Summary

制备及胸腺器官切片文化中的应用

Published: August 06, 2016
doi:

Summary

我们描述的是,在用流式细胞仪组合,可以被用来建模显影T细胞的阳性和阴性选择胸腺切片的制备方法。胸腺片也可以适用于胸腺细胞迁移,本地化的原位分析,并通过免疫荧光和双光子显微镜信令。

Abstract

在产生了功能性的,自我耐受T细胞库的产生一种独特的和高度有组织的胸腺微胸腺选择前进, 在体外模型来研究T系承诺和发展已经提供了有价值的见解这一过程。然而,这些系统缺乏必需的T细胞发育的完整三维胸腺环境,因此, 在体内胸腺选择的不完全近似。一些涉及到建模T细胞发育的挑战可以通过使用原位模型提供了一个完整的胸腺微环境完全支持显影性T细胞的胸腺选择来克服。胸腺器官切片培养补充现有的原位技术。胸腺切片保存胸腺皮质和髓质区的完整性,并提供了一​​个平台,研究一个限定的发育阶段的或内源T C的重叠胸腺细胞发育一个成熟的胸腺微环境中厄尔。鉴于生成每只小鼠〜20片的能力,胸腺切片进行高通量实验的可扩展性方面呈现出独特的优势。此外,在生成胸腺切片和电位从不同遗传背景叠加不同胸腺子集或其它的细胞群相对容易提高该方法的多功能性。这里,我们描述的胸腺片,隔离和胸腺细胞的叠加,以及流式细胞仪分析胸腺片分离制备协议。这个系统还可以适于研究非常规T细胞的发育以及可视胸腺细胞迁移,胸腺间质细胞相互作用,并通过双光子显微镜与胸腺选择相关联的TCR信号。

Introduction

T细胞分化,通过在此期间,他们遇到几个检查点,以确保一个功能,自我耐受T细胞库1-3的产生胸腺一系列发育中间体。阳性选择促进与T细胞受体(TCR),能够识别的,具有低到中度亲和力,肽由皮质胸腺上皮细胞(CTEC)2,3-主要组织相容性复合体分子(MHC)呈现胸腺细胞的存活。阴性选择和调节性T(T REG)细胞发育有助于通过消除或转移强烈由MHC 2,4提出自身肽反应胸腺细胞的建立自身耐受。未成熟CD4 + CD8 +双阳性(DP)胸腺细胞表达了通过选择过程分化成成熟T细胞亚群的TCR,其中大部分是MHC I类限制性CD8 +细胞毒性或MHC II类限制性CD4 +辅助单阳性(SP)T细胞中,离开胸腺在次级淋巴器官1-3执行效应器功能之前。

添加到T细胞发育的复杂性是动态迁移并在整个基质细胞网络5-9显影胸腺细胞的细胞接触。这些基质细胞在胸腺细胞发展中发挥不同的作用,并在那里阳性和阴性选择发生10胸腺皮质和髓质区之间被差异分布。虽然正选择主要发生在皮质,有越来越多的证据认为DP胸腺细胞迁移到髓质和继续需要的TCR信号它们分化成成熟T细胞暗示髓质可提供必要的正选择和谱系完成附加的信号之前分化11,12。进一步,尽管表达专门髓质胸腺上皮细胞(MTEC)和本组织限制性抗原促进自身反应性胸腺13,14的缺失的情况下,阴性选择的相当大的比例发生在响应皮层到遍在表达自身肽树突呈现细胞15,16。因此,T细胞发育的准确的模型必须提供高度组织胸腺微环境,具有完整皮层和髓质区,便于胸腺细胞和基质细胞之间的相互作用,并支持胸腺细胞迁移,因为这些细胞经历阳性和阴性选择。

为了补充体外胸腺细胞分析作为研究阳性和阴性选择,一些在体外,原位的手段,并 T细胞发育的体内模型已经开发17-22。它已经非常难以概括在体外的积极选择,但表达Notch配体干细胞群或基质细胞T细胞前体共培养,特别是OP9-DL1 / 4的细胞,具有支持T系的承诺和有限的正向选择的能力使其成为一个非常宝贵的体外模型研究T细胞发育23-25。本系统的限制,但是,包括这些细胞缺乏胸腺基质细胞上发现的唯一的肽加工机械和三维胸腺微环境的事实。

