Analysis of lymphocyte antigens

8.1 Analysis of lymphocyte antigens

Techniques used for the analysis of lymphocyte antigens are: (a) powerful analytical tools for the analysis of complex antigenic systems, including detergent-solubilized cells; and (b) sufficiently flexible and compatible to be capable of improvement and modification to facilitate novel experi- mental applications.

8.1.1 Radiolabelling of cells and their secreted products

Radiolabelling of cell components (proteins, glycoproteins, phosphoproteins, phospholipids, etc.) can provide convenient markers for analytical and preparative techniques (see Sections 8.1.2 and 8.1.3). Endogenous or biosynthetic labelling of secreted proteins is described in detail for monoclonal antibodies and hybridoma cell lines (see Chapter 2 and Section 8.4). Similar tech- niques are used for the labelling of other proteins or glycoproteins, e.g. cytokines secreted from cell lines in vitro or, indeed, for any constitutive cell protein which undergoes significant turnover during the labelling period (see also Chapter 10).

Chemical or exogenous labelling of the surface of a viable cell is a common starting point for many studies on cell-surface antigens (see Section 8.1.2). Differential exogenous labelling of the surface of intact, viable cells may be compared with labelling of the cells of the same type after detergent solubilization. This can provide valuable information on the relative distribution of an antigen between the cytoplasm and exterior of the surface membrane.

External labelling is usually achieved by confining the chemical coupling reaction to the outer surface of the membrane, e.g. using a molecule too large to cross the membrane (often a protein, as in lactoperoxidase labelling) (see Section 8.1.2), or by binding one of the essential components of the reaction to an insoluble support (the surface of a plastic macrobead or tube, as in Iodogen labelling) (see Section 4.9.3).

Success relies crucially on a population of cells with high (preferably uniform) viability and intact surface membranes.

C H A P T E R 8: Lymphocyte structure

8.1.2 Cell-surface iodination: lactoperoxidase technique

Lactoperoxidase, in the presence of hydrogen peroxide, catalyses the incorporation of iodine into tyrosine residues. This gentle and efficient method utilizes the action of glucose oxidase on glu- cose to generate hydrogen peroxide continuously during the reaction. As the enzymes are too big to be able to cross the plasma membrane the addition of iodine is confined to the cell surface.

MATERIALS AND EQUIPMENT Phosphate-buffered saline (PBS)

Cells for iodination (10 8 /ml in PBS) Lactoperoxidase (0.2 mg/ml in PBS) Glucose oxidase (2.0 IU/ml in PBS)

50 m M glucose in PBS Sodium 125

I, carrier free Gamma spectrometer

METHOD

1 Wash the cells three times in PBS by centrifugation (150 g for 10 min at room temperature)

to remove exogenous material, count and adjust to 10 8 cells/ml.

2 To 100 µl of cell suspension add 10 µl lactoperoxidase (0.2 mg/ml in PBS initial concentration), 10 µl glucose oxidase (2 IU/ml in PBS, initial concentration) and

18.5 × 10 6 Bq 125 I.

3 Initiate the reaction by the addition of 10 µl of 50 m M glucose in PBS and incubate for

10 min at room temperature.

4 Add 10 ml of ice-cold PBS to stop the reaction.

5 Wash the cells three times in PBS by centrifugation (150 g for 10 min at 4°C).

6 If required, the cells may be detergent solubilized, and their radioactive incorporation determined.

TECHNICAL NOTES • Because the lactoperoxidase cannot cross the plasma membrane of viable cells, only surface

proteins are iodinated. However, internal and external proteins are labelled if the cells are dead; therefore good cell viability is essential. See Section 6.4 for dead cell removal.

• The method is also useful for soluble proteins, but the enzymes will contaminate the protein

preparation. To avoid this, enzymes coupled to a solid phase should be used. Polyacrylamide beads coupled with lactoperoxidase and glucose oxidase are available commercially. The reac- tion may then be terminated by removal of the beads.

• Lactoperoxidase may be ‘poisoned’ by the addition of 10 mm sodium azide and the reaction

terminated precisely. In addition, be sure that the PBS does not contain sodium azide as a pre- servative, otherwise the reaction will never start.

• Lactoperoxidase catalyses its own iodination. In some systems, iodination artefacts have been

reported due to the adsorption of this material to surface of the cell being labelled. If this is a problem, cells may be iodinated with insolubilized Iodogen but with reduced efficiency.

• Methods using isotopes vary in the harshness (potential for alteration of the conformation of the labelled material) of the reaction required to achieve the desired result.

8.1ANALYSIS OF LYMPHOCYTE ANTIGENS

• It is essential that use of radiolabels is appropriately recorded and safety advice regarding the handling of radioisotopes is followed in accordance with the local guidelines of the institution where the work is carried out.

• For optimum results it is advisable to calculate the half-life decay of the isotopes used. Here are some useful websites that give comprehensive details on radioisotope health and safety procedures as well as useful information regarding detection and half-life: http://www.practicingsafescience.org http://www.hse.gov.uk

8.1.3 Tritium labelling of cell-surface glycoproteins

Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids. The aldehydes thus formed can be reduced easily with 3 H-sodium borohydride. At 0°C the peri- odate anion only penetrates the cell membrane very slowly and so oxidation will be restricted mainly to cell-surface sialic acid residues.

MATERIALS Phosphate-buffered saline (PBS)

Lymphocytes (3 × 10 7 /ml in PBS)

1 M sodium metaperiodate in PBS

0.1 M glycerol in PBS Tritiated sodium borohydride Ice Beta spectrometer

METHOD

1 Wash lymphocytes (3 × 10 7 ) twice with PBS by centrifugation (150 g for 10 min at 4°C).

2 Resuspend in 1 ml PBS and place on ice.

3 Add 0.1 ml 1 M sodium metaperiodate and incubate on ice for 5 min.

4 Quench the reaction by adding 0.2 ml glycerol (0.1 M ).

5 Wash the cells three times with PBS by centrifugation.

6 Resuspend cells in 0.5 ml PBS.

7 Add 18.5 × 10 6 Bq sodium 3 H-borohydride.

8 Incubate for 30 min at room temperature.

9 After washing in cold PBS, the cells may be solubilized in detergent if required and their radioactive incorporation determined.

8.1.4 Specificity of the labelling reaction

Early studies of cell-surface labelling demonstrated that the addition of the radiolabel was indeed limited to the surface membrane, e.g. by electron microscope autoradiography (Fig. 8.1). Techniques are now sufficiently established that, unless you are working with a totally novel or bizarre system, this type of evidence for the localization of labelling is not sought. However, it is necessary to bear in mind that cells can adsorb exogenous proteins (particularly from dead and dying cells) on to their surface and so have a well-developed propensity to trap the unwary.

C H A P T E R 8: Lymphocyte structure

Fig. 8.1 Electron microscopic autoradiography of 125 I-labelled Trypanosoma cruzi organisms.

Trypomastigotes of the protozoan Trypanosoma cruzi were labelled with 125

I by the lactoperoxidase technique and processed for ultramicrotome sectioning. Ultrathin sections were dipped in K5 nuclear emulsion, allowed to expose and finally processed photographically before being viewed in a transmission electron microscope. The photograph shows individual silver grains at or near the cell membrane, confirming that the majority of the radioiodine has indeed conjugated to cell-surface residues. Final magnification × 31 000.