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Glycoprotein methods protocols - biotechnology
Mucin Antigen Presentation Using Dendritic Cells 48748740Mucin Antigen Presentation Using Dendritic CellsJoy Burchell, Rosalind Graham, and Joyce Taylor-Papadimitriou1. IntroductionThe study of humoral and cellular responses to mucins requires many of the stan-dard immunologic techniques, although working with molecules as large as mucinssometimes leads to logistic problems. This chapter focuses on some of the techniquesthat may be used to analyze the immune response to mucins using dendritic cells topresent mucin peptides.Changes in the glycans, carried both on proteins and lipids, has long been associ-ated with the change to malignancy (1). In the case of proteins, many of these glycansare carried on mucins (2–4). This aberrant glycosylation may make the mucin anti-genically distinct to that expressed by normal cells, and it is relatively easy to envisagehow this would affect the humoral response to the molecule (5). However, there is anincreasing amount of evidence that glycosylated peptides can be presented to T-cellsvia the major histocompatibility complex (MHC) molecules (6,7). Galli-Stampino etal. have shown that peptides carrying simple O-linked core 1 sugars (Galβ1-3GalNAc)can bind to MHC class II molecules and be presented to T-cells, whereas the morecomplex core 2 sugars cannot. This is of particular relevance with regard to the immu-nogenicity of mucins because often the aberrant glycosylation of mucins that isobserved in carcinomas results in the expression of the simple core 1 structure and Tnand STn (8,9), and the revealing of normally cryptic peptide epitopes.The mucin that has been most extensively studied with regard to its immunogenic-ity is MUC1, a membrane-bound epithelial mucin. Much attention has been focusedon MUC1 as a potential target for active specific immunotherapy because this mucinis over- expressed and aberrantly glycosylated by many carcinomas but particularlythose of the breast and ovary (10). Like all mucins, MUC1 has a large domain oftandemly repeated amino acids allowing potential epitopes to be repeated many times.Humoral responses have been identified in breast cancer patients (5) and cytotoxicT-lymphnodes (CTLs) have been isolated from cancer patients that can kill MUC1-expressing target cells in a non-MHC-restricted manner (see Chapter 39). However,From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 488 Burchell et al.classic MHC class I epitopes have also been identified within the tandem repeat (TR)of MUC1 (11) and T-helper epitopes may also be present.Dendritic cells are the most potent antigen-presenting cells (APCs) of the body andare involved in the presentation of antigens to naive T-cells. Human dendritic cells canbe isolated from peripheral blood mononuclear cells (PBMCs) by culturing the adher-ent cells in interleukin-4 (IL-4) and GM-CSF (12,13), whereas mouse dendritic cellscan be obtained from bone marrow cells by culturing in the presence of granulocyte-macrophage colon-stimulating factor (GM-CSF) alone. These APCs can be used topresent peptides or glycopeptides to T-cells. The use of the mouse system obviouslyhas many advantages, but when studying human mucins the murine response to a for-eign antigen is being analyzed. This can be overcome, to a certain extent, by the use ofmice transgenic for human mucins, but only mice transgenic for MUC1 and MUC7 arecurrently available (14,15). However, even when using transgenic mice, the murineresponse to the immunogen is still being evaluated. The use of transgenic mice crossedwith MHC class I A2 molecules (11) may make the system more applicable to thehuman situation.This chapter describes the isolation of dendritic cells from human and murine sourcesand their use in presenting mucin peptides or glycopeptides to autologous