CD1d四聚体检测技术的初步改良
摘要
前言
NKT细胞TCR识别由非胸腺依赖抗原与CDld结合而成的复合物。α-GalCer为目前发现的NKT细胞结合效能最强的糖脂抗原,其可以装载入CDld分子抗原结合沟槽,应用此糖脂和分子所建立的CDld四聚体技术是检测NKT细胞的权威方法。
肝脏是机体最大的消化与代谢器官,然而其在免疫学领域的作用日渐清晰,甚至有人已把肝脏作为独立的免疫器官；NKT细胞在肝内单个核细胞比率达到8%-15%,为所有具备淋巴样器官中最多的器官。本试验即利用肝脏内NKT细胞比率较高的特性,以评定不同制备条件建立的四聚体检测效果,从而对其制备条件进行改良。不同肝脏单个核细胞制备方法可对NKT细胞比率产生影响,选择一种稳定的制备方法也是保证改良方案实施的重要基础。
Tween-20是常用的实验室有机助溶剂,属水包油型乳化剂,可用作增溶剂、扩散剂、稳定剂、润滑剂和抗静电剂。CDld四聚体在制备过程中用含有Tween-20的PBS分散糖脂抗原是能否建立成功四聚体的关键步骤,但其亦能对反应体系造成不利影响,因而在保证其分散效果的同时也应该控制Tween-20在整个反应体系里的量才能找到一个理想的平衡点。a-GalCer与融合蛋白的孵育时间是CDld四聚体制备过程的限速步骤,现今的制作流程中二者的孵育时间皆在12小时以上,这完全限制了CDld四聚体技术的快速应用。
本研究建立的CDld四聚体技术可对小鼠胸腺、脾脏、肝脏iNKT细胞进行检测,并对CDld四聚体制作过程的PBS所含Tween-20浓度及a-GalCer与融合蛋白的孵育时间进行摸索,希求应用最少的Tween-20及最短的孵育时间达到良好稳定的检测效果的目的。
方法
以肝脏内单个核细胞为检测对象,利用含Tween-20不同浓度PBS所建立CDld四聚体检测其NKT比率,通过对比Tween-20浓度不同对CDld四聚体检测效率的差异,确定最合理的Tween-20浓度；以肝脏内单个核细胞为检测对象,利用糖脂a-GalCer与CDld-IgG1 Fc融合蛋白经不同孵育时间所建立CDld四聚体对其NKT比率进行检测,通过对比孵育时间的不同对CDld四聚体检测效率的差异,确定最合理的孵育时间。
结果
CDld四聚体检测NKT方法的建立应用保守建立方法,即用含Tween-201%的PBS稀释a-GalCer糖脂储存液,并与CDld-IgG1 Fc融合蛋白孵育过夜12小时所建立的四聚体检测液对所提纯肝脏单个核细胞、胸腺细胞、脾脏单个核细胞进行检测,可检测到NKT细胞亚群,其检测阳性比率与国外文献相符,表明四聚体建立成功。
用含不同浓度Tween-20 PBS (0.00%、0.05%、0.10%、0.25%.0.50%、1.00%、2.00%)制备的CDld四聚体检测肝脏单个核细胞内NKT细胞,对其检测效率进行比较,在0.5%至2%范围内增加时,所测得NKT细胞比率稍有下降趋势或比率相当,统计学处理Tween-20浓度在0.5、1%、2%时,三者的检出能力无统计学差异,显示Tween-20在0.5%时即能制备出具有完整检出能力的CDld四聚体。
不同糖脂a-GalCer与CDld-IgG1 Fc融合蛋白装载时间(1h、2h、4h、6h、8h、12h、24h)所制备的CDld四聚体检测肝脏单个核细胞内NKT细胞,对其检测效率进行比较。数据表明,随装载时间在1h、2h、4h、6h、8h范围内不断增加,所测得NKT细胞比率不断增加；在8h、12h、24h范围内增加时,所测得NKT细胞比率稍有下降趋势或比率相当,统计学处理装载时间在8h、12h、24h时,三者的检出能力无统计学差异,显示装载时间8h时即能制备出具有完整检出能力的四聚体。
结论
建立CDld四聚体检测技术,可以对肝脏、胸腺、脾脏NKT细胞进行检测。在CDld四聚体制备过程中,用于分散a-CalCer糖脂抗原的PBS所含Tween-20的浓度可降低到0.5%。在CDld四聚体制备过程中,a-CalCer和CDld-IgG1 Fc融合蛋白的孵育时间可缩短到8h。
TCR of NKT cells recognize the combination of thymus independent antigen and CD1d molecule.a-GalCer is the strongest glycolipid of NKT cell with high binding efficacy at present, which can be loaded into the CD1d antigen-binding groove。The CD1d tetramer technology which is established using this glycolipid and CD1d molecule to detect the NKT cell is the authoritative method.
