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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).

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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

免疫学 > 免疫细胞分离 > 淋巴细胞
作者: Marco Lepore
Marco LeporeAffiliation: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Bio-protocol author page: a2266
Sebastiano Sansano
Sebastiano SansanoAffiliation: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Bio-protocol author page: a2267
Claudia de Lalla
Claudia de LallaAffiliation: Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
Bio-protocol author page: a2268
Paolo Dellabona
Paolo DellabonaAffiliation: Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
Bio-protocol author page: a2269
Giulia Casorati
Giulia CasoratiAffiliation: Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
Bio-protocol author page: a2270
Gennaro De Libero
Gennaro De LiberoAffiliation 1: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Affiliation 2: Singapore Immunology Network (SIgN), Agency for Science, Technology, and Research, Singapore
Bio-protocol author page: a2271
 and Lucia Mori
Lucia MoriAffiliation 1: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Affiliation 2: Singapore Immunology Network (SIgN), Agency for Science, Technology, and Research, Singapore
For correspondence: lucia.mori@unibas.ch
Bio-protocol author page: a2272
Vol 5, Iss 11, 6/5/2015, 1910 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1491

[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)

[Abstract]

Materials and Reagents

  1. Autoreactive CD1-restricted T cell clones (generated as described in de Lalla et al. 2011)
  2. 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)
  3. Antigen presenting cells (APCs) expressing relevant CD1 isoforms and per se poorly stimulating autoreactive T cell
  4. RPMI 1640 (Amimed, catalog number: 1-41F01-I )
  5. Stable glutamine (Amimed, catalog number: 5-10K50-H )
  6. Sodium pyruvate (Amimed, catalog number: 5-60F00-H )
  7. Non essential amino acids (Amimed, catalog number: 5-13K00-H )
  8. Kanamycin (Amimed, catalog number: 4-08F00-H )
  9. Fetal bovine serum (Lonza, catalog number: DE14-802F )
  10. PBS without Ca2+ and Mg2+ (Amimed, catalog number: 3-05F29-I )
  11. ELISA
    1. 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 )
    2. HRP-streptavidin (BioLegend, catalog number: 405210 )
    3. OPD SIGMAFAST (Sigma-Aldrich, catalog number: P9187-50SET )
    4. Cytokine standards: Recombinant human GM-CSF (BioLegend, catalog number: 572409 ), recombinant human IFN-γ (BioLegend, catalog number: 570209 )
  12. Anti-CD1 blocking antibodies (anti-CD1c mAb) (Abcam, catalog number: ab18216-100 )
  13. Anti-human CD19 mAbs (Miltenyi Biotec, catalog number: 130-097-055 )
  14. Methanol (Applichem, catalog number: A0688 , 2500PE)
  15. Chloroform (Applichem, catalog number: A1585 , 1000)
  16. Ethyl acetate (Merck KGaA, catalog number: 1.00868.1000 )
  17. 1-Butanol (Sigma-Aldrich, catalog number: 34867 )
  18. Diisopropyl ether (Sigma-Aldrich, catalog number: 3827-IL-F )
  19. Isopropanol (Applichem, catalog number: A1592.2500 )
  20. Acetic acid (Applichem, catalog number: A2354.0500 )
  21. Acetone (Applichem, catalog number: A1567.2500 )
  22. Acetonitrile (Riedel-De Haen, catalog number: 34967 )
  23. Hexane (Sigma-Aldrich, catalog number: 34994 )
  24. HCl 37% fuming (Merck KGaA, catalog number: 317.1 000)
  25. Formic acid (Merck KGaA, catalog number: 1.11670.1000 )
  26. Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
  27. H2O (Sigma-Aldrich, catalog number: 95304 )
  28. Water-saturated butanol (see Recipes)
  29. Elution solutions for lipid fractionation on amino-cartridge (see Recipes)
  30. Complete medium (see Recipes)

Equipment

  1. Aminopropyl cartridges (SEP-PAK Vac 6 cc, 500 mg NH2 cartridges) (Waters Corporation, catalog number: WAT200606)
  2. HPLC system (Jasco)
  3. Nucleodur C18 Pyramid end-capped column (3-μm particle size, 3-mm ID, 125-mm length) (Macherey-Nagel, catalog number: N9040986 )
  4. Automated fraction collector (Gilson, catalog number: FC203B )
  5. Glass conical tubes (30 ml and 1 ml volumes, Glass Keller)
  6. Glass pipettes (Pirex)
  7. 96 wells flat bottom culture plates (BD Biosciences, Falcon®, catalog number: 353075 )
  8. 96 wells ELISA immune-plates (Maxisorp, Nunc, catalog number: 439454 )
  9. Humidified CO2 cell culture incubator (Heraeus, Hera cell 150)
  10. Spectrophotometer/ELISA Reader (Synergy H1 Hybrid Reader, BioTek Instruments)
  11. Sonicator (Sonics, Vibra Cell)
  12. Rotating wheel (Labinco BV, catalog number: 76000 )
  13. Manometer-regulated N2 gas tank (Carba gas)

Procedure

  1. Lipid extraction and fractionation
    The following lipid extraction and fractionation procedure is adapted from (Facciotti et al., 2012; Folch et al., 1957) and it is optimized for 109 cells. For different numbers of cells, adjust the volumes accordingly. This protocol was used to extract lipids from mouse thymocytes or THP-1 cells (ATCC TIB-202TM). For other types of cells the protocol may require optimization.