T-cells.2. Materials1. Buffers and cell culture medium:a. Blood collection buffer: 1,400 mL of RPMI-1640 HEPES-buffered medium plus 336 mLof 3.3% trisodium citrate in distilled water and 14 mL 5 µM β-mercaptoethanol. Ali-quot 25 mL of this into sterile 50-mL conical screw-capped tubes and store at 4°Cuntil use.b. Hank’s buffered salt solution (HBSS).c. AIM V medium (Gibco-BRL, Gaithersburg, MD) containing 2 mM glutamine, 50 µMβ-mercaptoethanol.d. Minimal essential medium (MEM).e. IMDM medium with glutamax (Gibco-BRL) containing 50 µM β-mercaptoethanol, 5µg/mL of transferrin (Sigma, St. Louis, MO), 100 IU/mL of penicillin, and 100 µg/mlof streptomycin.f. OPTIMEM reduced serum medium (Gibco-BRL).2. Fetal calf serum (FCS).3. Ficoll-Paque (Pharmacia Biotech).4. Human cytokines: GM-CSF (Sandoz) and IL-4 (Genzyme), both made up in AIMV-medium and stored aliquoted at –20°C.5. Phytohemagglutinin (PHA) (Sigma), made up at 2 mg/mL and stored in aliquots at –20°C.6.3H thymidine stock at 1 mCi/mL (Amersham).7. Automated cell harvester such as a Micro 96 manufactured by Skatron, but a simplerversion will work just as well.8. X63 cells secreting murine GM-CSF.9. G418 (Gibco-BRL).10. Peptides: NeoSystems has been found to be an excellent manufacturer, particularly oflarge peptides. The production of glycopeptides is a very specialized procedure espe-cially if more than one sugar is required to be added. It is usually necessary to collaboratewith a chemist who is familiar with the synthesis of glycopeptides. Mucin Antigen Presentation Using Dendritic Cells 48911. Tissue culture plastics: 96-well flat-bottomed plates (Nunc, Nagle, UK), 33-mm dishes(Nunc), 15-mL conical sterile tubes (Falcon, Merck), and tissue culture flasks (Falcon).12. 37°C, 5% CO2 incubator.3. Methods3.1. Preparation and Culturing of Human Dendritic Cells(see Notes 1–4)Our method of preparing and culturing human dendritic cells is an adaptation of themethods published in refs. 12 and 13.1. Collect 20 mL of donor blood into 25 mL of blood collection buffer (see Subheading 2.,item 1a).2. Layer 22.5 mL of blood solution onto 18 mLof Ficoll (see Subheading 2., item 3). Spinat 400g for 20min.3. Remove buffy coat taking up as little Ficoll as possible. Make up the volume of the buffycoat to 100 mL with HBSS (see Subheading 2., item 1b) and spin at 400g for 15–20 min.Wash the pelleted cells twice in HBSS.4. Resuspend the cells in 10 mL AIM V medium (see Subheading 2., item 1c) and spin at1200 rpm for 10 min.5. Resuspend in 10 mL of AIM V medium (see Note 1), count and plate about 7 × 106 in 3mL of medium onto 33-mm tissue culture dishes (see Note 2). Incubate at 37°C for 2 h in5% CO2 incubator.6. Remove medium and nonadherent cells by pipetting up and then relatively gently wash-ing the medium over the dish (see Note 3). To each dish, add 3 mL AIM V mediumcontaining 800 U/mL of human GM-CSF and 500 U/mL of human IL-4 (see Subheading2., item 4).7. Culture for 7 d at 37°C under 5% CO2. Cells require feeding (by removal of 1 mL ofmedium and replace with 1 mL of medium containing fresh IL-4 and GM-CSF) every 2–3 d. On d 7, the cells should be ready for use (see Note 4).3.2. Proliferation Assay Using Autologous Human T-Cells(see Notes 5–8)1. Harvest the dendritic cells on d 7 of culture by vigorously pipetting the medium up anddown. Wash once in AIM V medium with no cytokines. Count (see Note 5) and incubate2 × 105cells in 1 mL of AIM V medium in 15-mL conical bottom tubes with or without100 µg/mL of test peptides or glycopeptides (see Note 6) at 37°C for 2 h.2. Count the nonadherent cells from Subheading 3.1., step 6 (see Note 7) and dispense2 × 105cells per well of a 96-well flat-bottomed tissue culture dish. Include enough wellsof PBLs to have six wells with no dendritic cells and six wells for a nonspecific stimulilike PHA (see Subheading 2., item 5).3. Add 100 µL per well of the peptide pulsed dendritic cells from Subheading 3.2., step 1.If possible have at least six wells per sample. Add 10 µg of the appropriate peptide orglycopeptide to the wells. To the wells with PBLs alone and PBLs with PHA make thevolume up to 200 µL with AIM V medium and where appropriate add 2 µg per well ofPHA. Incubate for 6 d at 37°C in a 5% CO2 incubator.4. On d 6, add 1 µC/well of 3H thymidine (see Subheading 2., item 6) and incubate at 37°Cfor 16–18 h.5. Harvest the cells and count the 3H thymidine incorporated (see Note 8, Subheading 2., item 7). 