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
Summary
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.
Abstract
体液免疫的特点是，以产生功能性的ASC，其合成和分泌特异性抗原（银），ABS等的病原体，并用于宿主防御。用于定量测定的个体的体液免疫应答的功能状态，既血清ABS和循环的ASC通常为功能读数测定。在人类中，外周血是可用于由主机B细胞引起的体液免疫应答的确定最方便和最容易获得的样本。不同B细胞亚群，包括的ASC，可直接从外周血通过选择与谱系特异性抗体共轭微珠或通过细胞仪具有流量分选分离。此外，纯化的幼稚和记忆B细胞可被激活并分化成在培养的ASCs。的ASCs促进抗体分泌的功能活性可以通过ELISPOT进行量化，这是收敛酶的测定法- 连接immunoabsorbance吸附试验（ELISA）和Western印迹技术，使个人的ASC的枚举在单细胞水平。在实践中，ELISPOT测定已越来越多地用于评估，因为易于大量血液样品的处理的疫苗效力。从外周血中分离的人类B细胞的方法中，B细胞的分化为携带者，和酶联免疫斑点的就业总IgM-和IgG-的ASC的定量将在这里描述。
Introduction
B细胞发挥体液免疫的发展中发挥中心作用。他们最初开发在骨髓和进入血液流中作为幼稚B细胞，其可以迁移到淋巴组织，诸如脾，淋巴结，扁桃体，为进一步发展。在银相遇，一些幼稚B细胞迁移到淋巴滤泡，其中生发中心B细胞可以分化成记忆B细胞和plasmablasts（PBS）/浆细胞（PC）的。虽然大多数PBS / PC的出口进入血流，少数最终驻留在骨髓经受终端分化为长寿命的计算机1。 B细胞在循环是异构的，并且在稳定状态下，PBS / PC是外周血2罕见。作为谱系特异性表面标志物的可用性的结果，流式细胞术已经成为外周血B细胞亚群的鉴定和表征的常用方法。流动cytometr的扩展应用y是在加入细胞分选仪的功能，其允许分离与B细胞的个别亚群的分离具有高纯度的。基于人类循环B细胞一般分为三个主要亚群特异性表面受体在不同的发育阶段的表达：幼稚B细胞（CD19 + CD27 – CD38 – ），记忆B细胞（CD19 + CD27 + CD38 – ）和PBS /电脑（CD19 + CD27 + CD38 +）3-4（ 图1）。普通B细胞的性质还没有遇到过AGS。然而，它们可以分化成的IgM + CD27 +记忆B细胞。虽然幼稚B细胞在表达B细胞抗原受体（BCR）相关分子（ 例如，CD19，CD20和CD22），他们在他们的免疫球蛋白库5异构均匀。大多数CD27 +记忆B细胞可以分化成CD27+ / HI CD38 + PBS /电脑6。此外，记忆B细胞和PBS / PC是多克隆并表现出发育和功能的异质性4-7。流通中的PB / PC通常短暂的，不表达CD138，但那些由落户骨髓将最终分化，成为长寿。终末分化的PC表达CD138，下调其表面8 CD27分子。因为两者的PB和PC是能够分泌腹肌的，在许多情况下，他们被共同表示为携带者。相比之下，无论是普通B细胞，也没有记忆B细胞能产生相当数量的绝对9-10。放置在适当的培养条件下6，11-15时10日-然而，分离时，两者幼稚和记忆B细胞可以分化成的ASCs 3。事实上，从携带者和CD27 CD38 在体外分化有着相似的表面衍生的表达与那些直接分离FROM外周血6。此外， 在体外分化的携带者表达CD20表面的较低水平，这同样循环PBS /个人电脑6。虽然培养衍生的ASCs都是短暂的，它们能分泌腹肌，这表明它们在功能上是胜任和能够向体液免疫。