    Lipid extraction
    1. Pellet the cells by centrifugation at room temperature for 5 min at 300 x g, resuspend them in 10 ml PBS and transfer them in a glass tube.
      After this step use glass tubes exclusively.
    2. Wash cells 2x with 10 ml PBS by centrifugation (5 min at 300 x g) and completely remove the PBS by aspiration with a glass Pasteur pipette.
    3. Resuspend the pellet in 8 ml of a mixture of water/methanol/chloroform (1:1:2 vol/vol/vol).
    4. Sonicate 2 x 30 sec (5 Hz).
    5. Incubate 3 h at room temperature in a rotating wheel.
    6. Centrifuge 5 min at 3,100 x g at room temperature.
    7. Collect the organic layer (bottom layer) with a glass pipet avoiding contamination with the aqueous phase (upper layer) and store at -20 °C.
    8. Add 4 ml of methanol/chloroform (1:2 vol/vol) to the remaining aqueous phase.
    9. Vortex 2 min and incubate 1 h at room temperature in rotation.
    10. Centrifuge 5 min at 3,100 x g.
    11. Collect the organic (bottom) layer with a glass pipet avoiding contamination with the aqueous phase (upper layer) and store at -20 °C until use.
    12. Repeat steps A8-11 one additional time.
    13. Pool the collected organic phases and dry for ~2 h under nitrogen flow delivered as a gas stream (~3 bars) through a Pasteur pipette connected to a N2 source. Dissolve in 2 ml methanol/chloroform (1:2 vol/vol) and store at -20 °C. We refer to this extraction as “apolar” because it contains most of the cellular lipids with exception of the highly polar ones.
    14. Measure the volume of the remaining aqueous phase and add 1 vol of water-saturated butanol (see Recipes).
    15. Vortex 2 min and incubate 1 h at room temperature in a rotating wheel.
    16. Centrifuge for 5 min at 3,100 x g.
    17. Collect the upper layer (butanol phase), dry under nitrogen flow, dissolve in 2 ml methanol and store at -20 °C. As this extraction contains highly polar lipids poorly soluble in methanol/chloroform we call it “polar”.

    Lipid fractionation
    The following fractionation procedure allows the separation of both apolar and polar lipid extractions in 10 individual fractions of different polarity using aminopropyl cartridges. Apply the same procedure described here to both the lipid extractions separately using two different cartridges.
    1. Equilibrate the cartridge with 6 ml hexane.
    2. Load the cartridge with the total volume of extracted lipids (2 ml, Figure 1 A).
    3. Sequentially apply the indicated volumes of the eluents (see Recipes) and collect the fractions individually eluted by gravity in glass conical tubes (Figure 1B; each letter represents an eluent and it also identifies individual fractions, e.g. the fraction eluted with solution “a” is called “a”, etc.):
      1. 4 ml
      2. 4 ml
      3. 3 ml
      4. 11 ml
      5. 9 ml
      6. 3 ml
      7. 4.5 ml
      8. 6 ml
      9. 4 ml
      10. 4 ml
    4. Dry eluted fractions under nitrogen flow and dissolve each fraction in the solutions and volumes indicated below:
      1. 280 µl methanol/chloroform (1:2 vol/vol)
      2. 280 µl methanol/chloroform (1:1 vol/vol)
      3. 280 µl methanol/chloroform (1:1 vol/vol)
      4. 280 µl methanol/chloroform (1:1 vol/vol)
      5. 280 µl methanol/chloroform (1:1 vol/vol)
      6. 280 µl methanol/chloroform (9:1 vol/vol)
      7. 280 µl methanol/chloroform (9:1 vol/vol)
      8. 280 µl methanol/chloroform (9:1 vol/vol)
      9. 280 µl methanol/chloroform (9:1 vol/vol)
      10. 500 µl methanol/chloroform (9:1 vol/vol)
    5. Store each resuspended fraction at -20 °C.


      Figure 1. Scheme illustrating the lipid fractionation procedure. A. Loading of apolar and polar extracted lipids on two separate amino (NH2) cartridges. B. Sequential elution of lipid fractions from the amino cartridges. Letters indicate eluents and corresponding fractions. Numbers indicate the temporal order of elution and fraction collection.

