Summary

考试流式细胞仪检测胸腺阳性和阴性选择

Published: October 08, 2012
doi:

Summary

我们提出了一种基于流式细胞仪的方法来研究T细胞发育<em在体内</em使用基因操纵的野生型小鼠的T细胞受体的转基因背景。

Abstract

健康的免疫系统需要对外来抗原的T细胞反应,而其余的宽容,以自身抗原。随机重排的T细胞受体(TCR)的α和β位点产生一个具有丰富的多样性,在抗原特异性T细胞库,无论是对自己和国外。安全和有用的T细胞在胸腺发育过程中的曲目的选择是至关重要的。胸腺选择的缺陷的发展,自身免疫性疾病和免疫缺陷病的1-4。

T细胞祖细胞进入胸腺双阴性(DN)胸腺细胞不表达CD4或CD8共受体。 αβTCR和两个共受体的表达发生在双阳性(DP)阶段。互动的自我肽-MHC(住房抵押贷款公司)提出的胸腺细胞的αβTCR确定DP胸腺细胞的命运。高亲和力相互作用导致负选择和消元重刑自反应的胸腺细胞。在积极的选择和发展的CD4或CD8单阳性(SP)T细胞能够识别外来抗原的自我MHC 5低亲和力相互作用的结果。

的阳性筛选可以研究在小鼠的多克隆(野生型)的TCR剧目通过观察成熟T细胞的产生。然而,他们研究的阴性选择,这涉及到删除的小抗原特定人群的理想选择。许多模型系统已被用于研究的阴性选择,但不同的能力复述的生理活动6。例如, 在体外刺激的胸腺细胞缺乏选择是密切参与胸腺环境,而施用外源性抗原可导致非特异性缺失胸腺细胞7-9。目前,最好的工具,研究在体内的负选择,表达一个transg的有老鼠ENIC TCR特异的内源性自身抗原。然而,许多经典的TCR转基因模型的特点是在DN阶段的过早表达的转基因TCRα链,导致过早的负选择。我们的实验室已经开发出了HY的CD4模型,其中转基因HY,TCRα是有条件表示在自行分配学位阶段,让阴性选择过程中发生的DP,SP转型发生在野生型小鼠10。

在这里,我们描述了一个基于流式细胞仪的协议在HY CD4小鼠模型,研究胸腺的阳性和阴性选择。虽然负选择HY CD4小鼠中是高度生理,这些方法还可以被应用到其他的TCR转基因模型。我们也将目前的一般策略分析正选择一个适用于任何转基因操作的小鼠多克隆剧目。

Protocol

请参阅图1为实验方案的总体方案。 1。解剖地方无菌钢丝网屏幕为60 x 15 mm的培养皿中。需要一个单元的每个组织样本。 每个培养皿中加入5ml的Hank氏平衡盐溶液(HBSS)。保持在冰上菜。 安乐死小鼠与CO 2。 安全小鼠剥离面,腹一面朝上。用70%乙醇进行杀菌,并确保皮草纠结向下喷雾鼠标。 用手术剪,开始清扫腹切口?…

Representative Results

在生理TCR转基因模型和野生型小鼠,正开始选择在DP 明亮的舞台前,后移动到DP 沉闷的阶段抗原相遇。 DP 沉闷的胸腺细胞,然后进入一个过渡CD4 + CD8 罗的舞台前,成为CD4SP或CD8SP胸腺细胞( 图2B)。成熟的SP胸腺细胞的特点是高的TCR表达和损失的CD24( 图2C)。虽然CD8 CD4配置文件可以发现缺陷,积极的选择,检查TCRβCD69或CD5可以提?…

Discussion

这里提出的协议可以被用来检查非TCR转基因和TCR转基因小鼠中的阳性和阴性选择。此协议描述了表面抗原染色。有关的分子机制的进一步的分析,这是经常需要进行细胞内染色。我们使用了BD Biosciences公司的Cytofix / Cytoperm工具包的大多数细胞内的蛋白质和BD Biosciences公司的Foxp3的染色试剂盒的转录因子。通常,我们获得了我们的样品后,立即染色。然而,样品可以被存储在FACS缓冲液中过夜用1%多?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

的作者想感谢张冰,他的技术援助。这项工作是由加拿大卫生研究院的研究(MOP-86595)。 TAB是一个CIHR的新研究者和AHFMR学术。 QH的支持,一个CIHR加拿大研究生奖学金 – 博士和一个AIHS的全日制学籍。 SAN是由英国女王伊丽莎白二世研究生奖学金的支持。 AYWS支持由NSERC – 博士研究生奖学金。

