隔离和不同的老年小鼠胸腺移植的移植。
Summary
This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.
Abstract
调节胸腺选择的有效性的机制仍然不明确。这里介绍的方法允许在体内特异于自我和外来抗原的T细胞的发展和选择的分析。该方法需要来自各种老年小鼠免疫缺陷SCID收件人胸腺移植物的植入。在一段相对较短的时间的收件人完全重构与来自植入胸腺移植物衍生的T细胞。只有播种的胸腺细胞在胸腺在隔离的时间进行选择，并发展成为成熟的T细胞。这样，改变在移植的T细胞的年龄依赖性胸腺事件的函数的性质和特异性可以进行评估。虽然技术专长需要成功胸腺移植，这种方法提供了以研究在体内的广泛的病理是由于异常胸腺功能和/或homeostas或结果的一个独特的战略是。
Introduction
胸腺是在T细胞发育的关键事件发生1器官。驻地胸腺细胞，在T细胞受体（TCR）α和β基因的重排，进行一系列与在胸腺2的皮质和髓质区lymphostromal和抗原呈递细胞（APC）的相互作用。胸腺阳性选择由皮质胸腺上皮细胞（TEC）介导，以产生胸腺细胞识别的抗原肽在宿主主要组织相容性复合体（MHC）I类和II类分子2-3的上下文中。随后胸腺阴性选择嗣继承吹扫自身反应的胸腺细胞，通过与髓TEC或树突细胞（DC）从自身蛋白质结合的MHC I类和II类分子3推导出本发明的肽的相互作用来驱动。这些处理的最终结果是建立成熟的CD4 +和CD8 + T细胞能够对广谱响应一池外来抗原的同时表现出最小的反应性的自身蛋白质4。
胸腺选择事件的效率是由许多因素，包括胸腺成熟，髓和皮质TEC，胸腺DC亚群组成的频率，和胸腺前体3的源的影响。值得注意的是，异常胸腺选择可导致自身免疫性5或免疫缺陷的病状，从受损的阴性或阳性选择，它们分别出现。的分子事件调节胸腺选择，然而，却知之甚少。 体外方法，如reaggregate胸腺器官培养（RTOC）6，已被证明是用于分析与胸腺选择相关联的基本事件是有用的，但不能完全概括的正在进行的动态在体内的活动。这样一来，这个胸腺移植为基础的方式成立，以便更好地研究胸腺细胞评选活动在体内 7 </s向上>。
本协议描述移栽从新生和成年供体小鼠thymi到免疫缺陷SCID受体小鼠。这种技术允许调节阳性和阴性胸腺选择，以及各种T细胞亚群的个体发育过程中胸腺输出机制的研究。最近，这种方法已被用于证明胸腺选择的效率出生的小鼠，导致自身反应性T细胞的增加发展后早期的限制，并降低T细胞剧目特异于外来抗原7。
Protocol
Representative Results
Discussion
Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by funding received from the National Institutes of Health (1R01AI083269).
Materials
| Ethanol | Decon Laboratories | 2705HC | |
| PBS | GIBCO | 14190-144 | pH 7.2 |
| Betadine Solution | Purdue Products | 67618-150-17 | |
| 18 gauge needle | BD | 305195 | |
| Sutures (1.0 metric) | Ethicon | J493G | 18" (45cm) |
| AUTOCLIP Wound Clips (9mm) | Clay Adams® Brand | 427631 | |
| Transfer pipette | Fisher Scientific | 13-711-20 | Sterile, disposable |
| Mouse anti-CD3 Ab | eBioscience | 11-0031-85 | Clone: 1452C11 |
| Mouse anti-CD4 Ab | eBioscience | 48-0042-82 | Clone: RM4-5 |
| Mouse anti-CD8 Ab | eBioscience | 25-0081-82 | Clone: 53-6.7 |
| Lancet | Medipoint | Goldenrod 4mm | |
| Pacific Orange Succinimidyl Ester, Triethylammonium Salt | Invitrogen | P30253 | |
| 96-well round botton polypropylene plates | Corning | 3365 | |
| 1.2mL polypropylene cluster tubes | Corning | 4401 | |
| 5mL polypropylene round-bottom tubes | BD | 352002 | |
| 40uM Nylon Cell Strainer | Falcon | 352340 | |
| 16% Paraformaldehyde Solution, EM Grade | Electron Microscopy Services | 15710 | Hazardous |
| Puralube Optical Ointment | Fisher Scientific | NC0138063 | |
| Lympholyte | Cedarlane | CL5030 | |
| 60 mm cell culture dish | Corning | 430196 | 60mm x 15mm, Sterile |
References
- Kappler, J. W., Roehm, N., Marrack, P. T cell tolerance by clonal elimination in the thymus. Cell. 49 (2), 273-280 (1987).
- Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat. Rev. Immunol. 6, 127-135 (2006).
- Klein, L., Kyewski, B., Allen, P. M., Hogquist, K. A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nat. Rev. Immunol. 14 (6), 377-391 (2014).
- Schwartz, R. H. Acquisition of immunologic self-tolerance. Cell. 57 (7), 1073-1081 (1989).
- Kishimoto, H., Sprent, J. A defect in central tolerance in NOD mice. Nat. Immunol. 2 (11), 1025-1031 (2001).
- Anderson, G., Jenkinson, E. J. Investigating central tolerance with reaggregate thymus organ cultures. Methods. Mol. Biol. 380, 185-196 (2007).
- He, Q., et al. Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner. J. Immunol. 191 (12), 5858-5866 (2013).
- Stewart, C. C., Stewart, S. J. Chapter 6. Immunophenotyping. Current Protocols in Cytometry. , (2001).
- Rice, H. E., et al. Thymic transplantation for complete DiGeorge syndrome: medical and surgical considerations. J. Pediatr. Surg. 39 (11), 1607-1615 (2004).
- Markert, M. L., et al. Transplantation of thymus tissue in complete DiGeorge Syndrome. N. Engl. J. Med. 341, 1180-1189 (1999).
- Market, M. L., Devlin, B. H., McCarthy, E. A. Thymus transplantation. Clin. Immunol. 135 (2), 236-246 (2010).
- Wells, A. D., Li, X., Strom, T. B., Turka, L. A. The role of peripheral T-cell depletion in transplantation tolerance. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356 (1409), 617-623 (2001).
- Albuquerque, A. S., et al. Human FOXN1-Deficiency is associated with ab double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation. PLoS One. 7 (5), e37042 (2012).
- Chinn, I. K., Milner, J. D., Scheinberg, P., Douek, D. C., Markert, M. L. Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly. Clin Exp Immunol. 173 (1), 140-149 (2013).
- Savino, W. The thymus is a common target organ in infectious diseases. PLoS Pathog. 2 (6), e62 (2006).