  2. T cell activation assay
    In this section we describe how to evaluate the T cell stimulatory capacity of lipid preparations. To maximize assay sensitivity, a series of essential points have to be considered.
    1. Autoreactive T cell clones recognizing the cells from which the lipids are extracted, should be highly responsive.
    2. APCs should express high levels of the restriction molecules for optimal antigen presentation.
    3. APCs should be chosen for their poorly stimulatory capacity in the absence of exogenously added antigen, in order to minimize the levels of background stimulation of autoreactive T cells in the absence of tumor-derived lipids.
    As the T cell clones we have used in the experiments were CD1c-restricted, we chose freshly purified circulating B cells from healthy donors as APCs. These cells display high levels of cell surface CD1c and weakly stimulated T cell clones in the absence of added antigens (De Libero et al., 2005). Isolation of B cells was achieved by sorting using magnetic MicroBeads coupled to anti-human CD19 mAbs according to the manufacturer protocol. In another set of experiments we used low numbers of fixed THP-1 cells (105/well) transfected with CD1c gene (De Libero et al., 2005). We observed that fixation for 20 sec with 0.05% glutaraldehyde significantly reduced the CD1c-dependent T cell clone stimulatory capacity of THP-1-CD1c cells, thus making detection of lipid-specific T cell stimulation possible after addition of active lipid fractions. The use of fixed APCs, however, has an important limitation. Fixed cells are metabolically inactive and are unable to process antigens. Thus, if the unknown lipid antigen(s) require(s) processing steps, the use of fixed APCs will not reveal the T cell stimulatory activity of lipid fractions containing unprocessed antigens. Note that all the above issues (choice of T cells and APCs, fixation of APCs, etc.) need to be optimized in preliminary experiments according to individual experimental settings and objectives.
    1. Plate 1-5 x 105 APCs/well in 96 wells flat bottom plates in 45 µl of complete medium.
    2. Prepare at least 3 dilutions of each lipid fraction. Tested dilutions should be in the range 1:10-1:1,000 of the original preparation. As the fractions are dissolved in organic solvents, which are toxic for cells (volumes and type of solvents are indicated in the step B4 of the paragraph “Lipid fractionation”), they need to be dried (under nitrogen, to avoid oxygen-induced lipid alterations) and offered to the cells in a solution, which is compatible with cell viability. Generally, 20 µl of each fraction are transferred into a new glass conical tube, dried, dissolved in 20 µl of PBS 20% methanol (vol/vol, vehicle) and sonicated. Dissolved fractions are then used to pulse APCs at various dilutions.
    3. Add fractions to plated APCs. As example, 5 µl of each dissolved fraction (undiluted or previously diluted 1:10 or 1:100 using PBS 20% methanol) are added to the wells containing APCs in 45 µl of complete medium. In this way each lipid fraction will be finally diluted 1:10, 1:100 or 1:1000, respectively in the culture wells. Importantly, the methanol contained in the vehicle in which the fractions are dissolved will also be diluted to a final concentration of 2%, which is compatible with APC viability and T cell activation. Include control wells in which only the vehicle (5 µl of PBS 20% methanol) is added to the 45 µl of APCs. Perform triplicate replicas of each experimental condition.
    4. Incubate 4 h at 37 °C in humidified CO2 incubator.
    5. Add 5 x 105 T cells/well in 50 µl of complete medium.
    6. Incubate 24-48 h at 37 °C in humidified CO2 incubator.
    7. Collect supernatants and measure cytokine release by standard sandwich ELISA (according to the manufacturing protocols; see Materials and Reagents). We measured release of GM-CSF (after 24 h) and IFN-γ (after 48 h) as read out of T cell activation. However, it is important to note that the choice of the cytokine needs to be done according to the cytokines more abundantly released by tested T cells and excluding those released by the APCs, which should be determined in preliminary experiments. In general, GM-CSF release represents a very sensitive and relatively fast read out for in vitro T cell clone activation. Measuring at least two different cytokines is recommendable to avoid false positive results. Lipid fractions are considered positive if they induce dose-dependent T cell cytokine release. Results are expressed as fold change over background (cytokine release in the presence of APCs incubated with lipid/cytokine release with APCs incubated with vehicle). Fold change ≥ 2 is considered relevant.
    8. The activity of stimulatory fraction(s) has to be confirmed in a second set of experiments, in which blocking antibodies specific for the CD1 isoform that restricts the response of the T cells used are included. Blocking antibodies and appropriate isotype control antibodies are used at a final concentration of 20 µg/ml and are added to APCs incubated with active lipid fractions at least 30 min before T cells.

  3. Sub-fractionation of active lipid fractions
    Once one or more T cell stimulatory lipid fractions are identified a second fractionation step is made to further purify the antigenic lipid molecules.
    The procedure described in this section uses reverse-phase HPLC performed with a Nucleodur C18 Pyramid end-capped column (3-μm particle size, 3-mm ID) and two mobile phases:
    Mobile phase A: methanol/water/formic acid (74:25:1, vol/vol/vol) (pH 4.0)
    Mobile phase B: methanol/formic acid (99:1, vol/vol) (pH 4.0)
    The following gradient was used to isolate polar lipids:
    1. B 20% from time 0 to min 1
    2. B from 20% to 50% from min 1 to min 2
    3. B 50% from min 2 to min 4
    4. B from 50% to 100% to min 4 to min 34
    5. B 100% from min 34 to min 54
    6. B from 100% to 20% from min 54 to min 55
    7. B 20% from min 55 to min 60
    The flow rate was 0.5 ml/min.
    1. Inject the active lipid fraction in the HPLC system.
    2. Collect individual sub-fractions every 30 sec using an automated collector connected to the HPLC system.
    3. Dry the sub-fractions under nitrogen flow and resuspend fractionated lipids in 10 µl methanol.
    4. Perform T cell activation assay as described above by testing all individual sub-fractions at various dilutions.
    5. Store the rest of the sub-fractions at -20 °C for further analyses.
    After identification of T cell stimulatory lipid fractions, structural analyses need to be performed to characterize the active lipid molecule(s). Liquid chromatography-tandem MS, high-resolution mass spectrometry and NMR are the techniques of choice (De Libero et al., 2005; Lepore et al., 2014).

