性T细胞Transinfection从树突状细胞捕捉细菌
Summary
这里的协议，提出通过该期间抗原呈递发生经由transinfection从预感染树突状细胞（DC）的CD4 + T细胞以测量细菌捕获。我们说明如何执行必要的步骤：原代细胞中，DC，DC / T细胞共轭形成感染和细菌的T细胞转染的测量隔离。
Abstract
Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.
Introduction
当病原体感染其宿主，通常的先天和适应性免疫反应所必需的细菌清除的活化。先天性免疫是防御的第一道防线，防止大多数感染。先天免疫区分以精确的方式元件被间微生物大类保守的（病原体相关分子模式）1。先天免疫的机制包括物理障碍如皮肤，化学品的障碍（抗菌肽，溶菌酶）和先天的白细胞，包括吞噬细胞（巨噬细胞，中性粒细胞和树突状细胞），肥大细胞，嗜酸性粒细胞，嗜碱性粒细胞，和天然杀伤细胞2。这些细胞识别和消灭病原体，无论是通过接触或通过细胞吞噬功能，其中包括病原体吞噬和杀害攻击他们。该系统不允许终身防守，而相比之下，适应性免疫，从而赋予针对p免疫记忆athogens。适应性免疫系统是防御的第二行，并且能够识别和反应以多种微生物和非微生物物质3的特定抗原。适应性免疫系统的主要组成部分是淋巴细胞，它包括B和T细胞。 B细胞参与了体液应答，分泌抗病原体或外源蛋白质。然而，T细胞代表了细胞介导的免疫力，调节细胞因子分泌或杀死病原体感染的细胞4的免疫应答。
抗原呈递细胞（APC），包括树突状细胞或巨噬细胞，先天免疫系统的组分，可以识别吞噬病原体和过程细菌成分成抗原，其由主要组织相容性复合物（MHC）5-7呈现在细胞表面上。后的APC已吞噬的病原体，它们通常迁移到引流淋巴结，在那里它们以T相互作用细胞。 T淋巴细胞可以通过它们的T细胞受体识别特异性的肽-MHC复合物。免疫突触（IS）发生在负载抗原的APC和淋巴细胞之间的界面中的抗原呈递8,9。有些细菌可以生存的吞噬功能，并在装甲运兵车系统传播。这种观点认为，受感染的APC作为细菌的水库或“特洛伊木马”，以促进细菌的蔓延10。装甲运兵车和采取的是在形成过程中发生淋巴细胞之间的亲密接触也可作为一个平台，交换膜，遗传物质和外来体的一部分，可以被劫持用于某些病毒感染的T细胞;这个过程被称为transinfection 11-13
一些病原性细菌（ 李斯特菌， 沙门氏菌和志贺氏菌 ）能够侵入T淋巴细胞在体内和修改其行为14-16。我们有最近描述了T淋巴细胞也能抗原呈递16的过程中捕获的细菌通过从以前感染树突细胞（DC）transinfection。 T细胞细菌捕获由transinfection极其比直接感染更有效（1,000-4,000x）。 T细胞捕获病原体和非病原菌显示比transinfection是由T细胞驱动的过程。引人注目的是，transinfected T（TIT）细胞迅速杀死细菌捕获和这样做的效率比专业的吞噬细胞16。这些结果，这打破免疫学的教条，表明适应性免疫的细胞可以执行是假想排他性的先天免疫的功能。此外，我们发现，乳头细胞分泌大量的促炎细胞因子和体内的细菌感染保护。
这里，我们提出用于研究细菌transinfection过程中的不同协议小鼠模型。该模型是基于使用CD4 + T细胞的转基因小鼠OTII，其中承担特异性的TCR的OVA（OVAp）的肽323-339中的I-17抗体的相互作用是特异性地与细菌感染的骨的上下文marrow-衍生的DC（DC培养上清）18,19装有OVAp，形成稳定的免疫突触。
T细胞transinfection可以可视化，并使用荧光显微镜进行跟踪。此外，流式细胞仪可用于通过取由细菌表达绿色荧光蛋白（GFP）16,20发出的荧光的优点检测感染的细胞。此外，T细胞transinfection可以通过更灵敏的方法，庆大霉素存活测定法，允许大量事件的测量来定量。庆大霉素是一种不能渗透的真核细胞的抗生素。因此，使用这种抗生素可以胞内细菌的分化幸存抗生素除了FROM外那些被打死21。
Protocol
Representative Results
Discussion
T细胞或T淋巴细胞是一种类型的白细胞中发挥在细胞介导的免疫中发挥中心作用，并属于适应性免疫反应26。 T细胞是难治被感染在体外 ，但一些报道表明，它们可以感染体内 14,15。 APC和T细胞的过程中免疫突触的紧密接触作为交换生物材料 13，包括某些病毒如HIV 11的平台。据最近表明，违背了教条，T细胞，适应性免疫细胞的范例，也能够有效地捕获…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cientìficas (CSIC).