The liver is the biggest digestion and metabolism organ, however it is clear now its domain function in the immunology, even some people have taken the liver the independent immune organ, taking participation in immune response; NKT cells in the liver mononuclear cells ratio is 8%-15%. Our test use this special quality considering the ratio of the NKT cell in the liver, to evaluate the different experimental condition when establishment of the CD1d tetramer process,to carries on the optimization to its preparation condition.
Tween-20 is commonly used laboratory organic flux, is the oil-in-water emulsion medicinal preparation, may serve as the solubilizing agent,stabilizer, lubricant and antistatic agent and so on. CD1d tetramer in preparation process including the Tween-20 PBS dispersion antigen whether to establish successful tetramer, but it can also have the adverse impact on the reacting system, thus while guaranteeing its dispersion effect should also control Tween-20 to find an ideal balance point in the entire reacting system quantity.a-GalCer and the fusion protein's bath time is rate-limiting step in the CD1d tetramer preparation process。nowadays bath time is all over 12 hours, this has limited the CD1d tetramer technology for fast application.
This research establishes the CD1d tetramer technology to be use in the detection for iNKT cell in mouse thymus, spleen and liver. Analyzing the Tween-20 concentration in PBS and a-GalCer fusion protein bath time, hoping for it applying least Tween-20 and shortest bath time achieves the goal of good stable detection effect.
Methods
Taking liver mononuclear cells as the examination object, using established CD1d tetramer involving Tween-20 different concentration in PBS to examine NKT ratio, through contrasting efficiency difference determines the most reasonable Tween-20 concentration.
Taking the liver mononuclear cell as the examination object, using established CD1d tetramer involving different bath time of the glycolipid and the CD1d-IgG1 Fc fusion protein to carry on the detection for NKT, through contrasting different CD1d tetramer efficiency difference, determines the most reasonable bath time.
Result
Detection of liver mononuclear cells of NKT cells With different concentration of Tween-20 PBS (0.00%,0.05%,0.10%,0.25%,0.50%,1.00%,2.00%) prepared process.Its detection efficiency in the 0.5 to 2%range increase, but the measured ratio of NKT cells decreased slightly when Tween-20 in 0.5,1%,2%.the detection ability of the three No statistically significant difference was found.0.5%Tween-20 can be used in prepare process and the detection ability of CD1d tetramer is good。
The CD1d tetramer detection involving different a-GalCer and CD1d-IgG1 Fc fusion protein loading time (1h,2h,4h,6h,8h,12h and 24h) is carried on for the comparison to its detection efficiency. The data indicated that along with the loading time in 1h,2h,4h,6h and in the 8h scope continually increases, the NKT cell ratio continually to increase; In 8h,12h and in the 24h scope increases, NKT cell ratio to having the declining trend or the ratio slightly quite,。loading time 8h,12h and 24h is non-statistics difference, demonstrated loading time 8h can prepare the ability completely CD1d tetramer.
Conclusion
Has established the CD1d tetramer detection technology, and its examination efficiency is stable, reliable. CD1d tetramer detection efficiency and Tween-20 concentration have the close connection, based on this its examination efficiency with its concentration present related tendency, but after surpassing 0.5%, the efficiency do not have the difference. CD1d tetramer examination efficiency and the bath time of a-GalCer and CD1d-IgG1 Fc fusion protein to have the close connection。The examination efficiency and the bath time present related tendency, but after surpassing 8h, efficiency do not have the difference.