Representative data



Figure 2. A. Response of a CD1-restricted human T cell clone to lipid fractions (A-J) extracted from THP-1 cells. Three dilutions of each fraction were tested. GM-CSF released in the supernatant was measured by ELISA and expressed as fold change over background. B. HPLC profile corresponding to the sub-fractionation of the T cell stimulatory lipid fraction G in panel A. Sub-fractions were collected every 30 sec. C. GM-CSF release by the CD1-restricted T cell clone to HPLC sub-fractions of fraction G. Each sub-fraction was tested at two dilutions (1:10, filled columns, and 1:100, open columns). Bars indicate sd.

For more representative data also refer to the following: T cell activation assay, Figure 3 (Lepore et al., 2014); Lipid sub-fractionation, Figure 1 (Facciotti et al., 2012) and Figure 2 (Lepore et al., 2014).

Recipes

  1. Water-saturated butanol
    1-Butanol/H2O 1:1, vol/vol
    Vigorously shake 5 min, let stand for at least 30 min to allow phase separation. Use the saturated butanol (upper phase).
  2. Eluents for lipid fractionation on amino-cartridges
    1. Ethyl acetate/hexane 15:85, vol/vol
    2. Chloroform/methanol 23:1 vol/vol
    3. Diisopropyl ether/ acetic acid 98:5, vol/vol
    4. Acetone/methanol 9:1.35, vol/vol
    5. Chloroform/methanol 2:1, vol/vol
    6. Methanol
    7. Isopropanol/3 N HCl in methanol 4:1, vol/vol
    8. Methanol/3 N HCl in methanol 9:1, vol/vol
    9. Chloroform/methanol 2:1, vol/vol
    10. Chloroform/methanol/3.6 M ammonium acetate in water 30/60/8, vol/vol/vol
  3. Complete medium
    RPMI 1640
    2 mM stable glutamine
    1 mM sodium pyruvate
    1 mM non-essential amino acids
    50 µg/ml kanamycin
    10% heat-inactivated fetal bovine serum

Acknowledgements

This work was supported by Grants of the Swiss National Science Foundation (NMS1813), A*STAR/Australian NHMRC (1201826277) and University of Basel (DMS2306). The protocols described here were used in (Facciotti et al., 2012) and (Lepore et al., 2014).

References

  1. Bodennec, J., Koul, O., Aguado, I., Brichon, G., Zwingelstein, G. and Portoukalian, J. (2000). A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J Lipid Res 41(9): 1524-1531.
  2. Chang, D. H., Deng, H., Matthews, P., Krasovsky, J., Ragupathi, G., Spisek, R., Mazumder, A., Vesole, D. H., Jagannath, S. and Dhodapkar, M. V. (2008). Inflammation-associated lysophospholipids as ligands for CD1d-restricted T cells in human cancer. Blood 112(4): 1308-1316.
  3. de Jong, A., Pena-Cruz, V., Cheng, T. Y., Clark, R. A., Van Rhijn, I. and Moody, D. B. (2010). CD1a-autoreactive T cells are a normal component of the human alphabeta T cell repertoire. Nat Immunol 11(12): 1102-1109.
  4. de Lalla, C., Lepore, M., Piccolo, F. M., Rinaldi, A., Scelfo, A., Garavaglia, C., Mori, L., De Libero, G., Dellabona, P. and Casorati, G. (2011). High-frequency and adaptive-like dynamics of human CD1 self-reactive T cells. Eur J Immunol 41(3): 602-610.
  5. De Libero, G., Moran, A. P., Gober, H. J., Rossy, E., Shamshiev, A., Chelnokova, O., Mazorra, Z., Vendetti, S., Sacchi, A., Prendergast, M. M., Sansano, S., Tonevitsky, A., Landmann, R. and Mori, L. (2005). Bacterial infections promote T cell recognition of self-glycolipids. Immunity 22(6): 763-772.
  6. Facciotti, F., Ramanjaneyulu, G. S., Lepore, M., Sansano, S., Cavallari, M., Kistowska, M., Forss-Petter, S., Ni, G., Colone, A., Singhal, A., Berger, J., Xia, C., Mori, L. and De Libero, G. (2012). Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus. Nat Immunol 13(5): 474-480.
  7. Folch, J., Lees, M. and Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1): 497-509.
  8. Lepore, M., de Lalla, C., Gundimeda, S. R., Gsellinger, H., Consonni, M., Garavaglia, C., Sansano, S., Piccolo, F., Scelfo, A., Haussinger, D., Montagna, D., Locatelli, F., Bonini, C., Bondanza, A., Forcina, A., Li, Z., Ni, G., Ciceri, F., Jeno, P., Xia, C., Mori, L., Dellabona, P., Casorati, G. and De Libero, G. (2014). A novel self-lipid antigen targets human T cells against CD1c(+) leukemias. J Exp Med 211(7): 1363-1377.