490 Burchell et al.3.3. Isolation of Murine Dendritic Cells (see Notes 9–11)1. Mouse femur and tibia are removed and placed in MEM (see Subheading 2., item 1d)plus 2% FCS in a 90-mm tissue culture dish. Using forceps and a scalpel, as much muscleand connective tissue as possible is removed from the bones.2. Transfer the bones into a fresh dish containing MEM plus 2% FCS and snip off the endsof each bone creating a hollow tube. Wash out the marrow with a fine needle and 5-mLsyringe containing MEM plus 2% FCS. Prepare a single cell suspension from the marrowusing the syringe and needle or vigorous pipetting.3. Transfer the marrow into a 20-mL universal. Allow any fragments of bone or muscle to fallto the bottom of the tube under gravity and the transfer the supernatant to a fresh universal.4. Count cells and resuspend at 3.33 × 105/mL in IMDM (see Subheading 2., item 1e) plus5%FCS and mouse GM-CSF (see Subheading 3.4. and Note 9). Cells are either culturedin T25 flasks (10 mL) or T75 flasks (30 mL) at 37°C, 5% CO2.5. After 2 d of culture, some clumps of adherent cells should be apparent. Nonadherent cellsand media are removed and replaced with fresh media containing GM-CSF (see Note 10).6. Replace one third of the media with fresh media on d 5 of culture.7. Harvested the nonadherent dendritic cells on d 7, resuspend in a smaller volume of freshmedia and cultured overnight in a tissue culture dish before use (see Note 11).3.4. Production of Murine GM-CSF by X63 Cells(see Note 12)1. X63 cells (see Subheading 2., item 8) are cultured in IMDM plus 5%FCS plus 1 mg/mLG418 (see Subheading 2., item 9).2. Grow up to about 3 × 107 cells in the above medium.3. Spin cells and wash twice in IMDM plus 5%FCS to remove the G418.4. Resuspend the cells in media without G418 at a concentration of 3 × 105cells/mL in oneT75 tissue culture flask. Incubate the flask upright at 37°C 5% CO2for 48 h.5. Pellet the cells and collect the GM-CSF containing supernatant (see Note 12).3.5. Immunization of Mice with Peptide Pulse Dendritic Cells(see Notes 6 and 11)1. Dendritic cells are pulsed with peptides (see Note 6) overnight d 7 to d 8 of the culture.On d 7 DCs are harvested and resuspended at 2 × 106cells/mL in OPTIMEM medium(see Subheading 2., item 1f) with or without 100 µg/mL peptide. 3 mL are then dis-pensed into 33-mm tissue culture dishes and incubated overnight at 37°C in a 5% CO2incubator (see Note 11).2. Harvest dendritic cells by vigorous pipetting, wash once in PBS, and suspend in PBS atbetween 1–5 × 106 cells/mL.3. Immunize mice subcutaneously with peptide pulse dendritic cells or control dendriticcells at 1–5 × 105/mouse in 100 µL of PBS.3.6. Murine Proliferation Assay Using Splenocytes(see Notes 8, 13, and 14)1. Seven to 14 d after the mice received the autologous dendritic cells, sacrifice the miceand remove the spleens into IMDM culture medium.2. Disperse the spleens by passing through a sterile grid and achieve a single cell suspensionby vigorous pipetting. Then count the cells, ignore the red blood cells (see Note 13), andresuspend at 2.5 × 106 cells/mL. Mucin Antigen Presentation Using Dendritic Cells 4913. Dispense 100 µL of the cells into the wells of a 96-well flat-bottomed tissue culture dishand add 5–50 µg of peptide per well (see Note 14). For each spleen, as a positive controlPHA (see Subheading 2., item 5) is added