NKT细胞TCR识别由非胸腺依赖抗原与CDld结合而成的复合物。α-GalCer为目前发现的NKT细胞结合效能最强的糖脂抗原,其可以装载入CDld分子抗原结合沟槽,应用此糖脂和分子所建立的CDld四聚体技术是检测NKT细胞的权威方法。
肝脏是机体最大的消化与代谢器官,然而其在免疫学领域的作用日渐清晰,甚至有人已把肝脏作为独立的免疫器官；NKT细胞在肝内单个核细胞比率达到8%-15%,为所有具备淋巴样器官中最多的器官。本试验即利用肝脏内NKT细胞比率较高的特性,以评定不同制备条件建立的四聚体检测效果,从而对其制备条件进行改良。不同肝脏单个核细胞制备方法可对NKT细胞比率产生影响,选择一种稳定的制备方法也是保证改良方案实施的重要基础。
Tween-20是常用的实验室有机助溶剂,属水包油型乳化剂,可用作增溶剂、扩散剂、稳定剂、润滑剂和抗静电剂。CDld四聚体在制备过程中用含有Tween-20的PBS分散糖脂抗原是能否建立成功四聚体的关键步骤,但其亦能对反应体系造成不利影响,因而在保证其分散效果的同时也应该控制Tween-20在整个反应体系里的量才能找到一个理想的平衡点。a-GalCer与融合蛋白的孵育时间是CDld四聚体制备过程的限速步骤,现今的制作流程中二者的孵育时间皆在12小时以上,这完全限制了CDld四聚体技术的快速应用。
本研究建立的CDld四聚体技术可对小鼠胸腺、脾脏、肝脏iNKT细胞进行检测,并对CDld四聚体制作过程的PBS所含Tween-20浓度及a-GalCer与融合蛋白的孵育时间进行摸索,希求应用最少的Tween-20及最短的孵育时间达到良好稳定的检测效果的目的。
方法
以肝脏内单个核细胞为检测对象,利用含Tween-20不同浓度PBS所建立CDld四聚体检测其NKT比率,通过对比Tween-20浓度不同对CDld四聚体检测效率的差异,确定最合理的Tween-20浓度；以肝脏内单个核细胞为检测对象,利用糖脂a-GalCer与CDld-IgG1 Fc融合蛋白经不同孵育时间所建立CDld四聚体对其NKT比率进行检测,通过对比孵育时间的不同对CDld四聚体检测效率的差异,确定最合理的孵育时间。
结果
CDld四聚体检测NKT方法的建立应用保守建立方法,即用含Tween-201%的PBS稀释a-GalCer糖脂储存液,并与CDld-IgG1 Fc融合蛋白孵育过夜12小时所建立的四聚体检测液对所提纯肝脏单个核细胞、胸腺细胞、脾脏单个核细胞进行检测,可检测到NKT细胞亚群,其检测阳性比率与国外文献相符,表明四聚体建立成功。
用含不同浓度Tween-20 PBS (0.00%、0.05%、0.10%、0.25%.0.50%、1.00%、2.00%)制备的CDld四聚体检测肝脏单个核细胞内NKT细胞,对其检测效率进行比较,在0.5%至2%范围内增加时,所测得NKT细胞比率稍有下降趋势或比率相当,统计学处理Tween-20浓度在0.5、1%、2%时,三者的检出能力无统计学差异,显示Tween-20在0.5%时即能制备出具有完整检出能力的CDld四聚体。
不同糖脂a-GalCer与CDld-IgG1 Fc融合蛋白装载时间(1h、2h、4h、6h、8h、12h、24h)所制备的CDld四聚体检测肝脏单个核细胞内NKT细胞,对其检测效率进行比较。数据表明,随装载时间在1h、2h、4h、6h、8h范围内不断增加,所测得NKT细胞比率不断增加；在8h、12h、24h范围内增加时,所测得NKT细胞比率稍有下降趋势或比率相当,统计学处理装载时间在8h、12h、24h时,三者的检出能力无统计学差异,显示装载时间8h时即能制备出具有完整检出能力的四聚体。
结论
建立CDld四聚体检测技术,可以对肝脏、胸腺、脾脏NKT细胞进行检测。在CDld四聚体制备过程中,用于分散a-CalCer糖脂抗原的PBS所含Tween-20的浓度可降低到0.5%。在CDld四聚体制备过程中,a-CalCer和CDld-IgG1 Fc融合蛋白的孵育时间可缩短到8h。
TCR of NKT cells recognize the combination of thymus independent antigen and CD1d molecule.a-GalCer is the strongest glycolipid of NKT cell with high binding efficacy at present, which can be loaded into the CD1d antigen-binding groove。The CD1d tetramer technology which is established using this glycolipid and CD1d molecule to detect the NKT cell is the authoritative method.