材料和试剂

  1. 自身反应性CD1限制性T细胞克隆(如de Lalla等人 2011中所述产生)
  2. THP-1细胞(ATCC TIB-202 TM或其它靶细胞,其能够以CD1依赖性方式和在不存在外源提供的抗原的情况下刺激T细胞克隆)
  3. 表达相关CD1同种型和本身刺激性自身反应性T细胞的抗原呈递细胞(APC)
  4. RPMI 1640(Amimed,目录号:1-41F01-I)
  5. 稳定的谷氨酰胺(Amimed,目录号:5-10K50-H)
  6. 丙酮酸钠(Amimed,目录号:5-60F00-H)
  7. 非必需氨基酸(Amimed,目录号:5-13K00-H)
  8. 卡那霉素(Amimed,目录号:4-08F00-H)
  9. 胎牛血清(Lonza,目录号:DE14-802F)
  10. PBS(不含Ca 2+和Mg 2+)(Amimed,目录号:3-05F29-I)。
  11. ELISA
    1. MAb对:纯化的抗人GM-CSF(BioLegend,目录号: 502202),生物素偶联的抗人GM-CSF(BioLegend,目录号:   502304); 纯化的抗人IFN-γ(BioLegend,目录号:50750), 生物素结合的抗-IFN-γ(BioLegend,目录号:502504)
    2. HRP-链霉亲和素(BioLegend,目录号:405210)
    3. OPD SIGMAFAST(Sigma-Aldrich,目录号:P9187-50SET)
    4. 细胞因子标准:重组人GM-CSF(BioLegend, 编号:572409),重组人IFN-γ(BioLegend,目录号: 570209)
  12. 抗CD1阻断抗体(抗CD1c mAb)(Abcam,目录号:ab18216-100)
  13. 抗人CD19mAbs(Miltenyi Biotec,目录号:130-097-055)
  14. 甲醇(Applichem,目录号:A0688,2500PE)
  15. 氯仿(Applichem,目录号:A1585,1000)
  16. 乙酸乙酯(Merck KGaA,目录号:1.00868.1000)
  17. 1-丁醇(Sigma-Aldrich,目录号:34867)
  18. 二异丙醚(Sigma-Aldrich,目录号:3827-IL-F)
  19. 异丙醇(Applichem,目录号:A1592.2500)
  20. 乙酸(Applichem,目录号:A2354.0500)
  21. 丙酮(Applichem,目录号:A1567.2500)
  22. 乙腈(Riedel-De Haen,目录号:34967)
  23. 己烷(Sigma-Aldrich,目录号:34994)
  24. HCl 37%发烟(Merck KGaA,目录号:317.1000)
  25. 甲酸(Merck KGaA,目录号:1.11670.1000)
  26. 乙酸铵(Sigma-Aldrich,目录号:A1542)
  27. H 2 O(Sigma-Aldrich,目录号:95304)
  28. 水饱和的丁醇(见配方)
  29. 氨基盒上脂质分馏的洗脱溶液(参见配方)
  30. 完整介质(见配方)

设备

  1. 氨基丙基柱(SEP-PAK Vac 6cc,500mg NH 2柱)(Waters Corporation,目录号:WAT200606)
  2. HPLC系统(Jasco)
  3. Nucleosur C18 Pyramid end-capped column(3-μmparticle size,3-mm ID,125-mm length)(Macherey-Nagel,目录号:N9040986)
  4. 自动馏分收集器(Gilson,目录号:FC203B)
  5. 玻璃锥形管(30ml和1ml体积,Glass Keller)
  6. 玻璃移液管(Pirex)
  7. 96孔平底培养板(BD Biosciences,Falcon ,目录号:353075)
  8. 96孔ELISA免疫板(Maxisorp,Nunc,目录号:439454)
  9. 加湿的CO 2细胞培养箱(Heraeus,Hera cell 150)
  10. 分光光度计/ELISA读数器(Synergy H1 Hybrid Reader,BioTek Instruments)
  11. 超声波仪(Sonics,Vibra Cell)
  12. 旋转轮(Labinco BV,目录号:76000)
  13. 压力计调节的N 2气罐(Carba气体)