Materials

Name of the reagent Company Catalogue number Comments (optional)
HyClone Hank’s balanced salt solution Thermo Scientific SH30030.02
Metal mesh screens Cedarlane CX-0080-E-01
Petri dishes (60 x 15 mm) Fisher Scientific 877221
Syringes (3 ml) BD Biosciences 309657
Conical tubes (15 ml) Sarstedt 62.554.205
Microscope Zeiss – Primo Star 415500-00XX-000
Hemocytometer Hausser Scientific 3110
96-well plate Sarstedt 82.1582.001
Multichannel pipette Fisherbrand 21-377-829
Fetal calf serum PAA A15-701
Phosphate buffered saline Fisher Scientific SH3025802
Sodium azide IT Baker Chemical Co. V015-05
FcR blocking reagent Clone 2.4G2
Anti-mouse HY TCR eBioscience XX-9930-YY* Clone T3.70
Anti-mouse CD4 eBioscience XX-0042-YY* Clone RM4-5
Anti-mouse CD8α eBioscience XX-0081-YY* Clone 53-6.7
Anti-mouse CD24 eBioscience XX-0242-YY* Clone M1/69
Anti-mouse TCRβ eBioscience XX-5961-YY* Clone H57-597
Anti-mouse CD69 Biotinylated eBioscience 13-0691-YY* Clone H1.2F3
Anti-mouse CD5 Biotinylated eBioscience 13-0051-YY* Clone 53-7.3
Streptavidin eBioscience XX-4217-YY*
Flow cytometer BD Biosciences – FACS Canto 338962
FACS tubes BD Biosciences 352052
Flow cytometry analysis software TreeStar – Flowjo FlowJo v7/9
HyClone RPMI – 1640 medium Thermo Scientific SH30027.01

*XX varies by fluorochrome and YY varies by vial size.

References

  1. Liston, A., Lesage, S., Wilson, J., Peltonen, L., Goodnow, C. C. Aire regulates negative selection of organ-specific T cells. Nat. Immunol. 4, 350-354 (2003).
  2. Liston, A. Gene dosage–limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity. J. Exp. Med. 200, 1015-1026 (2004).
  3. Hogquist, K. A., Baldwin, T. A., Jameson, S. C. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5, 772-782 (2005).
  4. Liston, A., Enders, A., Siggs, O. M. Unravelling the association of partial T-cell immunodeficiency and immune dysregulation. Nat. Rev. Immunol. 8, 545-558 (2008).
  5. Starr, T. K., Jameson, S. C., Hogquist, K. A. Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139-176 (2003).
  6. McCaughtry, T. M., Hogquist, K. A. Central tolerance: what have we learned from mice. Seminars in immunopathology. 30, 399-409 (2008).
  7. Zhan, Y. Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation. Proceedings of the National Academy of Sciences of the United States of America. 100, 1197-1202 (2003).
  8. Brewer, J. A., Kanagawa, O., Sleckman, B. P., Muglia, L. J. Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo. J. Immunol. 169, 1837-1843 (2002).
  9. Martin, S., Bevan, M. J. Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection. European journal of immunology. 27, 2726-2736 (1997).
  10. Baldwin, T. A., Sandau, M. M., Jameson, S. C., Hogquist, K. A. The timing of TCR alpha expression critically influences T cell development and selection. J. Exp. Med. 202, 111-121 (2005).
  11. Tung, J. W. Modern flow cytometry: a practical approach. Clinics in laboratory medicine. 27, 453-468 (2007).
  12. Aliahmad, P., Kaye, J. Development of all CD4 T lineages requires nuclear factor TOX. J. Exp. Med. 205, 245-256 (2008).
  13. Kastner, P. Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes. Eur. J. Immunol. 40, 2143-2154 (2010).
  14. Albu, D. I. BCL11B is required for positive selection and survival of double-positive thymocytes. J. Exp. Med. 204, 3003-3015 (2007).
  15. Van De Wiele, C. J. Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation. J. Immunol. 172, 4235-4244 (2004).
  16. Ueno, T. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. 200, 493-505 (2004).
  17. Saini, M. Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds. Science signaling. 3, ra23 (2010).
  18. Hu, Q., Sader, A., Parkman, J. C., Baldwin, T. A. Bim-mediated apoptosis is not necessary for thymic negative selection to ubiquitous self-antigens. J. Immunol. 183, 7761-7767 (2009).
  19. Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M., von Boehmer, H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature. 333, 742-746 (1988).
  20. 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).
  21. Derbinski, J., Schulte, A., Kyewski, B., Klein, L. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2, 1032-1039 (2001).
  22. Anderson, M. S. Projection of an immunological self shadow within the thymus by the aire protein. Science. 298, 1395-1401 (2002).
  23. Kurts, C. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 184, 923-930 (1996).
  24. Nitta, T., Nitta, S., Lei, Y., Lipp, M., Takahama, Y. CCR7-mediated migration of developing thymocytes to the medulla is essential for negative selection to tissue-restricted antigens. Proceedings of the National Academy of Sciences of the United States of America. 106, 17129-17133 (2009).
  25. Bouneaud, C., Kourilsky, P., Bousso, P. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity. 13, 829-840 (2000).
  26. Gallegos, A. M., Bevan, M. J. Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation. J. Exp. Med. 200, 1039-1049 (2004).
  27. Moon, J. J. Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity. 27, 203-213 (2007).
  28. Bouillet, P. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Nature. 415, 922-926 (2002).
  29. Suen, A. Y., Baldwin, T. A. Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens. Proceedings of the National Academy of Sciences of the United States of America. , (2012).
  30. Calnan, B. J., Szychowski, S., Chan, F. K., Cado, D., Winoto, A. A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection. Immunity. 3, 273-282 (1995).
  31. Zhou, T. Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells. J. Exp. Med. 183, 1879-1892 (1996).
  32. Baldwin, T. A., Hogquist, K. A. Transcriptional analysis of clonal deletion in vivo. J. Immunol. 179, 837-844 (2007).

Play Video

Cite This Article
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, doi:10.3791/4269 (2012).

View Video