Materials
| RPMI | Fisher Scientific | SH3025501 | |
| r-GMCSF | Peprotech | 315-03 | |
| LPS | SIGMA | L2630-10mg | |
| Na Pyruvate | Thermo Scientific | SH3023901 | |
| 2-ME | Gibco | 31350-010 | |
| OVAp OTII (323–339) | GenScript | — | |
| Cell Strainer 70uM | BD | 352350 | |
| 30uM Syringe Filcons Sterile | BD | 340598 | |
| AutoMacs Classic | Miltenyi Biotec | 130-088-887 | |
| Gentamicin | Normon | 624601.6 | |
| Transwell | Costar | 3415 | |
| LB | Pronadisa | 1231 | |
| Agar | Pronadisa | 1800 | |
| Paraformaldehyde 16% | Electron Microscopy Sciences | 15710 | |
| Triton X-100 | |||
| CD8 biot | BD Biosciences | 553029 | |
| IgM Biot | ImmunoStep | Clone RMM-1 | |
| B220 Biot | BD Biosciences | 553086 | |
| CD19 biot | BD Biosciences | 553784 | |
| MHC-II Biot (I-A/I-E) | BD Biosciences | 553622 | |
| CD11b biot | Immunostep | 11BB-01mg | |
| CD11c biot | Immunostep | 11CB3-01mg | |
| DX5 biot | BD Biosciences | 553856 | |
| Gr-1 biot | BD Biosciences | 553125 | |
| CD16/CD32 | ImmunoStep | M16PU-05MG | |
| anti Salmonella | ABD Serotec | 8209-4006 | |
| CD11cPE | BD Biosciences | 553802 | |
| CD4-APC | Tonbo Biosciences | 20-0041-U100 | |
| Gr-1 APC | BD Biosciences | 553129 | |
| MHC-II (I-A/I-E) FITC | BD Biosciences | 553623 | |
| Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed | Invitrogen | A-21245 | |
| CMAC (7-amino-4-chloromethylcoumarin) | Life technologies | C2110 | |
| BSA | SIGMA | A7030-100G | |
| Streptavidin MicroBeads | Miltenyi Biotec | 130-048-101 | |
| BD FACSCanto II | BD Biosciences | — |
Riferimenti
- Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature. 449 (7164), 819-826 (2007).
- Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. . Molecular biology of the cell. , (1989).
- Pancer, Z., Cooper, M. D. The evolution of adaptive immunity. Annual Review of Immunology. 24, 497-518 (2006).
- Rhoades, R. A., Bell, D. R. . Medical Phisiology. , (2012).
- Cossart, P. Bacterial Invasion: The Paradigms of Enteroinvasive Pathogens. Science. 304 (5668), 242-248 (2004).
- Kaufmann, S. H., Schaible, U. E. Antigen presentation and recognition in bacterial infections. Current Opinion in Immunology. 17 (1), 79-87 (2005).
- Pizarro-Cerdá, J., Cossart, P. Bacterial Adhesion and Entry into Host Cells. Cell. 124 (4), 715-727 (2006).
- Dustin, M. L. T-cell activation through immunological synapses and kinapses. Immunological Reviews. 221, 77-89 (2008).
- Calabia-Linares, C., Robles-Valero, J., et al. Endosomal clathrin drives actin accumulation at the immunological synapse. Journal of Cell Science. 124 (5), 820-830 (2011).
- Westcott, M. M., Henry, C. J., Cook, A. S., Grant, K. W., Hiltbold, E. M. Differential susceptibility of bone marrow-derived dendritic cells and macrophages to productive infection with Listeria monocytogenes. Cellular Microbiology. 9 (6), 1397-1411 (2007).
- Geijtenbeek, T. B., Kwon, D. S., et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 100 (5), 587-597 (2000).
- Izquierdo-Useros, N., Naranjo-Gòmez, M., et al. HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse. PLoS Pathogens. 6 (3), e1000740 (2010).
- Mittelbrunn, M., Sanchez-Madrid, F. Intercellular communication: diverse structures for exchange of genetic information. Nature Reviews. Molecular cell biology. 13 (5), 328-335 (2012).
- McElroy, D. S., Ashley, T. J., D’Orazio, S. E. Lymphocytes serve as a reservoir for Listeria monocytogenes growth during infection of mice. Microbial Pathogenesis. 46 (4), 214-221 (2009).
- Salgado-Pabon, W., Celli, S., et al. Shigella impairs T lymphocyte dynamics in vivo. Proceedings of the National Academy of Sciences. 110 (12), 4458-4463 (2013).
- Cruz Adalia, A., Ramirez-Santiago, G., et al. T cells kill bacteria captured by transinfection from dendritic cells and confer protection in mice. Cell Host and Microbe. 15 (5), 611-622 (2014).
- Barnden, M. J., Allison, J., Heath, W. R., Carbone, F. R. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunology and Cell Biology. 76 (1), 34-40 (1998).
- Matheu, M. P., Sen, D., Cahalan, M. D., Parker, I. Generation of bone marrow derived murine dendritic cells for use in 2-photon imaging. Journal of Visualized Experiments: JoVE. (17), (2008).
- Inaba, K., Inaba, M., et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. Journal of Experimental Medicine. 176 (6), 1693-1702 (1992).
- Thöne, F., Schwanhäusser, B., Becker, D., Ballmaier, M., Bumann, D. FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen. Journal of Microbiological Methods. 71 (3), 220-224 (2007).
- Vaudaux, P., Waldvogel, F. A. Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrobial Agents and Chemotherapy. 16 (6), 743-749 (1979).
- Zhang, X., Goncalves, R., Mosser, D. M., Coligan, J. E. Chapter 14, The isolation and characterization of murine macrophages. Current protocols in immunology. , Unit 14.1 (2008).
- Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. Isolation and th17 differentiation of naïve CD4 T lymphocytes. Journal of Visualized Experiments: JoVE. (79), e50765 (2013).
- Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. Purification of specific cell population by fluorescence activated cell sorting (FACS). Journal of Visualized Experiments: JoVE. (41), (2010).
- Foucar, K., Chen, I. M., Crago, S. Organization and operation of a flow cytometric immunophenotyping laboratory. Seminars in diagnostic pathology. 6 (1), 13-36 (1989).
- Itano, A. A., Jenkins, M. K. Antigen presentation to naive CD4 T cells in the lymph node. Nature Immunology. 4 (8), 733-739 (2003).