程序

  1. 脂质提取和分馏
    以下脂质提取和分级分离程序改编自(Facciotti等人,2012; Folch等人,1957),并且其针对10μM,/sup>细胞。 对于不同数量的单元格,请相应地调整卷。 该方案用于从小鼠胸腺细胞或THP-1细胞(ATCC TIB-202 TM)提取脂质。 对于其他类型的单元格,协议可能需要优化
    脂质提取
    1. 通过在室温下在300×下离心5分钟来沉淀细胞 g,将它们重悬于10ml PBS中并将其转移到玻璃管中 此步骤后,仅使用玻璃管。
    2. 通过离心(300×g /分钟)5分钟用10ml PBS洗涤细胞2次 并用玻璃巴斯德吸出完全除去PBS 吸管。
    3. 将沉淀重悬在8ml水/甲醇/氯仿(1:1:2体积/体积/体积)的混合物中。
    4. 超声2×30秒(5Hz)。
    5. 在室温下在旋转轮中孵育3小时。
    6. 在室温下以3,100×g离心5分钟。
    7. 用玻璃吸管收集有机层(底层)避免   污染水相(上层)并储存在-20℃
    8. 向剩余的水相中加入4ml甲醇/氯仿(1:2体积/体积)
    9. 涡旋2分钟,并在室温下旋转孵育1小时
    10. 在3,100×g离心5分钟。
    11. 收集有机(底部)层用玻璃吸管避免 用水相(上层)污染并储存在-20℃ 直到使用。
    12. 重复步骤A8-11一次。
    13. 收集收集的有机相,在氮气流下干燥〜2小时 作为气流(〜3巴)通过连接的巴斯德吸管输送 到N 2源。 溶于2ml甲醇/氯仿(1:2vol/vol)中 储存于-20°C。 我们将这种提取称为"非极性",因为它 包含除高度极性之外的大多数细胞脂质 那些。
    14. 测量剩余水相的体积,并加入1体积的水饱和的丁醇(见配方)
    15. 涡旋2分钟,并在室温下在旋转轮中孵育1小时
    16. 在3,100×g离心5分钟。
    17. 收集上层(丁醇相),在氮气流下干燥, 溶于2ml甲醇中,-20℃保存。 作为这种提取 含有难溶于甲醇/氯仿的高极性脂质 称为"极性"。

    脂质分馏
    以下分馏程序允许使用氨基丙基筒分离10个不同极性的单独级分中的非极性和极性脂质提取物。 应用这里描述的相同的程序两个脂质提取单独使用两个不同的墨盒。
    1. 用6ml己烷平衡滤筒。
    2. 用提取的脂质的总体积(2ml,图1A)装载筒
    3. 依次使用指定体积的洗脱液(见 食谱),并收集通过重力洗脱的级分 玻璃锥形管(图1B;每个字母表示洗脱液和它 也标识个别级分,例如洗脱的级分 解决方案"a"被称为"a",等。):
      1. 4 ml
      2. 4 ml
      3. 3 ml
      4. 11 ml
      5. 9 ml
      6. 3 ml
      7. 4.5 ml
      8. 6毫升
      9. 4 ml
      10. 4ml
    4. 在氮气流下干燥洗脱级分,并将每种级分溶解在如下所示的溶液和体积中:
      1. 280μl甲醇/氯仿(1:2vol/vol)
      2. 280μl甲醇/氯仿(1:1体积/体积)
      3. 280μl甲醇/氯仿(1:1体积/体积)
      4. 280μl甲醇/氯仿(1:1体积/体积)
      5. 280μl甲醇/氯仿(1:1体积/体积)
      6. 280μl甲醇/氯仿(9:1体积/体积)
      7. 280μl甲醇/氯仿(9:1体积/体积)
      8. 280μl甲醇/氯仿(9:1体积/体积)
      9. 280μl甲醇/氯仿(9:1体积/体积)
      10. 500μl甲醇/氯仿(9:1体积/体积)
    5. 将每个重悬浮的馏分储存在-20℃

      图1.说明脂质分级程序的方案。 A. 将非极性和极性提取的脂质加载到两个单独的氨基(NH 2)   墨盒。 B.从氨基序列洗脱脂质部分 墨盒。 字母表示洗脱液和相应的级分。 数字表示洗脱和馏分收集的时间顺序。