虽然在技术上更为繁琐,就地胸腺选择的体内模型可以克服一些与体外系统的障碍。 Reaggregate胸腺器官培养(RTOC)包含定义的胸腺细胞和胸腺基质细胞18,26,27的混合物。这些胸腺上皮细胞reaggregates保持MHC I类和II表达并且可以支持DEVELOPME常规T细胞亚群的NT,但仍然缺乏定义的皮质和髓质结构。胎儿胸腺器官培养(FTOC)是T细胞发育的一个流行的模型,可以通过lymphodepleted胸腺叶或通过胸腺细胞注射悬滴文化的胸腺细胞被接种到lymphoreplete胸腺叶和支持CD4 +和CD8 + T的高效发展在细胞培养18,28-31时间。在胎儿胸腺瓣培养开始有mTECs的缺乏,但根据条件定义皮质和髓质结构可以随时间发展。一个重要的考虑是,该模型可优先支持胎儿对成人T细胞的发展。最后,在成年小鼠胸腺中定义的前体胸腺内注射在技术上具有挑战性,但显然提供了一个环境,以支持 T细胞发育体内 。这些原位 和体内模型是优秀的快工具Ø研究T细胞发育,其使用应在一个实验按实验基础上加以考虑。

胸腺片,然而,最近已成为一个通用的,互补的模型,研究胸腺选择就地与可能性,以适应独特的,复杂的,并且通常更高的吞吐量实验。胸腺片保持皮质和髓质区的完整性并提供基质细胞的框架,开发以及高效阳性和阴性选择11,32-39期间支持胸腺细胞迁移。添加顶上胸腺切片胸腺细胞亚群迁移到组织到相应的微环境利基34,37。的重叠胸腺细胞可以从胸腺切片内源性细胞经由同类标记或荧光标记来区分,并可以在培养物中维持数天。胸腺器官切片文化,可以用来研究的各个方面T细胞发展,包括胸腺选择,胸腺细胞行为(迁移和细胞相互作用),和胸腺细胞定位,等等。给生成每只小鼠〜20胸腺切片的能力,实验的可扩展性通常比胸腺选择的原位模型其他更大。虽然胸腺切片的制备需要专门的设备,如vibratome,并且由于通过细胞死亡随着时间的推移细胞的损失和缺乏包封膜被限制在培养胸腺片的使用寿命,胸腺片提供了一个极好的模型对于一个成熟的胸腺微环境胸腺细胞同步群体的胸腺选择的分析。在这里,我们描述了胸腺片(包括收获的胸腺,胸腺裂片的琼脂糖嵌入和嵌入的组织的vibratome切片),隔离和胸腺细胞的重叠,并解离为流式细胞仪分析胸腺切片的制备方法。

Protocol

HOPITAL迈松内夫 – 罗斯芒特 – 适用于所有的动物研究协议是由动物护理委员会在该中心德RECHERCHE批准。 1.收获小鼠胸腺胸腺片的制备及单细胞悬浮液安乐死用CO 2鼠标颈椎脱位。 在层流罩,针鼠标腹侧到夹层板。喷用70%乙醇鼠标。用纱布涂抹,以防止乙醇进入胸腔和破坏组织取出多余的酒精。 解除在用一对镊子的胸骨的底部的皮肤,使通过皮肤切口。向上延伸?…

Representative Results

T细胞发育的不同方面,如阳性和阴性选择胸腺切片支持分析。对于成功的实验中,胸腺切片的质量是至关重要的。 因此,应研究胸腺切片以确保胸腺组织的完整性和周围胸腺切片琼脂糖是完好( 图1A)。当琼脂糖损坏导致在迁移进入组织胸腺细胞的数目显著减少表面张力就会受到损害。因此,具有组织损伤或刻痕的适应症胸腺片/眼泪在琼脂糖?…

Discussion

在这里,我们描述了胸腺切片并通过流式细胞术重叠预选择-MHC I类限制性TCR转基因的胸腺细胞的高效阳性和阴性选择的代表性结果的制备的协议。此系统已用于具有类似的成功支持从预选DP胸腺细胞32 MHC II型限制的CD4 + T细胞的阳性选择,并且,在激动剂抗原,阴性选择和胸腺T 发展11,12的存在, 36,38,39,43,44。该协议可以被修改为研究抑制剂,定义的肽的?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We would like to thank Marilaine Fournier for her comments on the manuscript and Josée Tessier for technical assistance. C57BL/6-Tg (OT-I)-RAG1<tmMom> #4175 were obtained through the NIAID Exchange Program, NIH. Support for this research is provided by a grant from the SickKids Foundation and CIHR-IHDCYN (NI15-002), an operating grant from the CIHR-III (MOP-142254), and start-up funds from the FRQS (Établissement de jeunes chercheurs) and Hôpital Maisonneuve-Rosemont Foundation to HJM. HJM is a junior 1 scholar of the FRQS, a CIHR New Investigator (MSH-141967), and a Cole Foundation Early Career Transition award recipient.

