Antibody-dependent cell-mediated cytotoxicity, lymphokine-activated and natural killer cells

7.4 Antibody-dependent cell-mediated cytotoxicity, lymphokine-activated and natural killer cells

In antibody-dependent cell-mediated cytotoxicity (ADCC), the apparent specificity of the killing reaction is superimposed on a non-specific effector cell by its acquisition of, or adsorption to, target cell-specific antibody. In the absence of antibody, a proportion of these non-specific killers can kill target cells directly and are referred to as natural killer (NK) cells. They are able to kill tumour cells without the need for previous immunization or passive antibody, and are greatly increased in numbers in mice carrying the nu/nu athymic mutation (where they presumably account for the resistance to spontaneous tumours shown by these T lymphocyte-deficient animals). They are virtually absent in the beige mutant of the C3H mouse.

Treatment of human or murine normal (non-immune) lymphocytes with IL-2 greatly enhances their non-specific killing capacity to many tumour targets. This led to the definition of

a third functional class of non-specific cytolytic cells known as lymphokine-activated killer or LAK cells. These cells may have clinical use for antitumour therapy. At present the lineage of cytolytic cells is confused but their functional classification is clear: (a) There is a ‘common pool’ of antigen-specific and non-specific cytolytic effector cells, com- posed of several cell lineages. (b) T lymphocytes are the only cytolytic effector cells known to date to have clonally distributed endogenous receptors for specific target-cell antigens. (c) The so-called large granular lymphocytes, identified by virtue of their morphology (Fig. 7.2c), are able to mediate NK- and LAK-cell activity in vitro. (d) Non-lymphoid cells are able to mediate NK and LAK activity. (e) LAK and NK cells may be functionally distinguished by the judicious choice of tumour target

cells; most tumour targets used to date have been LAK sensitive, but only a few are NK sensitive. (f) Most primary cultures of freshly excised tumours are NK-resistant but LAK-sensitive, as are hapten-modified normal cells.

7.4.1 Assay of LAK- and NK-cell activity

MATERIALS AND EQUIPMENT Heparinized, human venous blood Density gradient, e.g. Lymphoprep TM Tissue culture medium containing 5% fetal bovine serum (FBA) Recombinant IL-2

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

T-24 and K-562 cell lines Sodium 51 CrO 4 U-shaped microculture plates Plastic tubes (circa 2ml), e.g. LP3 Microplate carrier for centrifugation Gamma spectrometer

METHOD

1 Isolate peripheral blood mononuclear cells (PBMC) from heparinized venous blood by density gradient centrifugation and wash three times in tissue culture medium by centrifugation (150 g for 10 min at room temperature).

2 Adjust the PBMC to 5 × 10 6 cells/ml with tissue culture medium containing 5% FBA. For the assay of NK activity, no IL-2 or induction period is required: proceed to step 4.

3 For LAK-cell induction, supplement the PBMC suspension with recombinant IL-2 (500 U/ml) and dispense 100 µl aliquots into a U-shaped microculture tray as follows: allow for at least triplicate cultures at each dilution (see Protocol below), for each donor and each target (NK-resistant and -susceptible, see Technical notes), and leave five wells empty for each of the target cells for the determination of spontaneous isotope release.

Culture in a humidified 37°C incubator gassed with 5% CO 2 in air.

The time for optimum induction of LAK activity will vary with both donor and type of assay for which they are intended. Typically use cultures at 48–72 h after induction; however, LAK cells are still detectable at 7 days. It is conceivable that the early and late LAK activity might be due to varying proportions of the different cell types known to mediate this effector function.

4 Label the T-24 and K-562 target cells with 51 CrO 4 by mixing 37 × 10 5 Bq of isotope with

10 6 T-24 or K-562 targets and incubate in a 37°C water bath for 1.5 h, mixing every 30 min.

5 Wash the target cells three times by centrifugation, resuspend and count cells using a haemocytometer. Retain an aliquot of labelled cells for freeze–thaw determination of maximum isotope release. For accurate determination of LAK activity it is necessary to determine isotope release over a range of different effector : target-cell ratios.

6 Prepare a series of target-cell suspensions according to the Protocol; add l00 µl of each to separate assay wells.

7 Add l00 µl of the highest target suspension to each of the five empty wells allowed for the determination of spontaneous release and supplement with l00 µl of tissue culture medium.

8 Centrifuge the plate at 50 g for 15 min at room temperature in a microplate carrier.

Protocol.

Assay number

PBMC at 5 × 10 6 /ml l00 µl ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ Target cells/ml (use 100 µl per culture)

3 × 10 7 6 × 10 7 12.5 × 10 7 25 × 10 7 Effector : target-cell ratio

Continued on p. 216

7.4ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY

9 Incubate for 4 h at 37°C in a humidified incubator gassed with 5% CO 2 in air.

10 Remove 100 µl of supernatant into separate LP3 tubes, cap and count in a g spectrometer.

11 Calculate specific lysis of each experimental well as follows:

experimental c.p.m. − spontaneous c.p.m.

% specific lysis = × 100

maximum c.p.m. − spontaneous c.p.m.

12 Determine the mean ± standard error of each set of replicates and display each donor’s titration curve graphically for ease of comparison.

TECHNICAL NOTES • LAK and NK effector functions are distinguished by differential killing of the two target cells

used in the assay. T-24 cells are relatively NK-resistant, whereas K-562 cells are both NK- and LAK-susceptible. LAK activity varies widely between normal donors, between 30 and 100% specific lysis.

• As a guide, the maximum release from 10 4 target cells should be about 10 000 c.p.m. and the spontaneous release < 10% at 4 h. High spontaneous release is often due to the batch of FBS used as a tissue culture supplement; batches should be screened before purchase. FBA and autologous human serum give comparable results.

• It is also possible to generate LAK cells in bulk by culturing in flasks. • The relatively high concentration of IL-2 used to generate these cells excludes the use of cell

supernatants, e.g. from MLA 144, as an IL-2 source. • Over a short induction period, < 48 h, LAK activity is resistant to hydroxyurea, cyclosporin and steroid treatment. However, when longer induction periods are used, up to 7 days, there is

a decrease in the rate of LAK induction, suggesting that the cells participating in the early expression of LAK activity might be different to those involved at later time points. • This assay may be adapted for using the non-radioisotope technique of time-resolved fluores-

cence as an alternative to using 51 Cr.