  2. T细胞活化测定
    在本节中,我们描述如何评估脂质制剂的T细胞刺激能力。 为了最大化测定灵敏度,必须考虑一系列基本点
    1. 识别提取脂质的细胞的自身反应性T细胞克隆应当是高度响应的
    2. APC应该表达高水平的限制性分子,以获得最佳的抗原呈递
    3. 应选择APC的刺激能力差 不存在外源添加的抗原,以便使水平最小化  背景刺激的自身反应性T细胞缺失 肿瘤衍生的脂质。
    由于我们在实验中使用的T细胞克隆是CD1c限制性的,我们选择来自健康供体的新鲜纯化的循环B细胞作为APC。这些细胞在不存在添加的抗原的情况下显示高水平的细胞表面CD1c和弱刺激的T细胞克隆(De Libero等人,2005)。通过根据制造商方案使用偶联于抗人CD19mAb的磁性MicroBeads进行分选来实现B细胞的分离。在另一组实验中,我们使用用CD1c基因转染的低数量的固定THP-1细胞(10×10 6 /孔)(De Libero等人,2005)。我们观察到用0.05%戊二醛固定20秒显着降低THP-1-CD1c细胞的CD1c依赖性T细胞克隆刺激能力,从而使得在加入活性脂质级分后可能检测脂质特异性T细胞刺激。然而,固定APC的使用具有重要的限制。固定的细胞是代谢失活的,不能处理抗原。因此,如果未知的脂质抗原需要处理步骤,则使用固定的APC不会揭示含有未加工抗原的脂质级分的T细胞刺激活性。注意,根据个体实验设置和目标,需要在初步实验中优化所有上述问题(T细胞和APC的选择,APC的固定,等)。
    1. 在45μl完全培养基中的96孔平底板中铺板1-5×10 5个APC /孔。
    2. 准备每个脂质部分的至少3稀释。测试 稀释度应在原料的1:10-1:1,000的范围内 制备。当级分溶解在有机溶剂中时, 对细胞有毒性(溶剂的体积和类型在 段落"脂质分馏"的步骤B4),它们需要干燥 (在氮气下,以避免氧诱导的脂质改变)并提供 到溶液中的细胞,其与细胞活力相容。 通常,将20μl的每个级分转移到新的玻璃中 锥形管,干燥,溶于20μlPBS中20%甲醇(vol/vol, 车辆)并超声处理。然后将溶解的级分用于脉冲APC 。
    3. 添加分数到镀敷的APC。例如,5  μl的各溶解级分(未稀释或预先稀释1:10或 1:100,使用PBS 20%甲醇)加入到含有APC的孔中 45微升完全培养基。以这种方式,每个脂质部分将是 在培养物中分别以1:10,1:100或1:1000稀释 井。 重要的是,包含在载体中的甲醇, 级分溶解也将被稀释至终浓度   2%,其与APC存活力和T细胞活化相容。 包括对照孔,其中只有载体(5μlPBS 20% 甲醇)加入到45μl的APC中。 执行一式三份的副本 每个实验条件
    4. 在37℃在潮湿CO 2培养箱中孵育4小时
    5. 在50μl完全培养基中加入5×10 5个T细胞/孔
    6. 在37℃在潮湿CO 2培养箱中孵育24-48小时。
    7. 收集上清液并通过标准测量细胞因子释放 夹心ELISA(根据制造方案;参见材料 和试剂)。我们测量GM-CSF(24小时后)和IFN-γ的释放 (48小时后)作为T细胞活化读出。但是,这是很重要的 注意到细胞因子的选择需要根据 通过测试的T细胞更多地释放细胞因子并排除 那些由APC提供的,应该在初步确定 实验。一般来说,GM-CSF释放代表非常敏感和 相对快速地读出用于体外T细胞克隆激活。测量  至少两种不同的细胞因子是可以避免假的 阳性结果。如果脂质部分诱导,则认为它们是阳性的  剂量依赖性T细胞细胞因子释放。结果表示为fold 改变背景(在APC存在下的细胞因子释放) 与脂质/细胞因子释放与用载体孵育的APC孵育)。  倍数变化≥2被认为相关。
    8. 活动的 刺激级分必须在第二组中被证实 实验,其中特异性针对CD1同种型的阻断抗体 其包括限制所使用的T细胞的应答。 阻塞 抗体和合适的同种型对照抗体用于a 最终浓度为20μg/ml,并加入到与之孵育的APC中 活性脂质级分在T细胞之前至少30分钟。

  3. 活性脂质级分的亚分级分离 一旦鉴定一个或多个T细胞刺激性脂质级分,进行第二分级分离步骤以进一步纯化抗原性脂质分子。
    本节中描述的步骤使用用Nucleodur C18 Pyramid封端柱(3-μm粒径,3-mm ID)和两个流动相进行的反相HPLC:
    流动相A:甲醇/水/甲酸(74:25:1,体积/体积/体积)(pH4.0)
    流动相B:甲醇/甲酸(99:1,体积/体积)(pH4.0) 使用以下梯度分离极性脂质:
    1. B从时间0到min 1的20%
    2. B从20%到50%从min 1到min 2
    3. B 50%从min 2到min 4
    4. B从50%至100%至最小4至最小34
    5. B 100%从min 34到min 54
    6. B从100%到20%从最小54到最小55
    7. B 20%从min 55到min 60
    流速为0.5ml/min
    1. 将活性脂质级分注入HPLC系统中
    2. 每30秒使用连接到HPLC系统的自动收集器收集单独的子级分
    3. 在氮气流下干燥亚级分,并在10μl甲醇中重悬分级分离的脂质。
    4. 如上所述通过在各种稀释度下测试所有单独的亚级分来进行T细胞活化测定。
    5. 将其余的亚级分保存在-20°C用于进一步分析。
    在鉴定T细胞刺激性脂质级分后,需要进行结构分析以表征活性脂质分子。 液相色谱 - 串联质谱,高分辨率质谱和NMR 技术(De Libero等人,2005; Lepore等人,2014年)。