The liver is the biggest digestion and metabolism organ, however it is clear now its domain function in the immunology, even some people have taken the liver the independent immune organ, taking participation in immune response; NKT cells in the liver mononuclear cells ratio is 8%-15%. Our test use this special quality considering the ratio of the NKT cell in the liver, to evaluate the different experimental condition when establishment of the CD1d tetramer process,to carries on the optimization to its preparation condition.
Tween-20 is commonly used laboratory organic flux, is the oil-in-water emulsion medicinal preparation, may serve as the solubilizing agent,stabilizer, lubricant and antistatic agent and so on. CD1d tetramer in preparation process including the Tween-20 PBS dispersion antigen whether to establish successful tetramer, but it can also have the adverse impact on the reacting system, thus while guaranteeing its dispersion effect should also control Tween-20 to find an ideal balance point in the entire reacting system quantity.a-GalCer and the fusion protein's bath time is rate-limiting step in the CD1d tetramer preparation process。nowadays bath time is all over 12 hours, this has limited the CD1d tetramer technology for fast application.
This research establishes the CD1d tetramer technology to be use in the detection for iNKT cell in mouse thymus, spleen and liver. Analyzing the Tween-20 concentration in PBS and a-GalCer fusion protein bath time, hoping for it applying least Tween-20 and shortest bath time achieves the goal of good stable detection effect.
Methods
Taking liver mononuclear cells as the examination object, using established CD1d tetramer involving Tween-20 different concentration in PBS to examine NKT ratio, through contrasting efficiency difference determines the most reasonable Tween-20 concentration.
Taking the liver mononuclear cell as the examination object, using established CD1d tetramer involving different bath time of the glycolipid and the CD1d-IgG1 Fc fusion protein to carry on the detection for NKT, through contrasting different CD1d tetramer efficiency difference, determines the most reasonable bath time.
Result
Detection of liver mononuclear cells of NKT cells With different concentration of Tween-20 PBS (0.00%,0.05%,0.10%,0.25%,0.50%,1.00%,2.00%) prepared process.Its detection efficiency in the 0.5 to 2%range increase, but the measured ratio of NKT cells decreased slightly when Tween-20 in 0.5,1%,2%.the detection ability of the three No statistically significant difference was found.0.5%Tween-20 can be used in prepare process and the detection ability of CD1d tetramer is good。
The CD1d tetramer detection involving different a-GalCer and CD1d-IgG1 Fc fusion protein loading time (1h,2h,4h,6h,8h,12h and 24h) is carried on for the comparison to its detection efficiency. The data indicated that along with the loading time in 1h,2h,4h,6h and in the 8h scope continually increases, the NKT cell ratio continually to increase; In 8h,12h and in the 24h scope increases, NKT cell ratio to having the declining trend or the ratio slightly quite,。loading time 8h,12h and 24h is non-statistics difference, demonstrated loading time 8h can prepare the ability completely CD1d tetramer.
Conclusion
Has established the CD1d tetramer detection technology, and its examination efficiency is stable, reliable. CD1d tetramer detection efficiency and Tween-20 concentration have the close connection, based on this its examination efficiency with its concentration present related tendency, but after surpassing 0.5%, the efficiency do not have the difference. CD1d tetramer examination efficiency and the bath time of a-GalCer and CD1d-IgG1 Fc fusion protein to have the close connection。The examination efficiency and the bath time present related tendency, but after surpassing 8h, efficiency do not have the difference.