Materials

Vibratome Leica Biosystems VT1000S 
NuSieve GTG Agarose Lonza 50080 Low melting temperature agarose
Embedding Mold (Truncated – T12) Polyciences 18986 22mm x 22mm square, truncated to 12mm x 12mm
Double Edge Prep Blades Personna 74-0002
Tissue Adhesive 3M  1469SB
0.4 µm Cell Culture Inserts  BD Falcon 353090 Of several brands tested, these maintained the cells atop the slices the best
Dulbecco's Phosphate-Buffered Saline ThermoFisher 21600-010
RPMI-1640 with L-glutamine Wisent 350-000-CL
Fetal Bovine Serum Wisent 080-110 Heat inactivated
L-Glutamine, 200mM Wisent 609-065-EL
Penicillin/Streptomycin, 100X Wisent 450-201-EL
2-Mercaptoethanol Alfa Aesar A15890
15 ml Tenbroeck Tissue Grinders Wheaton 357426
Nylon Mesh Filter Component Supply U-CMN-255
Microcentrifuge Tube Sample Pestle Bel-Art F19922-0000
40 µm Nylon Cell Strainer BD Falcon 352340
Forceps Inox Tip Dumont  RS-5047 Fine tip curved forceps, size .17 X .10mm 
Micro Forceps Dumont  RS-5090 