代表数据



图A.A.CD1限制性人T细胞克隆对从THP-1细胞提取的脂质级分(A-J)的应答。测试每个级分的三个稀释度。通过ELISA测量上清液中释放的GM-CSF,并表示为相对于背景的倍数变化。 B.对应于图A中T细胞刺激性脂质部分G的亚分级分离的HPLC图谱。每30秒收集次级分。 C.通过CD1限制性T细胞克隆将GM-CSF释放到级分G的HPLC亚级分中。以两个稀释度(1:10,填充柱和1:100,空心柱)测试每个亚级分。栏表示sd。

对于更具代表性的数据还参考以下:T细胞活化测定,图3(Lepore等人,2014);脂质亚分级分离,图1(Facciotti等人,2012)和图2(Lepore等人,2014)。

食谱

  1. 水饱和的丁醇
    1-丁醇/H 2 O 1:1,体积/体积 剧烈摇动5分钟,静置至少30分钟以使相分离。 使用饱和丁醇(上相)。
  2. 氨基管上的脂质分馏洗脱液
    1. 乙酸乙酯/己烷15:85,体积/体积
    2. 氯仿/甲醇23:1体积/体积
    3. 二异丙醚/乙酸98:5,体积/体积
    4. 丙酮/甲醇9:1.35,体积/体积
    5. 氯仿/甲醇2:1,体积/体积
    6. 甲醇
    7. 异丙醇/3N HCl的甲醇溶液4:1,体积/体积
    8. 甲醇/3N HCl的甲醇溶液9:1,体积/体积
    9. 氯仿/甲醇2:1,体积/体积
    10. 氯仿/甲醇/3.6M乙酸铵水溶液30/60/8,vol/vol/vol
  3. 完成媒介
    RPMI 1640
    2mM稳定的谷氨酰胺 1mM丙酮酸钠 1 mM非必需氨基酸
    50μg/ml卡那霉素 10%热灭活的胎牛血清

致谢

这项工作是由瑞士国家科学基金会(NMS1813),A * STAR /澳大利亚NHMRC(1201826277)和巴塞尔大学(DMS2306)的拨款支持。 这里描述的方案用于(Facciotti等人,2012)和(Lepore等人,2014)。

参考文献

  1. Bodennec,J.,Koul,O.,Aguado,I.,Brichon,G.,Zwingelstein,G.and Portoukalian,J。(2000)。 通过氨基丙酸柱固相萃取法分离鞘脂类别的程序 < em> J Lipid Res 41(9):1524-1531。
  2. Chang,D.H.,Deng,H.,Matthews,P.,Krasovsky,J.,Ragupathi,G.,Spisek,R.,Mazumder,A.,Vesole,D.H.,Jagannath,S.and Dhodapkar, 炎症相关溶血磷脂作为人类癌症中CD1d限制性T细胞的配体。 em> Blood 112(4):1308-1316。
  3. de Jong,A.,Pena-Cruz,V.,Cheng,T.Y.,Clark,R.A.,Van Rhijn,I.and Moody,D.B。(2010)。 CD1a自身反应性T细胞是人类字母表T细胞库的正常组成部分。 Nat Immunol 11(12):1102-1109。
  4. de Lalla,C。,Lepore,M.,Piccolo,FM,Rinaldi,A.,Scelfo,A.,Garavaglia,C.,Mori,L.,De Libero,G.,Dellabona,P.and Casorati, (2011)。 人类CD1自身反应性T细胞的高频和适应性动力学。 Eur J Immunol 41(3):602-610。
  5. De Libero,G.,Moran,AP,Gober,HJ,Rossy,E.,Shamshiev,A.,Chelnokova,O.,Mazorra,Z.,Vendetti,S.,Sacchi,A.,Prendergast,MM,Sansano, S.,Tonevitsky,A.,Landmann,R。和Mori,L。(2005)。 细菌感染促进自身糖脂的T细胞识别。 免疫 em> 22(6):763-772。
  6. Facciotti,F.,Ramanjaneyulu,GS,Lepore,M.,Sansano,S.,Cavallari,M.,Kistowska,M.,Forss-Petter,S.,Ni,G.,Colone,A.,Singhal, ,Berger,J.,Xia,C.,Mori,L.and De Libero,G。(2012)。 Peroxisome衍生的脂质是刺激胸腺中不变的天然杀伤T细胞的自身抗原。 Nat Immunol 13(5):474-480
  7. Folch,J.,Lees,M。和Sloane Stanley,G.H。(1957)。 从动物组织中分离和纯化总脂质的简单方法 J Biol Chem 226(1):497-509
  8. Lepore,M.,de Lalla,C.,Gundimeda,SR,Gsellinger,H.,Consonni,M.,Garavaglia,C.,Sansano,S.,Piccolo,F.,Scelfo,A.,Haussinger, Montagna,D.,Locatelli,F.,Bonini,C.,Bondanza,A.,Forcina,A.,Li,Z.,Ni,G.,Ciceri,F.,Jeno,P., Mori,L.,Dellabona,P.,Casorati,G.and De Libero,G。(2014)。 一种新型自身脂质抗原靶向人类T细胞抗CD1c(+)白血病。 J Exp Med 211(7):1363-1377。
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How to cite this protocol: 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; Full Text



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