引文
1 Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu. Rev. Immunol.2007; 25:297-336.
2 Godfrey DI, MacDonald HR, Kronenberg M, et al. NKT cells:what's in a name? Nat Rev Immunol 2004; 4:231-237.
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24刘景华,窦立萍,王莉莉等.CDld四聚体检测NKT细胞,细胞与分子免疫学杂志,2009,25(1)：82-83
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20 Iwai T. Regulatory roles of NKT cells in the induction and maintenance of cyclophosphamide-induced tolerance. J. Immunol.2006; 177:8400-8409.
21 Thedrez A. CD4 engagement by CD1d potentiates activation of CD4+invariant NKT cells. Blood 2007.
22 Gumperz JE, Miyake S, Yamamura T, et al. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med.2002; 195:625-636.
23 Lee PT, Benlagha K, Teyton L, et al. Distinct functional lineages of human V[alpha]24 natural killer T cells. J. Exp. Med.2002; 195:637-641.
24 Crowe NY. Differential antitumor immunity mediated by NKT cell subsets in vivo. J. Exp. Med. 2005; 202:1279-1288.
25 Pellicci DG. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1-CD4+CDld-dependent precursor stage. J. Exp.Med.2002; 195:835-844.
26 Egawa T. Genetic evidence supporting selection of the V[alpha]14iNKT cell lineage from double-positive thymocyte precursors. Immunity 2005; 22:705-716.
27 Gapin L, Matsuda JL, Surh CD, et al. NKT cells derive from double-positive thymocytes that are positively selected by CDld. Nature Immunol.2001; 2:971-978.
28 Dao T. Development of CDld-restricted NKT cells in the mouse thymus. Eur. J. Immunol. 2004; 34:3542-3552.
29 Zhou D. Lysosomal glycosphingolipid recognition by NKT cells. Science 2004; 306:1786-1789.
30 Coles MC, Raulet DH. NK1.1+T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+cells. J. Immunol.2000; 164:2412-2418.
31 Ohteki T, MacDonald HR. Major histocompatibility complex class I related molecules control the development of CD4+8-and CD4-8-subsets of natural killer 1.1+T cell receptor-[alpha]/[beta]+cells in the liver of mice. J. Exp. Med.1994; 180:699-704.
32 Li W. An alternate pathway for CD4 T cell development:thymocyte-expressed MHC class II selects a distinct T cell population. Immunity 2005; 23:375-386.
33 Wei DG, Curran SA, Savage PB, et al. Mechanisms imposing the V[beta] bias of V[alpha]14 natural killer T cells and consequences for microbial glycolipid recognition. J. Exp. Med.2006; 203:1197-1207.
34 Schumann J, Mycko MP, Dellabona P, et al. Cutting edge:influence of the TCR V[beta] domain on the selection of semi-invariant NKT cells by endogenous ligands. J. Immunol.2006; 176:2064-2068.
35 Stanic AK. NF-[kappa]B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes. J. Immunol.2004; 172:2265-2273.
36 Chun T. CDld-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J. Exp. Med.2003; 197:907-918.
37 Tilloy F, Di Santo JP, Bendelac A, et al. Thymic dependence of invariant V[alpha]14+natural killer-T cell development. Eur. J. Immunol.1999; 29:3313-3318.
38 MC C, DH R. NK1.1+T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J. Immunol 2000,164:2412.
39 Hammond K, Cain W, Vandriel I, et al. Three day neonatal thymectomy selectively depletes NK1.1+T Cells. Int. Immunol.1998; 10:1491-1499.
40 Doisne JM, Bartholin L, Yan KP, et al. iNKT cell development is orchestrated by different branches of TGF-{beta} signaling. J Exp Med 2009; 206(6):1365-1378.
