Antibody-dependent cell-mediated cytotoxicity

7.3 Antibody-dependent cell-mediated cytotoxicity

In antibody-dependent cell-mediated cytotoxicity (ADCC) target cells coated with very small amounts of antibody are killed by non-immune effector cells. The effector cells (K cells) have receptors for the Fc regions of the antibody and appear to recognize immune complexes specifically. The exact killing mechanisms are unknown, but involve cell to cell contact and, in some of the effector cell types, may result from the release of lysosomal enzymes. With erythro- cyte targets the effector cells tend to be of the granulocyte–macrophage lineage; but with tumour target cells, cells of the lymphocyte lineage predominate.

There is evidence to suggest a large overlap between the progenitors of ADCC effector cells and those of lymphokine-activated killer (LAK) cells. Some of these progenitors also show natural killer (NK) cell activity; the overlap is almost certainly confined to cells of lymphoid lineage in this case. It is possible to distinguish between the NK and LAK non-specific (non-antigen-specific) cytotoxic cells by the use of NK-resistant or -susceptible target cells. ADCC effector cells may be detected by their antibody dependence.

7.3ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY

MATERIALS Mouse Chicken (has nucleated red blood cells) Rabbit anti-chicken erythrocyte serum (diluted 1 : 6000 in tissue culture medium plus 10% fetal

bovine serum) Tissue culture medium Fetal bovine serum (FBS)

Sodium 51 chromate ( 51 CrO 4 )

Sheep erythrocytes (SRBC) Eagle’s minimum essential medium (MEM) containing 1% v/v FBS

5% CO 2 in air

7.3.1 Target cells

METHOD

1 Take 0.2 ml of blood from the chicken into a heparinized syringe. The main wing vein is a convenient site for venepuncture to obtain small volumes of blood.

2 Dilute 0.1 ml of blood with 1.9 ml of Eagle’s MEM containing 10% FBS.

3 Use 0.1 ml of diluted blood and add 0.1 ml of sodium 51 CrO 4 .

4 Gas with 5% CO 2 in air.

5 Incubate at 37°C for 1 h.

6 Wash four times with medium containing 5% FBS. Centrifuge at 90 g for 7 min at 4°C.

7 Wash SRBC in tissue culture medium four times by centrifugation (450 g for 10 min).

8 Adjust SRBC concentration to 10 7 /ml.

9 Add 10 5 labelled chicken red cells to each ml of sheep red cells.

TECHNICAL NOTE The radioisotope 51 Cr has a half-life of around 28 days and is a g emitter. The recommended safety guidelines for handling isotopes must be adhered to when using this Protocol. Refer to the follow- ing websites for 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

7.3.2 Effector cells

METHOD

1 Remove the spleen from the mouse and prepare a single-cell suspension.

2 Adjust to 2.5 × 10 6 leucocytes/ml.

C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity

7.3.3 Cytotoxic assay

METHOD

1 Set up culture tubes according to the Protocol below.

Protocol. Tube

51 Cr-labelled chicken (in triplicate)

Spleen cells (µl)

Antibody (µl)

red cells (µl)

E 100 µl distilled water

100 µl distilled water

2 Cap the tubes and incubate, leaning at an angle of 30–45 degrees, in a gassed CO 2 incubator or a desiccator (5% in air) for 18 h.

3 Add 1 ml medium to each tube and then spin (90 g) for 10 min.

4 Remove 0.8 ml supernatant from each tube and assess this for 51 Cr release in a g spectrometer.

TECHNICAL NOTES • Tube A shows the 51 Cr release due to spleen cells plus antitarget antibody. The other cultures

are controls. Tube B gives the amount of release due to spleen cells alone, while C measures the release due to antibody. Spontaneous release of the label by the erythrocytes is monitored by tube D.

• Refer to the following websites for 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/hsehome.htm

Calculation Calculating the amount of cytotoxicity is complicated as there is some difficulty in choosing the

correct control value against which to calculate the experimental 51 Cr release. The reason for this is that spleen cells, in the absence of antibody, exert a protective effect over the chicken erythro- cytes. It will be seen that the 51 Cr release in tube B is usually less than the spontaneous release in tube D. Therefore, for the control culture, one may choose either effectors plus target cells (B) or target cells plus antibody (C).

The calculation of percentage cytotoxicity may then be as follows:

51 A − C % Cr release = × 100

7.3ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY

A = − B × 100 .

E − B Letters in formulae correspond to culture tubes in the Protocol (see p. 213). TECHNICAL NOTE

The assay for optimum conditions varies according to the ratio of effector to target cells.