to four wells and as a negative control mediumalone is added to four wells. Incubate at 37°C in a 5% CO2 incubator for 5 d.4. On d 5, add 1 µCi/well of 3H thymidine (see Subheading 2., item 6) and incubate at 37°Cfor 16–18 h.5. Wash and harvest the cells and count the 3H thymidine incorporated (see Note 8).4. Notes1. Human dendritic cells are cultured in serum-free medium, AIM V, as this reduces thebackground in the proliferation assay where the DCs are used as antigen presenting cells.2. From 20 mL of blood there is normally enough PBMCs to put up three to five 33-mm dishes.3. This dislodges the lymphocytes that can be collected and used as effector cells in theproliferation assay.4. The monocytes differentiate into dendritic cells which by d 7 will form about 20–30% ofthe cultured cells. In the isolation of dendritic cells from PBMCs no proliferation occursonly differentiation in the presence of IL-4 and GM-CSF. DCs come off the tissue culturedish in clumps and in addition isolated dendritic cells can often be seen floating in themedium. The cells are relatively large (compared to lymphocytes) and processes or den-drites can clearly be seen and the dendritic cells are often described as having “veils.” Byd 7, the dendritic cells should express high levels of MHC class I and class II, high levelsof CD40 and B7 and should be negative when stained for the monocyte marker CD14.The phenotype can be analysed by the use of FACscan. To show the DCs are functionallyactive they can be used as stimulators of an allogeneic mixed lymphocyte reaction.5. Accurate counting of the dendritic cells is difficult as they only form 20–30% of the cellpopulation. Where possible count the large cells and the cells that have processes.6. Peptides corresponding to any part of a mucin molecule can theoretically be used butusing peptides to a tandem repeat region will cover a large part of the molecule. In thecase of MUC1 the peptides have been confined to the tandem repeat and have consistedof one or more repeats. Up to three TR (60 amino acids) can be synthesised successfullyby commercial companies. Feeding large peptides, e.g., 60 mer, to the dendritic cellsfrom the outside makes the assumption that the peptide is taken up by endocytosis andenters the MHC class II pathway.7. The antigen presenting cell i.e. the dendritic cells, must be autologous to the nonadherentcells used as the responders.8. The use of an automated cell harvested is essential.9. The dendritic cells can be cultured in serum-free medium consisting of AIM V mediumcontaining mercaptoethanol. However, in tumour challenge experiments better resultshave been obtained when the cells are cultured in serum-containing medium althoughsome nonspecific protection may be observed.10. Over the next week, the clumps of adherent cells release nonadherent dendritic cells into themedium. Unlike the human dendritic cells cultured from PBMCs some proliferation does occur.11. During the overnight culture some cells will adhere to the tissue culture dish, these shouldnot be used.12. The supernatant from the X63 cells is used at a dilution of 1:10. Commercial murine GM-CSF is available but is rather expensive.13. Red blood cells can be lysed before setting up the proliferation assay which makes thelymphocytes easier to count. The protocol is as follows: 492 Burchell et al.a. Lysis buffer: 8.29 g NH4Cl, 1 g KHCO3,37.2 mg Na2EDTA. Add 800 mL H2O andadjust pH to 7.2–7.4 with 1 M HCl. Adjust volume to 1 L with water and filter steril-ize through 0.2-µm filter. Store at room temperature.b. Method: Spin splenocytes to pellet the cells and resuspend the cells from one spleenin 1 mL lysis buffer. Incubate at 4°C for 30 s to 1 min. Spin down cells and wash2 × 50 mL medium.14. The peptide used in the proliferation assay can be the same as that used for pulsing thedendritic cells or it can be a smaller peptide. For example when using MUC1 peptides a60 mer corresponding to three TR are used to pulse the dendritic cells whereas a 24 mer isused in the proliferation assay.References1. Hakomori, S.