106细胞/毫升补充与完整的 RPMI 1640 培养基含有20% 人血清 (热灭活在56°c), 2 毫米 l-谷氨酰胺, 和抗生素 (100 µg/毫升链霉素, 100 U/毫升青霉素)。种子1毫升 PBMC 悬浮每井入24井细胞培养板材。</li>
	<li>每井加 150 U rhIL-2。</li>
	<li>将文化板块放置在 5% CO2孵化器中37摄氏度。</li>
	<li>2天, 观察显微镜下的细胞形态学 (200X 倍放大);细胞在明亮和圆的时候处于良好的状态。</li>
</ol>2. 细胞的扩张和可存活细胞的编号
<ol><li>观察细胞形态学在显微镜下 (200X 放大), 并监测细胞生长的条件, 通过编号的细胞改变培养基: 增加2µL 的台盼蓝 (0.4%) 到18µL 细胞悬浮, 并计算细胞的浓度 (见 1.2, 图 2)。</li>
	<li>在24井板中丢弃500µL 的培养基, 并添加500µL 新鲜完整的培养基。每井加 150U rhIL-2。</li>
	<li>按照步骤 2.2, 每周更改两次媒体。</li>
	<li>绘制单元格增长曲线 (图 3)。</li>
	<li>当存活细胞浓度超过 5×106/毫升时, 在每个井中, 将细胞分成 1-2×106细胞/ml 浓度的新井。这个过程需要2-4 周时间。</li>
</ol>3. 细胞分型流式细胞术
<ol><li>当单元格展开时, 收集 1×106单元格以确定单元格线的表型。240 x g的离心细胞在室温下为5分钟 (NK/T 细胞将沉淀在管的底部)。</li>
	<li>放弃上清, 通过添加7毫升 PBS 洗涤沉淀。在室温下, 离心机在 240 x g处为5分钟。重复一次。</li>
	<li>在每管中加入200µL 人血清, 混合均匀, 在室温下孵育10分钟。</li>
	<li>在室温下, 离心细胞在 240 x g 处为5分钟。丢弃上清, 然后在 1×106/mL 上用冷 PBSA (PBS + 0.2%BSA) 重新挂起单元格。将单元格分成5管, 每个管包含 2×105单元格。</li>
	<li>离心细胞在 240 x g 5 分钟在4摄氏度。丢弃上清, 再用含有10µL PE (或 PE-Cy7) 和10µL FITC 的 PBSA 缓冲或 PBSA 的细胞重新挂上标记的抗体, 如图 4所示, 将 T 或 NK 细胞的细胞受体染色。使用 PBSA 缓冲区暂停的单元格用作负控制。</li>
	<li>在黑暗中用20-30 分钟的冰块在冰上孵化细胞。</li>
	<li>用冷 PBSA 洗两次细胞, 用300µL 冷 PBSA 重新悬浮细胞。</li>
	<li>用流式细胞仪分析红细胞表型。<ol>
<li>在 "创建工作表" 条件下设置流式细胞仪。使用显示正散射 (FSC) 与侧面散射 (SSC) 的点图建立实验模板。</li>
		<li>负载同种控制管, 以优化 fsc 和 SSC 电压, 并优化 fsc 阈值, 以消除碎片, 而不干扰细胞群体的利益。删除所有参数, 除 FSC, SSC, FITC, PE 和 PE-Cy7。</li>
		<li>使用同种控件和每个2色分析组中的单个正则控件执行补偿。</li>
		<li>加载样本和创建 HLA-DR vs CD19, CD4 vs CD8, CD56 vs CD16 和 CD3 VS CD16 点地块显示不同的细胞数量。 注: PBMC 用于使用负/同种控制和单阳性控制进行补偿。采用流式细胞仪对2色免疫荧光进行了常规的表面标记表达分析。以下抗体包括: 抗 HLA-DR, anti-CD4, anti-CD16 共轭异硫氰酸荧光素 (FITC), anti-CD8, anti-CD56, CD3 与藻红蛋白 (PE) 共轭, anti-CD19 与 PE-Cy7 共轭。</li>
	</ol>
</li>
</ol>4. NK/T 细胞的扩展和冷冻保存
<ol><li>当细胞表型分析完成时, 改变培养基。小心地取出一半的上清, 避免接触盘子底部的细胞。添加相同数量的新鲜培养基中含有300毫升 rhIL-2 的细胞板。</li>
	<li>每3天改变一半的培养基 (2.2), 直到细胞簇在显微镜下清晰可见 (图 2)。通常, 此过程需要2-3 周时间。</li>
	<li>在混合同一血统的不同水井后, 将细胞从24孔板转移到 T25 培养瓶中。当培养基的体积扩大到10-15 毫升时, 将培养基的体积加倍, 使其变黄。添加 rhIL-2 的浓度为 150 U/毫升。</li>
	<li>改变培养基24小时后冷冻后2-3 周生长, 当细胞质量可以观察肉眼。用细胞计数器测量细胞浓度。</li>
	<li>离心细胞悬浮5分钟在 240 x g, 并重新悬浮细胞颗粒在密度为 5-10 x 106细胞/毫升与冷冻库存解决方案, 其中含有90% 胎牛血清和 10% dimethylsulfoxide (亚砜)。以每分钟1摄氏度的速率冻结至-80 摄氏度, 然后直接转入液氮。</li>
</ol>

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D.Z.、X.Z. 和 X.C. 是在等待专利申请方面的共同发明者, 涵盖了此方法用于建立 NK/T 细胞系的潜在用途以及本研究建立的细胞系。D.Z.、X.Z. 和 X.C. 宣布在这项工作中没有竞争的财政利益。其余的作者宣布, 他们没有竞争的金融利益。

在该协议中, 建立了 CAEBV 患者全血 NK/T 细胞系的新方法。与现有方法相比, 该方法的主要优点是其简单性和对少量血液的要求, 同时具有较高的成功率和良好的细胞生存条件。此外, 可以通过培养 PBMC 来建立 NK/T 细胞系, 而不确定 eb 病毒潜伏预先感染的细胞类型, 因为这种测定在培养前会消耗更多的血液和时间。另外, 细胞系可以通过细胞系建立后的流式细胞术进行分析, 从而纯化细胞的不同表型。?...

eb 病毒是无处不在的, 不仅感染了 B 细胞, 而且传染了 T 和自然杀手 (nk) 细胞, 这导致了一些 eb 病相关的 NK/T 淋巴疾病 (LPD) 和淋巴瘤/白血病, 如 eb 联合噬血细胞lymphohistiocytosis, hydroa vacciniforme 淋巴瘤, 淋巴结外 nk/T 细胞淋巴瘤, 鼻型和侵袭性 NK 细胞白血病 1, 2, 3.其中包括严重的慢性主动 eb 病毒 (SCAEBV) 疾病, 主要发生在东亚, 现在被认为是由 eb 病毒感染的 T 或 NK 细胞的克隆扩张引起的 LPD4,5,6,7, 但没有明显的免疫缺陷存在于传染性传染性 (IM) 类似的症状, 包括发烧, 肿大, 淋巴结肿大, 肝功能障碍持续或反复, 以及高 eb 病毒-DNA 负荷在外围血液8,9。CAEBV 患者预后不佳10,11, 其发病机制和 eb 病毒的作用尚不清楚。因此, 从 eb 病毒相关的 nk/t 淋巴疾病和淋巴瘤的细胞系是非常有用的细胞模型, 以澄清 eb 病毒诱发 NK 或 t 细胞增殖机制及其与高发病率的白血病或淋巴.
到目前为止, 已经用不同的技术建立了几个单元格线12,13,14,15,16。人类 nk 细胞系由 nk 细胞淋巴瘤/白血病建立, 通过与小鼠基质细胞系进行共培养, 并在 rhIL-2 的浓度为每毫升15。KAI3 是由患有严重蚊子过敏或 SCAEBV 的患者与自体永生细胞系 (拼箱)、乙肝病毒转化的 B 细胞、作为饲养细胞以及在 100 u/毫升16中添加 rhIL-2 的病人建立的另一种 NK 细胞系。SNK6 和 SNT8 通过添加大剂量的 rhIL-2 (700 u/毫升)12, 从鼻 NK/T 细胞淋巴瘤患者的肿瘤组织中获得。与类似的技术, SNK-1 细胞是从 CAEBV 患者, 培养从 PBMC 通过去除 T 细胞和添加 700 u/毫升 rhIL-213,17。SNT13 和 SNT15 是通过删除 CD4+ 和 CD8+ 单元格18建立的。到目前为止, 其他 t 细胞和 nk 细胞系从 eb 病毒-NK/T LPD 患者都开发了此方法19。
上述现有方法的弊端包括: 饲养细胞的使用、高剂量 IL-2 的要求、多达10毫升全血的利用率, 或对 NK/T 细胞的净化, 这在临床上是非常有挑战性的, 因为在开始培养之前, 必须验证 eb 病毒潜伏感染的细胞类型。由于 CAEBV 主要发生在亚洲儿童身上, 所以在所有地区都不容易获得10毫升血液。在本研究中, 我们开发了一种新的简单的方法, 成功率高, 建立 NK 和/或 T 细胞系, 通过培养 PBMC 从 CAEBV 患者使用低剂量的 rhIL2 和2毫升的全血容量, 没有喂食细胞。结果表明, 该方法效率高, 节省时间。

<table><tbody><tr><td>Hu HLA-DR FITC</td><td>BD</td><td>560944</td><td>antibody for FACS</td></tr><tr><td>Hu CD4 FITC</td><td>BD</td><td>555346</td><td>antibody for FACS</td></tr><tr><td>Hu/NHP CD8 PE</td><td>BD</td><td>557086</td><td>antibody for FACS</td></tr><tr><td>Hu CD3 PE</td><td>BD</td><td>555340</td><td>antibody for FACS</td></tr><tr><td>Hu CD16 FITC 3G8</td><td>BD</td><td>555406</td><td>antibody for FACS</td></tr><tr><td>Hu/NHP CD56 PE MY31</td><td>BD</td><td>556647</td><td>antibody for FACS</td></tr><tr><td>hu/CD19 PE-Cy7</td><td>BD</td><td>560728</td><td>antibody for FACS</td></tr><tr><td>human IL-2</td><td>Roche</td><td>11147528001</td><td></td></tr><tr><td>human serum</td><td>MRC</td><td>CCC118-125</td><td></td></tr><tr><td>CO2 incubator</td><td>SANYO</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Techcomp</td><td>CT6T</td><td>Centrifugation</td></tr><tr><td>microscope</td><td>OLYMPUS CKX53</td><td>CKX53</td><td></td></tr><tr><td>Automated Cell Counter</td><td>Countstar</td><td>IC 1000</td><td>For cell counting</td></tr><tr><td>24 well cell culture cluster</td><td>Corning Incorporated</td><td>3524</td><td>Polystyrene plates</td></tr><tr><td>25cm2 cell culture flask</td><td>Nest</td><td>707001</td><td>Polystyrene</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270</td><td>Component of cell medium</td></tr><tr><td>RPMI Medium 1640</td><td>life</td><td>22400089</td><td>For cell medium</td></tr><tr><td>L-Glutamine</td><td>Amresco</td><td>374</td><td>Component of cell medium</td></tr><tr><td>100 x streptomycin penicillin solution BioRoYeeBRY-2309</td><td>BioRoYee</td><td>BRY-2309</td><td>Component of cell medium</td></tr><tr><td>Ficoll paque plus</td><td>GE Healthcare</td><td>17-1440-03</td><td>For in vitro isolation of lymphocyte</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td>For freezing cells</td></tr><tr><td>flow cytometer</td><td>BD</td><td>BD FACSAria II</td><td></td></tr><tr><td>soft ware</td><td>BD</td><td>BD FACSDiva soft ware</td><td>FACS analysis</td></tr></tbody></table>

an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection

从淋巴瘤或淋巴综合征患者中, 我们已经描述了一些方法来建立 NK/T 细胞系。这些方法使用饲养细胞, 纯化 NK 或 T 细胞多达10毫升的血液, 或高剂量的 IL-2。本研究提出了一种通过添加重组人 IL-2 (rhIL-2), 通过培养外周血单个核细胞 (PBMC) 来建立 NK 和 T 细胞系的新方法, 并使用2毫升的全血。细胞可以在两周内迅速增殖并维持3月以上。用该方法建立了 7 NK 或 T 细胞系, 成功率高。该方法简便、可靠, 适用于多例 CAEBV 或 NK/T 细胞淋巴瘤细胞株的建立。

在建立细胞系3天后, 多态细胞开始出现 (图 2)。7天后, 细胞生长迅速, 因为细胞的数量和生存能力都以较高的速率增加 (图 3)。当细胞浓度可超过 3-6 x 106时, 10-14 天后, 小簇细胞明显可见。在这一时期, 细胞应该被分成两到三口的培养板。大约一个月后, 一旦单元格号达到 3-5×107, 计数就足够高, 可以保存。
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Watch this Scientific Journal Video about An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection at JoVE.com

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

采用高效、少量外周血、低剂量 IL-2 的方法, 建立了 CAEBV 患者的 NK 和 T 细胞克隆的简便算法。

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

an-efficient-simple-method-to-establish-nk-t-cell-lines-from-patients

Research

JoVE Journal

Immunology and Infection

9.1K 次观看.  Chinese Academy of Sciences. 采用高效、少量外周血、低剂量 IL-2 的方法, 建立了 CAEBV 患者的 NK 和 T 细胞克隆的简便算法。

视频: An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

科研

免疫与感染

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Epstein - Barr病毒生长转化的淋巴细胞系的建立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

癌症研究

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

传染性单核细胞增多症儿童外周血样本中免疫细胞亚群的分离

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world&#39;s adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles.
Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 &#176;C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation.

This protocol permits the isolation of Epstein-Barr virus particles from the human P3HR1 cell line upon inducing the viral lytic cycle with phorbol 12-myristate 13-acetate. DNA is subsequently extracted from the viral preparation and subjected to real-time PCR to quantify the viral particle concentration.

从P3HR1细胞系分离和定量EB病毒

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.