Mitogenic response

9.5 Mitogenic response

When lymphocytes meet their specific antigen they are stimulated to undergo division. This mitogenic response is usually accompanied by a morphological change to a blast cell. The degree of lymphocyte activation may be assayed by either determining the percentage of blast cells in the culture, or measuring the amount of radioactive DNA analogue incorporated into newly synthesized DNA. It is important to note, however, that blast transformation, DNA synthesis and cell proliferation are not necessarily synonymous. Several instances have been reported where incorporation of DNA analogue has occurred without cell division. The in vitro mitotic response has been shown to have an approximate correlation with the in vivo situation, e.g. a normal indi- vidual would have a lower mitotic response to PPD (purified protein derivative of tubercle bacilli) than a Mantoux-positive individual. In addition, an immunodeficient individual with poor Mantoux reactivity would have a low in vitro mitotic response to PPD.

Lectins or phytomitogens, that are derived from plants, have the ability to induce blast-cell transformation and mitosis in a manner similar to antigen. The mitogen binds to a specific cell- surface receptor, as does antigen, the signal generated causes the nucleus to be derepressed and the lymphocyte enters the cell cycle. However, unlike antigens, the mitogen can stimulate a large proportion of lymphocytes. Again, as for antigen stimulation of lymphocytes in vitro, it has been possible to show an approximate correlation between the in vitro response to mitogens and the immune status of the individual. Phytohaemagglutinin (PHA) is the most extensively studied of the phytomitogens. Evidence suggests that soluble PHA stimulates only T cells, although the

9.5MITOGENIC RESPONSE

We describe two in vitro techniques for the assay of the response of human peripheral blood to PHA: (i) a ‘low-tech’ tube-based macro-assay that uses a lot of cells and is not convenient for large numbers of experimental groups but requires very little specialized equipment; and (ii) the microassay based upon microculture wells which uses very small quantities of cells and reagents and has been semiautomated, thus enabling many experimental groups with three to five replic- ates per group to be tested.

9.5.1 Tube radioassay for cell proliferation

MATERIALS AND EQUIPMENT Human peripheral blood Phytohaemagglutinin (PHA) Density gradient for lymphocyte isolation Tissue culture medium containing antibiotics Fetal bovine serum

3 H-thymidine

5 ml plastic tubes, Falcon 37°C incubator

Cylinder of 5% CO 2 in air

All procedures must be carried out under sterile conditions.

METHOD

1 Mix the blood with an equal volume of serum-free tissue culture medium.

2 Layer an equal volume of defibrinated blood onto the density gradient and centrifuge at 400 g for 20 min at 4°C. (For economy, it is possible to use a 2 : 1 ratio of diluted blood to density gradient.)

3 Most of the leucocytes will be found as a fuzzy white band at the serum–density gradient interface. Insert a Pasteur pipette into this band and aspirate the cells.

4 Wash the cells once with serum-free medium (250 g for 15 min at room temperature) and twice with medium containing 5% fetal bovine serum (150 g for 10 min at room temperature) by centrifugation.

5 Count lymphocytes and adjust to 2 × 10 6 /ml.

6 Set up lymphocyte cultures with PHA according to the Protocol.

7 Incubate the tubes in a 37°C CO 2 incubator. The maximum uptake of 3 H-thymidine occurs about 72 h after PHA stimulation. If you intend to conduct a complete experiment, it is essential that you investigate both the full dose–response curve and the kinetics of the response in your own culture system.

8 Four hours before harvesting, add 37 × 10 3 Bq of 3 H-thymidine to each culture.

Continued

C H A P T E R 9: Lymphocyte function

Protocol. Tube number (3–5 replicates of each tube)

1 : 80 Volume of lymphocytes (ml)

1 ml PHA diluted to:

1⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ (2 × 10 6 /ml initial conc.) Final PHA concentration

TECHNICAL NOTE Anti-CD3 may also be used as a polyclonal activator.

Cell harvesting and counting

MATERIALS AND EQUIPMENT Filter papers, Whatman 3 MM, 2.1-cm circle

Phosphate-buffered saline (PBS) Chloroform Trichloroacetic acid (TCA), 10% w/v aqueous solution Scintillation fluid Scintillation vials Beta spectrometer

METHOD

1 Wash cells two to three times in PBS by centrifugation.

2 Resuspend cell pellet in 0.4 ml PBS.

3 Support filter discs (one for each culture tube and numbered in pencil) on a pin in a cork board.

4 Place 0.2 ml of cell suspension onto the corresponding disc.

5 Air dry discs with a fan.

6 Wash all discs in 10% cold TCA to precipitate the protein. (At this stage all the discs may be combined.)

7 Wash discs in PBS and then absolute alcohol.

8 Rinse in the chloroform and allow to dry.

9 Place each disc in a scintillation vial containing scintillation fluid and count emissions in a scintillation counter.

Assessment of results Calculate the geometric mean c.p.m. for each group of replicates (because the researcher is con-

sidering cell doubling in populations). There are basically two ways of recording data:

1 by simply giving the mean c.p.m. for stimulated and unstimulated cultures or their difference (∆ c.p.m.);

9.5MITOGENIC RESPONSE

2 as an index of stimulation. This is calculated by the following equation: Index of stimulation =

c.p.m. PHA cultures c.p.m. unstimulated cultures

To compare different types of cells, each having their own unstimulated control, the situation is more complex. Cells from different tissues may have varying numbers of cells undergoing spon- taneous division and often the serum supplement used for culture is itself mitogenic, sometimes more on some tissues than others. Unstimulated (i.e. not PHA stimulated, in this case) or ‘back- ground’ radioisotope incorporation may be abnormally high in some cultures but not others. Spleen cultures, for example, show a much higher background incorporation than blood lym- phocyte cultures. In this case an index of stimulation would not be a useful way in which to pre- sent the data as the background variation would be hidden.

TECHNICAL NOTES • It may be necessary to test several batches of fetal bovine serum as they vary in their ability to

‘support’ in vitro cultures. • 131 Iodo-deoxyuridine may be used instead of 3 14 H- or C-thymidine. This DNA analogue has the advantage that it is not re-utilized in a culture and so is a measure of incorporation alone, with- out the complication of turnover. In addition, as it is a g emitter, it does not require scintillation fluid for counting.

• In the experiment above, we used only a 4-h pulse with 3 H-thymidine instead of the 16–20 h (overnight) pulse used by some investigators. We do this not only to shorten the time in cul- ture after isotope addition, thus reducing any effect of bacterial infection, but also to avoid re-utilization of isotope released from cells. This latter consideration is, however, of minimal importance under these conditions as there is a vast excess of free thymidine.

• Occasionally a high ‘background’ incorporation may be encountered when culturing cells from penicillin-sensitive individuals due to the antibiotic in the culture medium. Under these con- ditions use gentamycin alone.

• The potency of the mitogen needs to be considered and this may be deduced by initially performing a dose–response curve to assess mitogen stimulation.

9.5.2 Colorimetric microassay for cell proliferation

This is an alternative method for measuring cell proliferation that does not require a radioisotope and counting equipment.

MATERIALS AND EQUIPMENT IL-2-dependent T-cell line or mitogen-induced T-cell blasts

Tissue culture medium Fetal bovine serum (FBS) Phenol red-free tissue culture medium Phosphate-buffered saline (PBS) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), stock solution at 5 mg/ml

in PBS (stored in the dark) Isopropyl alcohol

C H A P T E R 9: Lymphocyte function

96-well microtitre plates, flat wells Centrifuge carriers for microplates ELISA reader

METHOD

1 Wash the cells three times by centrifugation to remove residual mitogen or IL-2 and

resuspend at 1 × 10 5 /ml in complete tissue culture medium (plus 10% FBS).

2 Dispense 100 µl aliquots into individual wells of a microtitre plate.

3 Add 100 µl aliquots of the IL-2 test samples to individual culture wells and incubate the plates at 37°C for 48 h. Controls should include cells, alone and with a positive standard IL-2 preparation.

4 After 48 h incubation, centrifuge the plates at 90 g for 10 min and remove the medium by rapidly inverting the plates with a firm flick.

5 Add 100 µl of MTT (1 mg/ml in tissue culture medium without phenol red) and incubate the plates for a further 3–4 h. Centrifuge the plate and remove medium as before.

6 Add 100 µl of isopropyl alcohol to each well to solubilize the formazan dye.

7 Read the plates on an ELISA reader using the following settings: test wavelength 570 nm, reference wavelength 630 nm.

Plot a curve of concentration versus optical density (equivalent to cell proliferation) for the standard preparation of IL-2 and use this to determine your unknown samples by interpolation. If a standard preparation of IL-2 is not available, arbitrary units should be assigned to the dilution of IL-2 that gives 50% maximal stimulation.