Extraction and Identification of T Cell Stimulatory Self-lipid Antigens
提取和鉴定可激活T淋巴细胞的自身脂质抗原
发布: 2015年06月05日第5卷第11期 DOI: 10.21769/BioProtoc.1491 浏览次数: 9798
评审: Savita NairRamalingam BethunaickanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
Abstract
Autoreactive T cells restricted to CD1 molecules and specific for endogenous lipids are abundant in human blood (de Jong et al., 2010; de Lalla et al., 2011). A few self-lipid molecules recognized by diverse individual T cell clones and accumulated within APCs following stress signals or cell transformation have been identified so far (de Jong et al., 2010; Chang et al., 2008; Lepore et al., 2014). These findings suggested that auto-reactive CD1-restricted T cells display broad lipid specificities and may play critical roles in different types of immune responses including cancer immune surveillance, autoimmunity and antimicrobial immunity. Therefore, the identification of the repertoire of self-lipid molecules recognized by T cells is important to study the physiologic functions of this T cell population and to assess their therapeutic potential (Lepore et al., 2014). Here we describe the protocol we established to isolate and identify endogenous lipids derived from leukemia cells, which stimulate specific autoreactive CD1c-restricted T lymphocytes (Lepore et al., 2014). This protocol can be applied to isolate lipid antigens from any type of target cells and to investigate the self-lipid antigen specificity of autoreactive T cells restricted to all CD1 isoforms (Facciotti et al., 2012).
Keywords: Self-lipid antigens (自脂质抗原)
CD1 (CD1)
Autoreactive T cells (自身反应性T细胞)
Materials and Reagents
- Autoreactive CD1-restricted T cell clones (generated as described in de Lalla et al. 2011)
- THP-1 cells (ATCC TIB-202TM, or other target cells able to stimulate the T cell clones in a CD1-dependent manner and in the absence of exogenously provided antigens)
- Antigen presenting cells (APCs) expressing relevant CD1 isoforms and per se poorly stimulating autoreactive T cell
- RPMI 1640 (Amimed, catalog number: 1-41F01-I )
- Stable glutamine (Amimed, catalog number: 5-10K50-H )
- Sodium pyruvate (Amimed, catalog number: 5-60F00-H )
- Non essential amino acids (Amimed, catalog number: 5-13K00-H )
- Kanamycin (Amimed, catalog number: 4-08F00-H )
- Fetal bovine serum (Lonza, catalog number: DE14-802F )
- PBS without Ca2+ and Mg2+ (Amimed, catalog number: 3-05F29-I )
- ELISA
- MAb pairs: Purified anti-human GM-CSF (BioLegend, catalog number: 502202 ), biotin-conjugated anti-human GM-CSF (BioLegend, catalog number: 502304 ); purified anti-human IFN-γ (BioLegend, catalog number: 50750 ), biotin-conjugated anti-IFN-γ (BioLegend, catalog number: 502504 )
- HRP-streptavidin (BioLegend, catalog number: 405210 )
- OPD SIGMAFAST (Sigma-Aldrich, catalog number: P9187-50SET )
- Cytokine standards: Recombinant human GM-CSF (BioLegend, catalog number: 572409 ), recombinant human IFN-γ (BioLegend, catalog number: 570209 )
- MAb pairs: Purified anti-human GM-CSF (BioLegend, catalog number: 502202 ), biotin-conjugated anti-human GM-CSF (BioLegend, catalog number: 502304 ); purified anti-human IFN-γ (BioLegend, catalog number: 50750 ), biotin-conjugated anti-IFN-γ (BioLegend, catalog number: 502504 )
- Anti-CD1 blocking antibodies (anti-CD1c mAb) (Abcam, catalog number: ab18216-100 )
- Anti-human CD19 mAbs (Miltenyi Biotec, catalog number: 130-097-055 )
- Methanol (Applichem, catalog number: A0688, 2500PE )
- Chloroform (Applichem, catalog number: A1585, 1000 )
- Ethyl acetate (Merck KGaA, catalog number: 1.00868.1000 )
- 1-Butanol (Sigma-Aldrich, catalog number: 34867 )
- Diisopropyl ether (Sigma-Aldrich, catalog number: 3827-IL-F )
- Isopropanol (Applichem, catalog number: A1592.2500 )
- Acetic acid (Applichem, catalog number: A2354.0500 )
- Acetone (Applichem, catalog number: A1567.2500 )
- Acetonitrile (Riedel-De Haen, catalog number: 34967 )
- Hexane (Sigma-Aldrich, catalog number: 34994 )
- HCl 37% fuming (Merck KGaA, catalog number: 317.1000 )
- Formic acid (Merck KGaA, catalog number: 1.11670.1000 )
- Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
- H2O (Sigma-Aldrich, catalog number: 95304 )
- Water-saturated butanol (see Recipes)
- Elution solutions for lipid fractionation on amino-cartridge (see Recipes)
- Complete medium (see Recipes)
Equipment
- Aminopropyl cartridges (SEP-PAK Vac 6 cc, 500 mg NH2 cartridges) (Waters Corporation, catalog number: WAT200606)
- HPLC system (Jasco)
- Nucleodur C18 Pyramid end-capped column (3-μm particle size, 3-mm ID, 125-mm length) (Macherey-Nagel, catalog number: N9040986 )
- Automated fraction collector (Gilson, catalog number: FC203B )
- Glass conical tubes (30 ml and 1 ml volumes, Glass Keller)
- Glass pipettes (Pirex)
- 96 wells flat bottom culture plates (BD Biosciences, Falcon®, catalog number: 353075 )
- 96 wells ELISA immune-plates (Maxisorp, Nunc, catalog number: 439454 )
- Humidified CO2 cell culture incubator (Heraeus, Hera cell 150)
- Spectrophotometer/ELISA Reader (Synergy H1 Hybrid Reader, BioTek Instruments)
- Sonicator (Sonics, Vibra Cell)
- Rotating wheel (Labinco BV, catalog number: 76000 )
- Manometer-regulated N2 gas tank (Carba gas)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Lepore, M., Sansano, S., Lalla, C. D., Dellabona, P., Casorati, G., Libero, G. D. and Mori, L. (2015). Extraction and Identification of T Cell Stimulatory Self-lipid Antigens. Bio-protocol 5(11): e1491. DOI: 10.21769/BioProtoc.1491.
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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
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