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
1Unité d'Immunopathologie Virale, Centre de Recherche du CHU Sainte-Justine, Department of Microbiology, Infectiology & Immunology, Faculty of Medicine,Université de Montréal, 2Infectious Diseases Service, CHU Sainte-Justine, Faculty of Medicine,Université de Montréal, 3Department of Paediatrics,Université de Montréal
Summary
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）。
Abstract
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。
Introduction
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。
Protocol
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，涂料的板通过加入到每个孔中20微升35％乙醇中1分钟，透化聚偏…
Representative Results
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VZ…
Discussion
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="j…Disclosures
The authors have nothing to disclose.
Acknowledgements
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'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。
Materials
| Leucocep tube | VWR | 89048-936/89048-932 | 12 ml or 50 ml tubes may be used depending on the volume of blood. |
| Ficoll-Paque | GE Healthcare | 17-1440-02 | Protect from light. |
| Benzonase nuclease | Novagen | 70746-3 | Keep at -20 C. |
| MultiScreenHTS-IP Filter Plate | Millipore | MSIPS4W10 | Sterile with pore size of 0.45 µm. |
| Mouse anti-human IFN-γ capture antibody | BD Biosciences | 551221 | NIB42 clone. |
| Pepmix VZV IE63 | JPT Peptide Technologies | PM-VZV-IE63 | Dissolve contents of one vial in 40 μL of DMSO. Use within 6 months. |
| Biotin-conjugated anti-IFN-γ monoclonal antibody | BD Biosciences | 554550 | 4SB3 clone. |
| Streptavidin conjugated with alkaline phosphatase | Bio-Rad Life Science | 170-3554 | Dilute for use on the same day. |
| BCIP/NBT | Bio-Rad Life Science | 170-6432 | Protect from light. |
References
- Ballen, K. K., et al. Umbilical cord blood transplantation: the first 25 years and beyond. Blood. 122 (4), 491-498 (2013).
- Vandenbosch, K., et al. Varicella-zoster virus disease is more frequent after cord blood than after bone marrow transplantation. Biol. Blood Marrow Transplant. 14 (8), 867-871 (2008).
- Barker, J. N., et al. Serious infections after unrelated donor transplantation in 136 children: impact of stem cell source. Biol. Blood Marrow Transplant. 11 (5), 362-370 (2005).
- Merindol, N., et al. Reconstitution of protective immune responses against cytomegalovirus and varicella zoster virus does not require disease development in pediatric recipients of umbilical cord blood transplantation. J. Immunol. 189 (10), 5016-5028 (2012).
- Feldman, S., et al. Varicella in children with cancer: Seventy-seven cases. Pediatrics. 56 (3), 388-397 (1975).
- Arvin, A. M. Varicella-Zoster virus: pathogenesis, immunity, and clinical management in hematopoietic cell transplant recipients. Biol. Blood Marrow Transplant. 6 (3), 219-230 (2000).
- Wiegering, V., et al. Varicella-zoster virus infections in immunocompromised patients – a single centre 6-years analysis. BMC Pediatr. 11, 31 (2011).
- Boeckh, M., et al. Long-term acyclovir for prevention of varicella zoster virus disease after allogeneic hematopoietic cell transplantation–a randomized double-blind placebo-controlled study. Blood. 107 (5), 1800-1805 (2006).
- Boeckh, M. Prevention of VZV infection in immunosuppressed patients using antiviral agents. Herpes. 13 (3), 60-65 (2006).
- Tomblyn, M., et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol. Blood Marrow Transplant. 15 (10), 1143-1238 (2009).
- Ljungman, P., et al. Long-term acyclovir prophylaxis in bone marrow transplant recipients and lymphocyte proliferation responses to herpes virus antigens in vitro. Bone Marrow Transplant. 1 (2), 185-192 (1986).
- Selby, P. J., et al. The prophylactic role of intravenous and long-term oral acyclovir after allogeneic bone marrow transplantation. Br. J. Cancer. 59 (3), 434-438 (1989).
- Distler, E., et al. Recovery of varicella-zoster virus-specific T cell immunity after T cell-depleted allogeneic transplantation requires symptomatic virus reactivation. Biol. Blood Marrow Transplant. 14 (12), 1417-1424 (2008).
- Levin, M. J., et al. Decline in varicella-zoster virus (VZV)-specific cell-mediated immunity with increasing age and boosting with a high-dose VZV vaccine. J. Infect. Dis. 188 (9), 1336-1344 (2003).
- Jones, L., et al. Phenotypic analysis of human CD4+ T cells specific for immediate-early 63 protein of varicella-zoster virus. Eur. J. Immunol. 37 (12), 3393-3403 (2007).
- Czerkinsky, C., et al. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J. Immunol. Methods. 110 (1), 29-36 (1988).
- Hutchings, P. R., et al. The detection and enumeration of cytokine-secreting cells in mice and man and the clinical application of these assays. J. Immunol. Methods. 120 (1), 1-8 (1989).
- De Castro, N., et al. Varicella-zoster virus-specific cell-mediated immune responses in HIV-infected adults. AIDS Res. Hum. Retroviruses. 27 (10), 1089-1097 (2011).
- Jones, L., et al. Persistent high frequencies of varicella-zoster virus ORF4 protein-specific CD4+ T cells after primary infection. J. Virol. 80 (19), 9772-9778 (2006).
- Malavige, G. N., et al. Viral load, clinical disease severity and cellular immune responses in primary varicella zoster virus infection in Sri Lanka. PLoS One. 3 (11), (2008).
- Sadaoka, K., et al. Measurement of varicella-zoster virus (VZV)-specific cell-mediated immunity: comparison between VZV skin test and interferon-gamma enzyme-linked immunospot assay. J. Infect. Dis. 198 (9), 1327-1333 (2008).
- Smith, J. G., et al. Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin. Diagn. Lab. Immunol. 8 (5), 871-879 (2001).
- Ouwendijk, W. J., et al. T-cell immunity to human alphaherpesviruses. Curr. Opin. Virol. 3 (4), 452-460 (2013).
- Rowland-Jones, S. L., et al. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J. Clin. Invest. 102 (9), 1758-1765 (1998).
- Alter, G., et al. Human immunodeficiency virus (HIV)-specific effector CD8 T cell activity in patients with primary HIV infection. J. Infect. Dis. 185 (6), 755-765 (2002).
- Lechner, F., et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 191 (9), 1499-1512 (2000).
- Fournillier, A., et al. A heterologous prime/boost vaccination strategy enhances the immunogenicity of therapeutic vaccines for hepatitis C virus. J. Infect. Dis. 208 (6), 1008-1019 (2013).
- Adetifa, I. M., et al. Interferon-γ ELISPOT as a biomarker of treatment efficacy in latent tuberculosis infection: a clinical trial. Am. J. Respir. Crit. Care Med. 187 (4), 439-445 (2013).
- Lalvani, A., Pareek, M. A 100 year update on diagnosis of tuberculosis infection. Br. Med. Bull. 93, 69-84 (2010).
- Berger, R., et al. A dose-response study of a live attenuated varicella-zoster virus (Oka strain) vaccine administered to adults 55 years of age and older. J. Infect. Dis. 178 Suppl. 1, (1998).
- Trannoy, E., et al. Vaccination of immunocompetent elderly subjects with a live attenuated Oka strain of varicella zoster virus: a randomized, controlled, dose-response trial. Vaccine. 18 (16), 1700-1706 (2000).
- Brunner, K. T., et al. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology. 14 (2), 181-196 (1968).
- Moretta, A., et al. Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations. J. Exp. Med. 158 (2), 571-585 (1983).
- Jung, T., et al. Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods. 159 (1-2), 197-207 (1993).
- Maecker, H. T., et al. Standardization of cytokine flow cytometry assays. BMC Immunol. 6, 13 (2005).
- Nomura, L., et al. Standardization and optimization of multiparameter intracellular cytokine staining. Cytometry A. 73 (11), 984-991 (2008).
- Letsch, A., Scheibenbogen, C. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods. 31 (2), 143-149 (2003).
- Merindol, N., et al. Umbilical cord blood T cells respond against the Melan-A/MART-1 tumor antigen and exhibit reduced alloreactivity as compared with adult blood-derived T cells. J. Immunol. 185 (2), 856-866 (2010).
- Altman, J. D., et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 274 (5284), 94-96 (1996).
- Scriba, T. J., et al. Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining. J. Immunol. 175 (10), 6334-6343 (2005).
- Stone, J. D., et al. Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs. J. Immunol. 187 (12), 6281-6290 (2011).
- Pantaleo, G., et al. Evidence for rapid disappearance of initially expanded HIV-specific CD8+ T cell clones during primary HIV infection. Proc. Natl Acad. Sci. USA. 94 (18), 9848-9853 (1997).
- Wherry, E. J. T cell exhaustion. Nat. Immunol. 12 (6), 492-499 (2011).
- Boulet, S., et al. A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses. J. Immunol. Methods. 320 (1-2), 18-29 (2007).
- Ahlborg, N., Axelsson, B. Dual- and triple-color fluorospot. Methods Mol. Biol. 792, 77-85 (2012).
- Precopio, M. L., et al. Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8(+) T cell responses. J. Exp. Med. 204 (6), 405-1416 (2007).
- Sadzot-Delvaux, C., et al. Recognition of the latency-associated immediate early protein IE63 of varicella-zoster virus by human memory T lymphocytes. J. Immunol. 159 (6), 2802-2806 (1997).
- Malavige, G. N., et al. IE63-specific T-cell responses associate with control of subclinical varicella zoster virus reactivation in individuals with malignancies. Br. J. Cancer. 102 (4), 727-730 (2010).
Cite This Article
Salem Fourati, I., Grenier, A., Jolette, É., Merindol, N., Ovetchkine, P., Soudeyns, H. Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation. J. Vis. Exp. (89), e51643, doi:10.3791/51643 (2014).