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7 Bai L, Sagiv Y, Liu Y, Freigang S, et al. Lysosomal recycling terminates CDld-mediated presentation of short and polyunsaturated variants of the NKT cell lipid antigen{alpha}GalCer. Proc Natl Acad Sci U S A 2009; 106(25):10254-10259
8 Watarai H, Nakagawa R, Omori-Miyake M, et al. Methods for detection, isolation and culture of mouse and human invariant NKT cells. Nat Protoc 2008; 3:70-78.
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24刘景华,窦立萍,王莉莉等.CDld四聚体检测NKT细胞,细胞与分子免疫学杂志,2009,25(1)：82-83
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18 Godfrey DI, Berzins SP. Control points in NKT-cell development. Nat Rev Immunol 2007; 7:505-518.
19 Berzins SP, McNab FW, Jones CM, et al. Long-term retention of mature NK1.1+NKT cells in the thymus. J. Immunol.2006; 176:4059-4065.
20 Iwai T. Regulatory roles of NKT cells in the induction and maintenance of cyclophosphamide-induced tolerance. J. Immunol.2006; 177:8400-8409.
21 Thedrez A. CD4 engagement by CD1d potentiates activation of CD4+invariant NKT cells. Blood 2007.
22 Gumperz JE, Miyake S, Yamamura T, et al. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med.2002; 195:625-636.
23 Lee PT, Benlagha K, Teyton L, et al. Distinct functional lineages of human V[alpha]24 natural killer T cells. J. Exp. Med.2002; 195:637-641.
24 Crowe NY. Differential antitumor immunity mediated by NKT cell subsets in vivo. J. Exp. Med. 2005; 202:1279-1288.
25 Pellicci DG. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1-CD4+CDld-dependent precursor stage. J. Exp.Med.2002; 195:835-844.
26 Egawa T. Genetic evidence supporting selection of the V[alpha]14iNKT cell lineage from double-positive thymocyte precursors. Immunity 2005; 22:705-716.
27 Gapin L, Matsuda JL, Surh CD, et al. NKT cells derive from double-positive thymocytes that are positively selected by CDld. Nature Immunol.2001; 2:971-978.
28 Dao T. Development of CDld-restricted NKT cells in the mouse thymus. Eur. J. Immunol. 2004; 34:3542-3552.
29 Zhou D. Lysosomal glycosphingolipid recognition by NKT cells. Science 2004; 306:1786-1789.
30 Coles MC, Raulet DH. NK1.1+T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+cells. J. Immunol.2000; 164:2412-2418.
31 Ohteki T, MacDonald HR. Major histocompatibility complex class I related molecules control the development of CD4+8-and CD4-8-subsets of natural killer 1.1+T cell receptor-[alpha]/[beta]+cells in the liver of mice. J. Exp. Med.1994; 180:699-704.
32 Li W. An alternate pathway for CD4 T cell development:thymocyte-expressed MHC class II selects a distinct T cell population. Immunity 2005; 23:375-386.
33 Wei DG, Curran SA, Savage PB, et al. Mechanisms imposing the V[beta] bias of V[alpha]14 natural killer T cells and consequences for microbial glycolipid recognition. J. Exp. Med.2006; 203:1197-1207.
34 Schumann J, Mycko MP, Dellabona P, et al. Cutting edge:influence of the TCR V[beta] domain on the selection of semi-invariant NKT cells by endogenous ligands. J. Immunol.2006; 176:2064-2068.
35 Stanic AK. NF-[kappa]B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes. J. Immunol.2004; 172:2265-2273.
36 Chun T. CDld-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J. Exp. Med.2003; 197:907-918.
37 Tilloy F, Di Santo JP, Bendelac A, et al. Thymic dependence of invariant V[alpha]14+natural killer-T cell development. Eur. J. Immunol.1999; 29:3313-3318.
38 MC C, DH R. NK1.1+T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J. Immunol 2000,164:2412.
39 Hammond K, Cain W, Vandriel I, et al. Three day neonatal thymectomy selectively depletes NK1.1+T Cells. Int. Immunol.1998; 10:1491-1499.
40 Doisne JM, Bartholin L, Yan KP, et al. iNKT cell development is orchestrated by different branches of TGF-{beta} signaling. J Exp Med 2009; 206(6):1365-1378.