References

  1. Carpenter, A. C., Bosselut, R. Decision checkpoints in the thymus. Nat Immunol. 11, 666-673 (2010).
  2. Starr, T. K., Jameson, S. C., Hogquist, K. A. Positive and negative selection of T cells. Annu Rev Immunol. 21, 139-176 (2003).
  3. Vrisekoop, N., Monteiro, J. P., Mandl, J. N., Germain, R. N. Revisiting thymic positive selection and the mature T cell repertoire for antigen. Immunity. 41, 181-190 (2014).
  4. Stritesky, G. L., Jameson, S. C., Hogquist, K. A. Selection of self-reactive T cells in the thymus. Annu Rev Immunol. 30, 95-114 (2012).
  5. Bousso, P., Bhakta, N. R., Lewis, R. S., Robey, E. Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science. 296, 1876-1880 (2002).
  6. Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol. 6, 127-135 (2006).
  7. Halkias, J., Melichar, H. J., Taylor, K. T., Robey, E. A. Tracking migration during human T cell development. Cell Mol Life Sci. 71, 3101-3117 (2014).
  8. Yin, X., Chtanova, T., Ladi, E., Robey, E. A. Thymocyte motility: mutants, movies and migration patterns. Curr Opin Immunol. 18, 191-197 (2006).
  9. Ladi, E., Yin, X., Chtanova, T., Robey, E. A. Thymic microenvironments for T cell differentiation and selection. Nat Immunol. 7, 338-343 (2006).
  10. 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, 377-391 (2014).
  11. Ross, J. O., et al. Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns. Proc Natl Acad Sci U S A. 111, E2550-E2558 (2014).
  12. Hu, Z., Lancaster, J. N., Sasiponganan, C., Ehrlich, L. I. CCR4 promotes medullary entry and thymocyte-dendritic cell interactions required for central tolerance. J Exp Med. 212, 1947-1965 (2015).
  13. Anderson, M. S., et al. Projection of an immunological self shadow within the thymus by the aire protein. Science. 298, 1395-1401 (2002).
  14. Takaba, H., et al. Fezf2 Orchestrates a Thymic Program of Self-Antigen Expression for Immune Tolerance. Cell. 163, 975-987 (2015).
  15. McCaughtry, T. M., Baldwin, T. A., Wilken, M. S., Hogquist, K. A. Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla. J Exp Med. 205, 2575-2584 (2008).
  16. Stritesky, G. L., et al. Murine thymic selection quantified using a unique method to capture deleted T cells. Proc Natl Acad Sci U S A. 110, 4679-4684 (2013).
  17. Anderson, G., Jenkinson, E. J. Review article: thymus organ cultures and T-cell receptor repertoire development. Immunology. 100, 405-410 (2000).
  18. Hare, K. J., Jenkinson, E. J., Anderson, G. In vitro models of T cell development. Semin Immunol. 11, 3-12 (1999).
  19. de Pooter, R., Zuniga-Pflucker, J. C. T-cell potential and development in vitro: the OP9-DL1 approach. Curr Opin Immunol. 19, 163-168 (2007).
  20. Lian, Z., et al. Intrathymically injected hemopoietic stem cells can differentiate into all lineage cells in the thymus: differences between c-kit+ cells and c-kit < low cells. Stem Cells. 15, 430-436 (1997).
  21. Manna, S., Bhandoola, A. Intrathymic Injection. Methods Mol Biol. 1323, 203-209 (2016).
  22. Goldschneider, I., Komschlies, K. L., Greiner, D. L. Studies of thymocytopoiesis in rats and mice. I. Kinetics of appearance of thymocytes using a direct intrathymic adoptive transfer assay for thymocyte precursors. J Exp Med. 163, 1-17 (1986).
  23. Schmitt, T. M., Zuniga-Pflucker, J. C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity. 17, 749-756 (2002).
  24. de Pooter, R. F., Schmitt, T. M., Zuniga-Pflucker, J. C. In vitro generation of T lymphocytes from embryonic stem cells. Methods Mol Biol. 330, 113-121 (2006).
  25. Dervovic, D. D., Ciofani, M., Kianizad, K., Zuniga-Pflucker, J. C. Comparative and functional evaluation of in vitro generated to ex vivo CD8 T cells. J Immunol. 189, 3411-3420 (2012).
  26. White, A., Jenkinson, E., Anderson, G. Reaggregate thymus cultures. J Vis Exp. (18), (2008).
  27. Anderson, G., Owen, J. J., Moore, N. C., Jenkinson, E. J. Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro. J Exp Med. 179, 2027-2031 (1994).
  28. Anderson, G., Jenkinson, E. J. Fetal thymus organ culture. CSH Protoc. , (2007).
  29. Mazda, O., Watanabe, Y., Gyotoku, J., Katsura, Y. Requirement of dendritic cells and B cells in the clonal deletion of Mls-reactive T cells in the thymus. J Exp Med. 173, 539-547 (1991).
  30. Ceredig, R., Jenkinson, E. J., MacDonald, H. R., Owen, J. J. Development of cytolytic T lymphocyte precursors in organ-cultured mouse embryonic thymus rudiments. J Exp Med. 155, 617-622 (1982).
  31. Fairchild, P. J., Austyn, J. M. Developmental changes predispose the fetal thymus to positive selection of CD4+CD8 T cells. Immunology. 85, 292-298 (1995).
  32. Bhakta, N. R., Oh, D. Y., Lewis, R. S. Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment. Nat Immunol. 6, 143-151 (2005).
  33. Le Borgne, M., et al. The impact of negative selection on thymocyte migration in the medulla. Nat Immunol. 10, 823-830 (2009).
  34. Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes. Immunity. 31, 986-998 (2009).
  35. Ueda, Y., et al. Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus. Nat Commun. 3, 1098 (2012).
  36. Dzhagalov, I. L., Chen, K. G., Herzmark, P., Robey, E. A. Elimination of self-reactive T cells in the thymus: a timeline for negative selection. PLoS Biol. 11, e1001566 (2013).
  37. Halkias, J., et al. Opposing chemokine gradients control human thymocyte migration in situ. J Clin Invest. 123, 2131-2142 (2013).
  38. Au-Yeung, B. B., et al. Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol. 15, 687-694 (2014).
  39. Melichar, H. J., Ross, J. O., Herzmark, P., Hogquist, K. A., Robey, E. A. Distinct temporal patterns of T cell receptor signaling during positive versus negative selection in situ. Sci Signal. 6, (2013).
  40. Hu, Q., Nicol, S. A., Suen, A. Y., Baldwin, T. A. Examination of thymic positive and negative selection by flow cytometry. J Vis Exp. (68), e4269 (2012).
  41. Mombaerts, P., et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 68, 869-877 (1992).
  42. Hogquist, K. A., et al. T cell receptor antagonist peptides induce positive selection. Cell. 76, 17-27 (1994).
  43. Weist, B. M., Kurd, N., Boussier, J., Chan, S. W., Robey, E. A. Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition. Nat Immunol. 16, 635-641 (2015).
  44. Melichar, H. J., Ross, J. O., Taylor, K. T., Robey, E. A. Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection. J Immunol. 194, 1057-1061 (2015).
  45. Hadjantonakis, A. K., Macmaster, S., Nagy, A. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2, (2002).
  46. Schaefer, B. C., Schaefer, M. L., Kappler, J. W., Marrack, P., Kedl, R. M. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell Immunol. 214, 110-122 (2001).

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Sood, A., Dong, M., Melichar, H. J. Preparation and Applications of Organotypic Thymic Slice Cultures. J. Vis. Exp. (114), e54355, doi:10.3791/54355 (2016).

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