-I. (1989) Aberrant glycosylation in tumors and tumor-associated carbohy-drate antigens. Adv. Cancer Res. 52, 257–331.2. Kim, Y. S., Gum, J., and Brockhausen, I. (1996) Mucin glycoproteins in neoplasia.Glycocon. J. 13, 693–707.3. Brockhausen, I., Yang, J. M., Burchell, J. M., Whitehouse, C., and Taylor-Papadimitriou,J. (1995 ) Mechanisms underlying aberrant glycosylation of MUC1 mucin in breast cancercells. Eur. J. Biochem. 233, 607–617.4. Corfield, A. P., Myerscough, N., Gough, M., Brockhausen, I., Schauer, R., and Paraskeva, C.(1995) Glycosylation patterns of mucins in colonic disease. Biochem. Soc. Trans. 23, 840–845.5. von Mensdorff-Pouilly, S., Gourevitch, M. M., Kenemans, P., Verstraeten, A. A., Litvinov,S. V., van Kamp, G. J., Meijer, S., Vermorken, J., and Hilgers, J. (1996) Humoral immuneresponses to polymorphic epithelial mucin (MUC1) inpatients with benign and malignantbreast tumours. Eur. J. Cancer 32, 1325–1331.6. Galli-Stampino, L., Meinjohanns, E., Frische, K., Meldal, M., Jensen, T., Werdeelin, O.,and Mouritsen, S. (1997) T-cell recognition of tumour-associated carbohydrates: Thenature of the glycan moiety plays a decisive role in determining glycopeptide Immunoge-nicity. Cancer Res. 57, 3214–3222.7. Haurum, J. S., Arsequell, G., Lellouch, A. C., Wong, S. Y. C., Dwek, R., McMichael, A. J.,and elliott, T. (1994) Recognition of carbohydrate by major histocompatibility complex classI-restricted, glycopeptide-specific cytotoxic T lymphocytes. J. Exp. Med. 180, 739–744.8. Campbell, B. J., Finnie, I. A., Hounsell, E. F., and Rhodes, J. (1995) Direct demonstration ofincreased expression of Thomsen-Friedenreich (TF) antigen in colonic adenocarcinoma andulcerative colitis mucin and its concealment in normal mucin. J. Clin. Invest. 95, 571–576.9. Lloyd, K. O., Burchell, J. M., Kudryashov V., Yin, B. W. T., and Taylor-Papadimitriou, J.(1996) Comparison of O-linked carbohydrate chains in MUC 1 mucin from Normal BreastEpithelial Cell Lines and Breast Carcinoma cell lines - Demonstration of a simpler andfewer glycan chains in tumor cells. J. Biol. Chem. 271, 33,325–33,334.10. Graham, R. A., Burchell, J. M., and Taylor-Papadimitriou, J. (1996) The PolymorphicEpithelial Mucin: Potential as an Immunogen for a Cancer Vaccine. Cancer Immunol.Immunother. 42, 71–80.11. Apostolopoulos, V., Karanikas, V., Haurum, J., and McKenzie, I. F. C. (1997) Inductionof HLA-A2-restricted CTLs to the Mucin 1 human breast cancer antigen. J. Immuno. 159,5211–5218.12. Romani, N., Gruner, S., Brang, D., Kampgen, E., Lenz, A., Trockenbacher, B., Konwalinka,G., Fritsch. P. O., Steinman, R. M., and Schuler, G. (1994) Proliferating dendritic cellprogenitors in human blood. J. Exp. Med. 180, 83–93. Mucin Antigen Presentation Using Dendritic Cells 49313. Sallusto, F. and Lanzavecchia, A. (1994) Efficient presentation of soluble antigen by cul-tured human dendritic cells is mantained by granulocyte/macrophage colony-stimulatingfactor plus interleukin 4 and downregulated by tumour necrosis factor α. J. Exp. Med. 179,1109–1118.14. Peat, N., Gendler, S. J., Lalani, E.-N., Duhig, T., and Taylor-Papadimitriou, J. (1992)Tissue-specific expression of a human polymorphic epithelial mucin (MUC1) in transgenicmice. Cancer Res. 52, 1954–1960.15. Bobek, L. A. Li, H., Rojstaczer, N., Jones, C., Gross, K. W., and Levine, M. J. (1998)Tissue-specific expression of human salivary mucin gene, MUC7 in transgenic mice.Transgenic Res. 7, 195–204. . (Gibco-BRL).2. Fetal calf serum (FCS).3. Ficoll-Paque (Pharmacia Biotech).4. Human cytokines: GM-CSF (Sandoz) and IL-4 (Genzyme), both made up in AIMV-medium. non-MHC-restricted manner (see Chapter 39). However,From :Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield