Glycoprotein methods protocols - biotechnology 048-9-487-493.pdf

Glycoprotein methods protocols - biotechnology

40

Mucin Antigen Presentation Using Dendritic Cells

Joy Burchell, Rosalind Graham, and Joyce Taylor-Papadimitriou

1 Introduction

The study of humoral and cellular responses to mucins requires many of the stan-dard immunologic techniques, although working with molecules as large as mucins sometimes leads to logistic problems This chapter focuses on some of the techniques that may be used to analyze the immune response to mucins using dendritic cells to present 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 glycans are 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 envisage

how this would affect the humoral response to the molecule (5) However, there is an

increasing amount of evidence that glycosylated peptides can be presented to T-cells

via the major histocompatibility complex (MHC) molecules (6,7) Galli-Stampino et

al have shown that peptides carrying simple O-linked core 1 sugars (Gal β1-3GalNAc) can bind to MHC class II molecules and be presented to T-cells, whereas the more complex 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 is observed in carcinomas results in the expression of the simple core 1 structure and Tn

and 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 focused

on MUC1 as a potential target for active specific immunotherapy because this mucin

is over- expressed and aberrantly glycosylated by many carcinomas but particularly

those of the breast and ovary (10) Like all mucins, MUC1 has a large domain of

tandemly repeated amino acids allowing potential epitopes to be repeated many times.

Humoral responses have been identified in breast cancer patients (5) and cytotoxic

T-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 Mucins

Edited by: A Corfield © Humana Press Inc., Totowa, NJ

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 and are involved in the presentation of antigens to naive T-cells Human dendritic cells can

be 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 cells

can 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 to present peptides or glycopeptides to T-cells The use of the mouse system obviously has 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 of mice transgenic for human mucins, but only mice transgenic for MUC1 and MUC7 are

currently available (14,15) However, even when using transgenic mice, the murine

response to the immunogen is still being evaluated The use of transgenic mice crossed

with MHC class I A2 molecules (11) may make the system more applicable to the

human situation.

This chapter describes the isolation of dendritic cells from human and murine sources and their use in presenting mucin peptides or glycopeptides to autologous T-cells.

2 Materials

1 Buffers and cell culture medium:

a Blood collection buffer: 1,400 mL of RPMI-1640 HEPES-buffered medium plus 336 mL

of 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°C until use

b Hank’s buffered salt solution (HBSS)

c AIM V medium (Gibco-BRL, Gaithersburg, MD) containing 2 mM glutamine, 50 µM

β-mercaptoethanol

d Minimal essential medium (MEM)

e IMDM medium with glutamax (Gibco-BRL) containing 50 µM β-mercaptoethanol, 5

µg/mL of transferrin (Sigma, St Louis, MO), 100 IU/mL of penicillin, and 100 µg/ml

of 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 AIM V-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 simpler version 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 of large 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 collaborate with a chemist who is familiar with the synthesis of glycopeptides

11 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 Methods

3.1 Preparation and Culturing of Human Dendritic Cells

( see Notes 1–4)

Our method of preparing and culturing human dendritic cells is an adaptation of the

methods 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) Spin

at 400g for 20min.

3 Remove buffy coat taking up as little Ficoll as possible Make up the volume of the buffy

coat 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 at

1200 rpm for 10 min

5 Resuspend in 10 mL of AIM V medium (see Note 1), count and plate about 7 × 106 in 3

mL of medium onto 33-mm tissue culture dishes (see Note 2) Incubate at 37°C for 2 h in 5% 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 medium containing 800 U/mL of human GM-CSF and 500 U/mL of human IL-4 (see Subheading

2., item 4).

7 Culture for 7 d at 37°C under 5% CO2 Cells require feeding (by removal of 1 mL of medium 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 and

down Wash once in AIM V medium with no cytokines Count (see Note 5) and incubate

2× 105cells in 1 mL of AIM V medium in 15-mL conical bottom tubes with or without

100µg/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 dispense

2× 105cells per well of a 96-well flat-bottomed tissue culture dish Include enough wells

of PBLs to have six wells with no dendritic cells and six wells for a nonspecific stimuli

like 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 or glycopeptide to the wells To the wells with PBLs alone and PBLs with PHA make the volume up to 200 µL with AIM V medium and where appropriate add 2 µg per well of PHA 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°C for 16–18 h

5 Harvest the cells and count the 3H thymidine incorporated (see Note 8, Subheading 2., item 7).

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 muscle and 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 ends

of each bone creating a hollow tube Wash out the marrow with a fine needle and 5-mL syringe containing MEM plus 2% FCS Prepare a single cell suspension from the marrow using the syringe and needle or vigorous pipetting

3 Transfer the marrow into a 20-mL universal Allow any fragments of bone or muscle to fall

to 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) plus 5%FCS and mouse GM-CSF (see Subheading 3.4 and Note 9) Cells are either cultured

in 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 cells

and 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 fresh

media 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/mL G418 (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 one T75 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% CO2

incubator (see Note 11).

2 Harvest dendritic cells by vigorous pipetting, wash once in PBS, and suspend in PBS at between 1–5 × 106 cells/mL

3 Immunize mice subcutaneously with peptide pulse dendritic cells or control dendritic cells 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 mice and remove the spleens into IMDM culture medium

2 Disperse the spleens by passing through a sterile grid and achieve a single cell suspension

by vigorous pipetting Then count the cells, ignore the red blood cells (see Note 13), and

resuspend at 2.5 × 106 cells/mL

3 Dispense 100 µL of the cells into the wells of a 96-well flat-bottomed tissue culture dish and add 5–50 µg of peptide per well (see Note 14) For each spleen, as a positive control PHA (see Subheading 2., item 5) is added to four wells and as a negative control medium

alone 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°C for 16–18 h

5 Wash and harvest the cells and count the 3H thymidine incorporated (see Note 8).

4 Notes

1 Human dendritic cells are cultured in serum-free medium, AIM V, as this reduces the background 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 the proliferation assay

4 The monocytes differentiate into dendritic cells which by d 7 will form about 20–30% of the cultured cells In the isolation of dendritic cells from PBMCs no proliferation occurs only differentiation in the presence of IL-4 and GM-CSF DCs come off the tissue culture dish in clumps and in addition isolated dendritic cells can often be seen floating in the medium 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.” By

d 7, the dendritic cells should express high levels of MHC class I and class II, high levels

of 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 functionally active 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 cell population 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 but using peptides to a tandem repeat region will cover a large part of the molecule In the case of MUC1 the peptides have been confined to the tandem repeat and have consisted

of one or more repeats Up to three TR (60 amino acids) can be synthesised successfully

by commercial companies Feeding large peptides, e.g., 60 mer, to the dendritic cells from the outside makes the assumption that the peptide is taken up by endocytosis and enters the MHC class II pathway

7 The antigen presenting cell i.e the dendritic cells, must be autologous to the nonadherent cells 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 medium containing mercaptoethanol However, in tumour challenge experiments better results have been obtained when the cells are cultured in serum-containing medium although some nonspecific protection may be observed

10 Over the next week, the clumps of adherent cells release nonadherent dendritic cells into the medium 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 should not 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 the lymphocytes easier to count The protocol is as follows:

a Lysis buffer: 8.29 g NH4Cl, 1 g KHCO3,37.2 mg Na2EDTA Add 800 mL H2O and

adjust 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 spleen

in 1 mL lysis buffer Incubate at 4°C for 30 s to 1 min Spin down cells and wash

2× 50 mL medium

14 The peptide used in the proliferation assay can be the same as that used for pulsing the dendritic cells or it can be a smaller peptide For example when using MUC1 peptides a

60 mer corresponding to three TR are used to pulse the dendritic cells whereas a 24 mer is used in the proliferation assay

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