既ELISA和酶联免疫斑点是目前最常用的方法，用以获得关于体液免疫应答功能信息。 ELISA是一个96孔基于板的测定法，它经常用于测量血清抗原特异性ABS和其他分析物（ 例如，细胞因子）的效价。这是方便和可扩展性。 ELISA被设计为使用固相酶测定来检测ABS或其它物质，如血清存在下，液体样品16英寸从血清的ELISA所述读数已被广泛地用于表示身体的免疫反应。必要收购重的工具从ELISA检测adouts是酶标仪。读者可以决定最终产品通常由辣根过氧化物酶（HRP）缀合的检测ABS和其特定的底物17的反应所得的光密度（OD）。对于报告的体液免疫应答，通过ELISA测定血清抗体水平表示在身体的ASC的集体，但不是个别，性能。此外，酶联免疫吸附未能顾及由记忆B细胞，其不分泌腹肌的参与。
像酶联免疫吸附，酶联免疫斑点是用于检测和监测外周血样品17-18中免疫应答的广泛使用的方法。酶联免疫斑点是涉及夹心ELISA的技术。在它，将细胞放置到聚偏二氟乙烯（PVDF）的96孔膜支持孔微孔板用于短期培养。 ELISPOT试验类似于上微孔板和developin进行免疫印迹克，以每孔中的PVDF膜的斑点。一种自动化的ELISpot读取器系统或人工计数立体显微镜是必需的。酶联免疫斑点的检测的免疫应答的主要优点是在携带者和细胞因子分泌细胞的定量其极好的灵敏度。它分别报告它们的功能活动，体液免疫和细胞免疫。在体液免疫功能的测定中，通过ELISA和通过ELISPOT列举的ASC的数量来确定血清抗体水平通常相关，但来自这两个测定法中的数据读出有功能的影响19-20一些差异。酶联免疫斑点的主要优点是它的方法的灵敏度。血清抗体滴度通过ELISA报告的水平呈现半定量作为OD读数，表示相对抗体水平，或更定量地，作为集中读数当包括参考腹肌的适当的同种型的已知量。与此相反，酶联免疫斑点的结果是presente-d作为携带者的在感兴趣的细胞池的绝对数量（ 例如，未分级的外周血单核细胞（PBMC），并从PBMC中纯化的B细胞）。酶联免疫斑点法可以检测单个ASC，但ELISA需要来自的ASCs抗体达之前测量达到优化的测定依赖浓度。因此，酶联免疫斑点是定量的灵敏度明显优于ELISA。此外，酶联免疫斑点法也适用于量化从激活记忆B细胞的体外分化的携带者。记忆B细胞不分泌腹肌但能分化成在激活时的ASC;于是他们就用ELISA法检测血清中绝对没有任何贡献。因此，酶联免疫斑点是选择在培养活化后循环记忆B细胞的免疫应答的测定方法。它允许对维持长期体液免疫的监测。
Protocol
Representative Results
Discussion
分离和人外周血B细胞的分离纯化
通常情况下，红细胞可以有效地破裂并通过裂解缓冲液（步骤1.2）清零。它不孵育用RBC裂解缓冲超过5分钟长的PBMC是重要的，因为细胞生存力可能由氯化铵的影响。可替代地，RBC和血小板可以同时通过以下方案除去。
1，血液到缓冲液：在9:1的体积比;混合新鲜全血酸 – 柠檬酸盐 – 葡萄糖（ACD）缓冲液（pH 7.4 39 mM柠檬酸，75mM柠…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study was supported by a research grant from the Ministry of Science and Technology of the Executive Yuan of Taiwan (NSC99-2320-B-002-011). I would like to acknowledge the excellent service provided by the Flow Cytometric Analyzing and Sorting Core of the First Core Laboratory in College of Medicine of National Taiwan University.
