作者要感谢研究参与者和他们的父母。我们还要感谢Réjean拉波因特博士（覃巴黎圣母院，蒙特利尔，加拿大）访问他的酶联免疫斑点读者，鲁波亚历山德罗夫进行统计分析博士和丹尼斯布莱，桑德拉·卡隆，Silvie瓦卢瓦和马丁卡蒂的专家技术援助。支持由乐全宗D&#39;操作倒莱PROJETS去RECHERCHE倩碧等科特迪瓦评价; DES技术（朱圣 - 和Justine）到HS和PO，由La基金会中心去cancérologie查尔斯 - 布鲁诺，并通过对白血病和淋巴瘤协会补助加拿大。 ISF是由来自LA基金会朱圣 - 和Justine和乐全宗德拉RECHERCHE魁北克 - 桑特（FRQS）奖学金支持。 AJG奖学金是从微生物学，传染病学与免疫学，蒙特利尔大学（加布里埃尔 - 侯爵奖学金），FRQS部的收件人，以及加拿大卫生研究院ŘESEARCH（CIHR）。 NM是由拉朱基金会圣 - 和Justine，科尔基金会和FRQS支持。

本研究方案经朱圣 - 和Justine，蒙特利尔，魁北克，加拿大，那里的研究进行的制度伦理审查委员会。知情同意书，并寻求从所有研究参与者，他们的父母或法定监护人获得。在第一天进行的所有程序和2必须是无菌的条件下（ 即在层流罩）下进行。标准的安全程序来处理人体血液必须严格遵守。 1，涂料的板<ol><li>通过加入到每个孔中20微升35％乙醇中1分钟，透化聚偏氟乙烯（PVDF）膜上，在96孔MultiScreen IP白色的ELISpot板的底部。膜应该成为稍透亮。 </li><li>使用MUL;立即用200μl1X磷酸盐缓冲盐水（137 mM氯化钠，2.7 mM的氯化钾，10毫米的Na 2 HPO 4，2mM的KH 2 PO 4，pH 7.4的PBS）洗涤各孔3次声道音吸管或类似的装置。由于乙醇的残基可以影响细胞的活力和捕获抗体结合这一步是至关重要的。酶联免疫斑点法板是脆弱的，必须小心处理:使用自动洗板机，不推荐。 </li><li>涂层的孔与10微升纯化的小鼠抗人IFN-γ捕获抗体稀释在1×PBS中的10微克/毫升的最终浓度。覆盖板与普通保鲜膜孵育过夜，4°C。 </li></ol> 2，挡板<ol><li>空井，窃听他们干，并用200微升的1×PBS洗板5次。 </li><li>填充孔用200μl的完全RPMI 1640培养基中孵育板2小时，在37℃（分块步骤）。 </li><li>洗涤板5倍用200μl的1×PBS中并保持在孔中充满的1×PBS中，直到细胞准备进行电镀。 </li></ol> 3，电池和电镀刺激<ol><li>准备peripheral血单核细胞（PBMC）从人的静脉血样离心上使用标准方法的Ficoll-Paque上的梯度。备选地，使用标准方法，解冻PBMC的等分试样在液氮下冷冻。在水套培养箱在5％CO 2气氛下后，最后离心后，重新悬浮细胞以2×10 6个细胞/ ml的终浓度，孵育过夜，在37℃。 </li><li>从培养箱中取出PBMC重悬他们于5ml RPMI 1640完全培养基中的终体积。 </li><li>在10微升从粘质沙雷氏菌 5分钟，在室温下遗传工程内切核酸酶的存在下培养PBMC。 </li><li>除去PBMC的一个50μl的等分试样，并用50μl的台盼蓝染料混合。计数细胞，并用血球和显微镜检查（染色排除法）估算％的生存能力。各种自动化方法进行细胞计数和细胞活力罐人评价所以可以使用。 PBMC应&gt; 95％存活。丢弃当样品生存能力低于95％。 </li><li>离心机的PBMC，在700×g离心5分钟，弃去上清，重悬细胞以2×10 6个细胞/ ml的终浓度添加2％（V / V）灭活的人血清（HIS）的AIM-V培养基。保持在37°C，直到刺激混合物加入到平板。 </li><li>加至孔中，1滴一次，加入100μl适当的刺激混合物。三刺激的混合物，应使用如下所述: <ol><li>用AIM-V培养基补充有2％（V / V）灭活的人血清（IHS）作为阴性对照。 </li><li>使用抗CD3单克隆抗体（克隆OKT3）稀释在AIM-V培养基补充有2％（V / V）的IHS至0.5微克/毫升作为阳性对照）的终浓度。 </li><li>使用VZV抗原，无论是γ-辐射的形式（10,000戈瑞; 30分钟的曝光时间）减毒活疫苗VZV（1,350斑块形成单位[PFU] /毫升）的稀释泰德1:200在AIM-V培养基补充有2％（V / V）IHS）或1微克67肽（15个氨基酸残基）的基础上，VZV的顺序立即早期（IE63）磷蛋白合成的等摩尔混合物的。通过监测缺乏的细胞病变效应明显迹象后PBMC培养4天控制照射疗效。 </li><li>准备一式两份的所有井。 </li></ol></li><li>加至各孔，一减少的时间，将100μl制备步骤5.4至适当的孔中的细胞。两个井应保持无细胞（阴性对照）。在水套培养箱在5％CO 2气氛下孵育20小时，在37℃。不要摇晃，移动或堆叠在另一个孵化过程中顶部的板块。 </li></ol> 4，景点开发与检测<ol><li>弃去细胞用1×PBS洗涤板10倍的补充有0.05％（V / V）吐温20（PBST）。吐温20有助于分离已经坚持了我的PVDF细胞mbrane以下孵育过夜。 </li><li>使用多通道移液管加入100微升的0.5毫克/毫升生物素标记的抗IFN-γ单克隆抗体（克隆4S.B3）稀释在1×PBS中含有0.5％（重量/体积）牛血清白蛋白（BSA）。孵育板2小时在室温下。 </li><li>洗涤各孔6倍用PBST和添加每孔加入100μl链霉亲和有机共轭的碱性磷酸酶稀释1:1,000在1x PBS中含有0.5％（重量/体积）BSA中。覆盖板并孵育1小时，在室温下进行。 </li><li>洗涤板3倍用PBST和3X用1×PBS。添加100μl的5 - 溴-4 - 氯-3 - 吲哚磷酸（BCIP）/硝基蓝四唑氯化物（NBT）底物溶液孵育5分钟，在室温下进行。 </li><li>下运行蒸馏水冲洗板。取出的ELISpot板的底部部分，并用蒸馏水洗膜的两侧。空气干燥该板。 </li><li>检查各孔，并用立体ð列举的斑点issection显微镜或使用自动点计数器扫描板。保持板避光，如果他们不能检查在同一天。 <ol><li>平均斑点在相应的复孔计数的数量和从斑点中的测试孔计数的数目减去斑点在阴性对照孔计数的数目（无细胞）。 </li><li>表达IFN-γ的生产为斑点形成单位的数量（SFU）每10 6个PBMC。 </li><li>认为试验样品是阳性如果SFU的数量为&gt; 50每10 6个PBMC和2个标准差（SD）以上的负对照（细胞+ AIM-V培养基中补充了2％（V / V）IHS）。 </li><li>使用每孔400 SFU的值时，斑点更是不胜枚举进行计数。 </li></ol></li><li>测试是否酶联免疫斑点法数据是正态分布的，或不使用Kolmogorov-Smirnov检验。当数据是正态分布，采用学生t TES进行统计比较T（2组）或单因素方差分析和Bonferroni法检测后（多重比较）。如果数据不是正态分布，用曼-惠特尼U检验（2组）或Kruskal-Wallis检验和邓恩的后测（多重比较）。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

修改和故障排除:IFN-γ酶联免疫斑点测定法已被用于检测细胞介导的针对多种微生物病原体，包括人类免疫缺陷病毒1型（HIV-1）的24,25，丙型肝炎病毒（HCV）26免疫反应， 27，和结核杆菌 28,29，只是仅举几例。在这里，我们描述的IFN-γ酶联免疫斑点法的发展来衡量细胞对免疫，用在定义儿科复苏脐血移植受者的VZV特异性免疫重建相互关系的希望。 <p class="jov...

在1989年第一次演出，脐血移植是越来越多地用作各种肿瘤和非肿瘤性血液疾病的儿童1治疗的一部分。水痘是一种细胞病变人力alphaherpesvirus这会导致两种不同的疾病，水痘（原发感染后），带状疱疹（激活后）。继原发感染，水痘持续整个背根神经节的感觉神经内避风的主机的使用寿命。其中最有威胁的感染性并发症以下UCBT是与水痘2-4关联。在我们的临床中心，在没有水痘预防，水痘水痘病的发病在3年累积发病率postUCBT为46％2。在这些患者中， 从头感染或VZV的激活通常与内脏传播到中枢神经系统，肺和肝5-7相关联。因此，阿昔洛韦，伐昔洛韦或泛昔洛韦预防通常给药至UBCT收件人8,9。然而，这种治疗策略不考虑VZV特异性T淋巴细胞或VZV特异性T细胞反应重建动力学的保护电位。随着国家扩大利用长期抗疱疹预防相关的潜在问题包括:a）病人过度治疗;二）抗病毒抗药性10,11的发展;和c）的VZV特异性免疫重建12,13减值。因为检测的功能VZV特异性T淋巴细胞相关性。和VZV感染长期保护的存在，改善临床结果4,14,15，监测细胞介导的针对水痘的免疫反应在移植后的时期，可能会导致更合理地使用抗病毒药物通过治疗使医生来区分谁的病人将受益于水痘预防从那些免疫系统能够控制VZV复制4,13的。 的IFN-γ酶联免疫斑点测定法被广泛地用于在多种试验系统及临床情况的监测细胞介导的​​免疫反应。斑产生下列显色底物的裂解，产生一个可见的和稳定的沉淀物在反应的位点。每个单独的点，从而代表一个单独的细胞因子产生细胞的足迹。 IFN-γ酶联免疫斑点法不仅测量单个细胞在体外的产生IFN-γ响应于体外刺激同源抗原的能力，但它也提供了在一个给定的细胞群体16,17响应单元的频率的估计。除了其高灵敏度，IFN-γ酶联免疫斑点法是直接执行，使得它的使用可能在旨在指导开始抗病毒治疗或终止个性化临床治疗方案的上下文。该过程详述如下describES是专门设计由外周血单核细胞下列体外刺激用VZV的抗原检测和测量产生IFN-γ的ELISPOT测定。

<table><tbody><tr><td>Leucocep tube</td><td>VWR</td><td>89048-936/89048-932</td><td>12 ml or 50 ml tubes may be used depending on the volume of blood.&amp;nbsp;</td></tr><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>17-1440-02</td><td>Protect from light.</td></tr><tr><td>Benzonase nuclease</td><td>Novagen</td><td>70746-3</td><td>Keep at -20 &amp;#176;C.</td></tr><tr><td>MultiScreenHTS-IP Filter Plate</td><td>Millipore</td><td>MSIPS4W10</td><td>Sterile with pore size of 0.45 &amp;micro;m.&amp;nbsp;</td></tr><tr><td>Mouse anti-human IFN-&amp;gamma; capture antibody</td><td>BD Biosciences</td><td>551221</td><td>NIB42 clone.&amp;nbsp;</td></tr><tr><td>Pepmix VZV IE63&amp;nbsp;</td><td>JPT Peptide Technologies</td><td>PM-VZV-IE63</td><td>Dissolve contents of one vial in 40 &amp;#956;l of DMSO. Use within 6 months.</td></tr><tr><td>Biotin-conjugated anti-IFN-&amp;gamma; monoclonal antibody</td><td>BD Biosciences</td><td>554550</td><td>4SB3 clone.</td></tr><tr><td>Streptavidin conjugated with alkaline phosphatase&amp;nbsp;</td><td>Bio-Rad Life Science</td><td>170-3554</td><td>Dilute for use on the same day.</td></tr><tr><td>BCIP/NBT</td><td>Bio-Rad Life Science</td><td>170-6432</td><td>Protect from light.</td></tr></tbody></table>

development of an ifn-&#947; elispot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation

水痘 - 带状疱疹病毒（VZV）是发病率和死亡率以下脐血移植（UCBT）的显著原因。出于这个原因，抗疱疹预防系统地施用给小儿脐血移植​​接受者，以防止水痘病毒感染相关的并发症，但没有强大的，基于证据的共识，它定义了最佳治疗时间。因为T细胞介导的​​免疫是负责水痘感染的控制，评估的VZV特异性T细胞反应的重建下列UCBT可以表示出对预防是否应保持或可停药。为此，一个特定的VZVγ干扰素（IFN-γ）酶联免疫斑点（ELISPOT）测定法的开发是由T淋巴细胞响应于体外刺激与照射减毒活水痘疫苗来表征的IFN-γ的产生。此法提供了水痘特定的C的快速，重复性好，灵敏的测量埃尔介导的免疫适于监测VZV特异性免疫的重构在临床环境和评估免疫反应对VZV抗原。

上面详述的IFN-γ酶联免疫斑点协议被开发，并在我们的实验室优化测量的幅度和针对VZV 4细胞介导的免疫应答的质量。可用于刺激步骤的VZV抗原的不同来源。这些措施包括:a）由水痘市售清洁剂灭活提取物感染的Vero细胞18;从具体的VZV编码的蛋白质，包括IE63 15和19的ORF4重叠合成肽二）池;三）减毒活水痘带状疱疹疫苗20;及d）VZV紫外线灭活抗原准备从破碎的VZV?...

Watch this Scientific Journal Video about Development of an IFN-ÃŽÂ³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation at Jo

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

新世代的功能测定法如γ干扰素（IFN-γ）的酶联免疫斑点法，其检测细胞因子的产生在单细胞水平，并提供T细胞应答的定量和定性特征，可用于评估细胞介导的​​针对水痘 - 带状疱疹免疫应答病毒（VZV）。

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

development-an-ifn-elispot-assay-to-assess-varicella-zoster-virus

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  Université de Montréal. 新世代的功能测定法如γ干扰素（IFN-γ）的酶联免疫斑点法，其检测细胞因子的产生在单细胞水平，并提供T细胞应答的定量和定性特征，可用于评估细胞介导的​​针对水痘 - 带状疱疹免疫应答病毒（VZV）。 

视频: Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

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 ...

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 ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

<meta charset="utf8"></head><body>用于分析真菌和病毒免疫的优化全血免疫检测

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

<meta charset="utf8"></head><body>作者聚焦:使用全血检测推进重症监护患者的免疫监测

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...