Materials
| BD Vacutainer K2E | BD Biosciences | 367525 | 10 ml tube |
| Ficoll-Paque Plus | GE Healthcare | 17-1440-02 | endotoxin-free |
| Trypan blue 0.5% solution | Biological Industries | 03-102-1B | |
| IMag Human B lymphocyte enrichment set | BD Biosciences | 558007 | |
| Biotinylated CD27 mAb | Biolegend | 302804 | clone O323 |
| Streptavidin magnetic microbeads | BD Biosciences | 9000810 | |
| 15 ml Falcon tubes | BD Falcon | 352196 | |
| Blue nylon mesh cell strainer, 40 μm | BD Falcon | 352340 | |
| Anti-human CD19-APC | Biolegend | 302212 | clone HIB19 |
| Anti-human CD27-eFluor 450 | eBioscience | 48-0279-42 | clone O323 |
| Anti-human CD38-PE-Cy7 | Biolegend | 303516 | clone HIT2 |
| Anti-human CD38-PE-Cy7 | BD Biosciences | 560677 | clone HIT2 |
| Anti-human CD45-FITC | Biolegend | 304006 | clone HI30 |
| Anti-human CD45-FITC | BD Biosciences | 555482 | clone HI30 |
| Anti-mouse/rat/human CD27-PerCP Cy5.5 | Biolegend | 124213 | clone LG.3A10 |
| Anti-human CD27-PerCP Cy5.5 | BD Biosciences | 65429 | clone L128 |
| Anti-human CD19-FITC | Miltenyi Biotec | 130-098-064 | clone LT19 |
| Anti-human CD19-FITC | GeneTex | GTX75599 | clone LT19 |
| Anti-human CD20-FITC | BD Biosciences | 555622 | clone 2H7 |
| biotinylated anti-human CD27 | Biolegend | 302804 | clone O323 |
| biotinylated anti-human CD27 | eBioscience | 13-0279-80 | clone O323 |
| 7-aminoactinomycin D (7-AAD) | BD Biosciences | 559925 | |
| CpG (ODN 2006) | InvivoGen | tlrl-2006 | type B CpG |
| Recombinant human IL-2 | PeproTech | 200-02 | |
| Recombinant human IL-10 | PeproTech | 200-10 | |
| Recombinant human IL-21 | PeproTech | 200-21 | |
| Recombinant human sCD40L | PeproTech | 310-02 | |
| Protein A of S. aureus Cowan (SAC) | Sigma-Aldrich | 82526 | |
| Pokeweed mitogen (PWM) | Sigma-Aldrich | L9379 | |
| MultiScreen filter plates, 0.45 µm pore size | Merck Millipore | MSIPS4510 | sterile, clear 96-well filter plate with hydrophobic PVDF membrane |
| BCIP/NBT solution | Sigma-Aldrich | B6404 | |
| BCIP/NBT single reagent, alkaline phosphatase substrate | Merck Millipore | ES006 | |
| Human IgG | Jackson ImmunoResearch | 009-000-003 | |
| Human IgG, Fc fragment | Jackson ImmunoResearch | 009-000-008 | |
| F(ab')2 fragment of goat anti-human Ig (IgG+IgM+IgA) | Jackson ImmunoResearch | 109-006-127 | |
| Goat anti-human IgG-alkaline phosphatase, Fcγ fragment specific | Jackson ImmunoResearch | 109-055-008 | |
| Goat anti-human IgM-alkaline phosphatase, Fcµ fragment specific | Jackson ImmunoResearch | 109-055-095 | |
| Goat anti-human IgG-peroxidase, Fcγ fragment specific | Jackson ImmunoResearch | 109-035-008 | |
| Goat anti-human IgM-peroxidase, Fcµ fragment specific | Jackson ImmunoResearch | 109-035-095 | |
| BD ELISPOT AEC substrate kit | BD Biosciences | 551951 | |
| C.T.L. ImmunoSpot analyzer | C.T.L. |
Riferimenti
- Bemark, M. Translating transitions – how to decipher peripheral human B cell development. J. Biomed. Res. 29 (4), 264-284 (2015).
- Odendahl, M., et al. Generation of migratory antigen-specific plasmablasts and mobilization of resident plasma cells in a secondary immune response. Blood. 105 (4), 1614-1621 (2005).
- Jackson, S. M., Wilson, P. C., James, J. A., Capra, J. D. Human B cell subsets. Adv. Immunol. 98, 151-224 (2008).
- Sanz, I., Wei, C., Lee, F. E., Anolik, J. Phenotypic and functional heterogeneity of human memory B cells. Semin. Immunol. 20 (1), 67-82 (2008).
- Perez-Andres, M., et al. Human peripheral blood B-cell compartments: A crossroad in B-cell traffic. Cytometry B Clin. Cytom. 78 (Suppl 1), S47-S60 (2010).
- Huggins, J., et al. CpG DNA activation and plasma-cell differentiation of CD27- naïve human B cells. Blood. 109 (4), 1611-1619 (2007).
- Wu, Y. C., Kipling, D., Dunn-Walters, D. K. The relationship between CD27 negative and positive B cell populations in human peripheral blood. Front. Immunol. 2, 81 (2011).
- Maïga, R. I., Bonnaure, G., Rochette, J. T., Néron, S. Human CD38hiCD138⁺ plasma cells can be generated in vitro from CD40-activated switched-memory B lymphocytes. J. Immunol. Res. 2014, 635108 (2014).
- Wienands, J., Engels, N. The memory function of the B cell antigen receptor. Curr. Top. Microbiol. Immunol. 393, 107-121 (2016).
- Kurosaki, T., Kometani, K., Ise, W. Memory B cells. Nat. Rev. Immunol. 15 (3), 149-159 (2015).
- Tzeng, S. J., Li, W. Y., Wang, H. Y. FcγRIIB mediates antigen-independent inhibition on human B lymphocytes through Btk and p38 MAPK . J. Biomed. Sci. 22, 87-98 (2015).
- Ettinger, R., et al. IL-21 induces differentiation of human naïve and memory B cells into antibody-secreting plasma cells. J. Immunol. 175 (12), 7867-7879 (2005).
- Bekeredjian-Ding, I., Foermer, S., Kirschning, C. J., Parcina, M., Heeg, K. Poke weed mitogen requires Toll-like receptor ligands for proliferative activity in human and murine B lymphocytes. PLoS One. 7 (1), e29806 (2012).
- Bernasconi, N. L., Traggiai, E., Lanzavecchia, A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science. 298 (5601), 2199-2202 (2002).
- Defrance, T., Vanbervliet, B., Brière, F., Durand, I., Rousset, F., Banchereau, J. Interleukin 10 and transforming growth factor beta cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. Med. 175 (3), 671-682 (1992).
- Hornbeck, P., Fleisher, T. A., Papadopoulos, N. M., Coligan, J. E. Chapter 2, Unit 2.2, Isotype determination of antibodies. Current Protocols in Immunology. , (2001).
- Tanguay, S., Killion, J. J. Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine Cytokine Res. 13 (4), 259-263 (1994).
- Crotty, S., Aubert, R. D., Glidewell, J., Ahmed, R. Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J. Immunol. Methods. 286 (1-2), 111-122 (2004).
- Zhang, Y., Wang, Y., Zhang, M., Liu, L., Mbawuike, I. N. Restoration of retarded influenza virus-specific immunoglobulin class switch in aged mice. J. Clin. Cell. Immunol. 7 (2), 403 (2016).
- Doedée, A. M., Kannegieter, N., Öztürk, K., van Loveren, H., Janssen, R., Buisman, A. M. Higher numbers of memory B-cells and Th2-cytokine skewing in high responders to hepatitis B vaccination. Vaccine. 34 (19), 2281-2289 (2016).
- Heine, G., Sims, G. P., Worm, M., Lipsky, P. E., Radbruch, A., Coligan, J. E. Chapter 7, Unit 7.5, Isolation of human B cell populations. Current Protocols in Immunology. , (2011).
- Safarík, I., Safaríková, M. Use of magnetic techniques for the isolation of cells. J. Chromatogr. B Biomed. Sci. Appl. 722 (1-2), 33-53 (1999).
- Morbach, H., Eichhorn, E. M., Liesem, J. G., Girschickm, H. J. Reference values for B cell subpopulations from infancy to adulthood. Clin. Exp. Immunol. 162 (2), 271-279 (2010).
- Thornton, A. M., et al., Coligan, J. E., et al. Chapter 3, Unit 3.5A, Fractionation of T and B cells using magnetic beads. Current Protocols in Immunology. , (2003).
- Klein, U., Rajewsky, K., Küppers, R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated memory)Bcells. J.Exp. Med. 188 (9), 1679-1689 (1998).
- Bohnhorst, J. &. #. 2. 1. 6. ;., Bjørgan, M. B., Thoen, J. E., Natvig, J. B., Thompson, K. M. Bm1-Bm5 classification of peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and disturbance in the B cell subpopulations in patients with primary Sjögren’s syndrome. J. Immunol. 167 (7), 3610-3618 (2001).
- Bleesing, J. J., et al., Coligan, J. E., et al. Chapter 7, Unit 7.35, Assays for B cell and germinal center development. Current Protocols in Immunology. , (2003).
- Horvatinovich, J. M., Sparks, S. D., Mann, K. P. Establishing a pure lymphocyte gate for subset analysis by flow cytometry. Cytometry. 26 (2), 172-177 (1996).
- Ruprecht, C. R., Lanzavecchia, A. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur. J. Immunol. 36 (4), 810-816 (2006).
- Smith, K., et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat. Protoc. 4 (3), 372-384 (2009).
- Caraux, A., et al. Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138- and CD138+ plasma cells. Haematologica. 95 (6), 1016-1020 (2010).
- Kuchen, S., Robbins, R., Sims, G. P., Sheng, C., Phillips, T. M., Lipsky, P. E., Ettinger, R. Essential role of IL-21 in B cell activation, expansion, and plasma cell generation during CD4+ T cell-B cell collaboration. J. Immunol. 179 (9), 5886-5896 (2007).
- Pinna, D., Corti, D., Jarrossay, D., Sallusto, F., Lanzavecchia, A. Clonal dissection of the human memory B-cell repertoire following infection and vaccination. Eur. J. Immunol. 39 (5), 1260-1270 (2009).
- Jahnmatz, M., et al. Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses. J. Immunol. Methods. 391 (1-2), 50-59 (2013).
- Weiss, G. E., et al. High efficiency human memory B cell assay and its application to studying Plasmodium falciparum-specific memory B cells in natural infections. J. Immunol. Methods. 375 (1-2), 68-74 (2012).
- Leehan, K. M., Koelsch, K. A. T Cell ELISPOT: For the identification of specific cytokine-secreting T cells. Methods. Mol. Biol. 1312, 427-434 (2015).
- Karahan, G. E., et al. Quantification of HLA class II-specific memory B cells in HLA-sensitized individuals. Hum. Immunol. 76 (2-3), 129-136 (2015).
- Hadjilaou, A., Green, A. M., Coloma, J., Harris, E. Single-cell analysis of B cell/antibody cross-reactivity using a novel multicolor FluoroSpot assay. J. Immunol. 195 (7), 3490-3496 (2015).
- Janetzki, S., Rueger, M., Dillenbeck, T. Stepping up ELISpot: multi-level analysis in FluoroSpot assays. Cells. 3 (4), 1102-1115 (2014).
- Alatrakchi, N., Graham, C. S., He, Q., Sherman, K. E., Koziel, M. J. CD8+ cell responses to hepatitis C virus (HCV) in the liver of persons with HCV-HIV coinfection versus HCV monoinfection. J. Infect. Dis. 191 (5), 702-709 (2005).
- Smith, S. G., et al. Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis. PLoS. One. 4 (11), e7972 (2009).
- Sundararaman, S., et al. High reproducibility of ELISPOT counts from nine different laboratories. Cells. 4 (1), 21-39 (2015).
- Janetzki, S., et al. Guidelines for the automated evaluation of Elispot assays. Nat. Protoc. 10 (7), 1098-1115 (2015).
