Propagation of antigen-responsive normal T-lymphocyte lines

6.7 Propagation of antigen-responsive normal T-lymphocyte lines

Under the conditions described below, normal T lymphocytes may be expanded into lines capable of extensive in vitro proliferation and yet still retain their effector capacity and antigen selectivity.

Immunogens used range from allogeneic cells to processed antigen expressed by MHC- compatible antigen-presenting cells or viral antigens expressed on infected autologous host cells.

There are many different methods for isolating antigen-specific T-cell clones in vitro. We will describe here methods that we have used for the isolation of murine and, in Section 6.8, human antigen-reactive T-cell lines and clones.

6.7.1 Dose–response curve for antigen

MATERIALS AND EQUIPMENT Inbred mice for immunization (it is important to use inbred mice of known haplotype as

Ia-identical animals have to be used as a source of feeder cells) Antigen Freund’s complete adjuvant Tissue culture medium Fetal bovine serum (FBS) 2-mercaptoethanol Antibiotics astreptomycin and penicillin 96-well culture plates

3 H-thymidine Automated cell harvester

Beta spectrometer

METHOD

1 Immunize mice by a subcutaneous injection at the base of the tail with antigen emulsified in Freund’s complete adjuvant (100 µl total volume per injection site).

2 Anaesthetize the mice 3– 4 days after the last injection and bleed for serum.

3 Kill the mice by cervical dislocation and remove the para-aortic and inguinal lymph nodes (draining the base of the tail) using aseptic technique.

4 Prepare a sterile single-cell suspension as described in Section 6.3.

5 Wash the cell suspension twice by centrifugation and resuspend the cells in complete medium (containing 10% FBS, 2 × 10 –5 M 2-mercaptoethanol, 100 U/ml penicillin and

100 µg/ml streptomycin) and resuspend at 2 × 10 6 cells/ml.

A control population should be prepared in an identical manner using cells from mice immunized with Freund’s complete adjuvant alone.

6 Dispense 100 µl aliquots of cells into the wells of a 96-well microtitre tray (U-shaped wells) and add a range of antigen concentrations in triplicate, each in 100 µl of medium.

7 Incubate for 4–6 days, depending on the peak of the mitogenic response, and during the

last 18 h of incubation add 37 × 10 3 Bq per well of 3 H-thymidine.

Continued on p. 194

6.7PROPAGATION OF ANTIGEN-RESPONSIVE NORMAL T-LYMPHOCYTE LINES

8 Harvest with an automated cell harvester and measure radioactive incorporation using a b scintillation counter.

9 Calculate the proliferation index according to the equation: c.p.m. cells with antigen c.p.m. cells without antigen −

Stimulation index =

c.p.m. cells without antigen

Repeat the calculations for cells from animals immunized with Freund’s adjuvant alone to check that the antigen being used reacts specifically with the cells primed with antigen in vivo.

10 Plot a dose–response curve for the antigen to determine the optimum concentration for in vitro stimulation.

TECHNICAL NOTE The immunization protocol must be varied to take account of the antigen being used. A single injection of a highly immunogenic antigen is often sufficient. However, for some antigens a booster injection 7 days after the first (or an even more extensive boosting) may be advantageous. In any case harvest the lymph nodes 3–4 days after the last injection.

6.7.2 Murine T-lymphocyte line production

METHOD

1 Immunize four to six mice as described above and prepare a single-cell suspension of lymph-node cells (see Section 6.3).

2 Resuspend the cells in tissue culture medium containing 10% FBS and aliquot at 2–5 × 10 6 cells per well in 24-well culture plates.

3 Add antigen at the optimum concentration (determined above) to achieve a total culture volume of 1.5 ml and incubate for 4–6 days at 37°C in a humidified incubator gassed with

5% CO 2 in air.

4 Harvest the cells by centrifugation (150 g for 10 min at room temperature) and isolate the blast cells by density gradient centrifugation (Section 6.2).

5 Wash the cells twice by centrifugation, resuspend in tissue culture medium containing 5% FBS and count in a haemocytometer.

6 Prepare a single-cell suspension from the spleens of syngeneic mice (see Section 6.3) for use as feeder cells.

7 Irradiate the normal spleen cells with 25 Gy irradiation, wash and resuspend in tissue culture medium containing 5% FBS.

8 Plate 1–2 × 10 6 irradiated feeder cells with 1 × 10 5 T-cell blasts per well in a total volume of

2 ml.

9 Incubate for 7–14 days.

10 Repeat steps 3–6 and plate 1–2 × 10 6 irradiated feeder cells with 1–2 × 10 5 T cells plus antigen at the optimum concentration.

11 Incubate for 4 days.

12 Repeat steps 3–7.

C H A P T E R 6: Isolation of cells

T-cell lines can be maintained long-term by this regime of regular stimulation and ‘rest’. When larger numbers of cells are required for assays, the cells can be expanded in tissue culture flasks

(start with 25 cm 2 growth area flasks). To maintain maximum cell density at the beginning of an expansion phase, incubate the flasks upright or at a slight angle to vertical. It is possible to main- tain antigen-reactive T-cell lines in culture for up to about 14 days without antigen stimulation by adding exogenous interleukin 2 (IL-2) to the medium. Activated (antigen-stimulated) T-cell lines which express IL-2 receptors will respond by vigorous growth and proliferation. However, growth declines rapidly within a few days because of a decrease in the expression of the IL-2 receptor. Restimulation with the appropriately presented antigen or a mitogen is then required to induce re-expression of high levels of the Il-2 receptor.

Laboratories vary in their techniques for production of T-cell lines and many factors can influence the outcome; persistence is obligatory and ‘green fingers’ are helpful.

6.7.3 T-lymphocyte cloning

MATERIALS AND EQUIPMENT Antigen-reactive T-cell line Syngeneic irradiated feeder cells Antigen Tissue culture medium containing 5% fetal bovine serum 96-well microculture plates (flat wells) Interleukin 2 (IL-2) (see Technical note)

METHOD

1 Prepare T-cell blasts as described in previous section.

2 Dispense 5 × 10 5 irradiated feeder cells in tissue culture medium containing IL-2 and the optimum concentration of antigen into the wells of the microtitre trays.

3 Prepare suspensions of the T-cell blasts at: (a) 100 cells/ml; (b) 33 cells/ml; (c) 10 cells/ml; and (d) 3.3 cells/ml.

4 Dispense aliquots of 100 µl of each suspension, preparing one plate each for (a) and (b), and three plates each for (c) and (d). The higher concentrations are used to check that the cells will grow.

5 Examine the plates under an inverted microscope with phase-contrast optics after 7 days.

6 Wells in which T lymphocytes have grown can easily be identified, as the phase-bright T cells grow as a clump amongst the dying, phase-dark feeder cells.

7 Positive wells should be transferred into 24-well culture plates containing fresh, irradiated syngeneic feeder cells (2 × 10 6 per well), the optimum concentration of antigen and IL-2.

8 To ensure that monoclonality is obtained the selected ‘cloned lines’ should be recloned by plating at 1 cell/ml.

Once cloned populations have been selected and expanded, aliquots should be cryopreserved against accidental loss or clonal exhaustion. By this stage the T lymphocytes will have adapted (probably been intensively selected) to the in vitro conditions and so can be maintained relatively

6.7PROPAGATION OF ANTIGEN-RESPONSIVE NORMAL T-LYMPHOCYTE LINES

TECHNICAL NOTE It is preferable to use recombinant IL-2 for technical convenience: titrate the units of activity to determine the optimum concentration in your system. This is likely to be around 2000 U/ml. Alternatively, IL-2-containing supernatants can be prepared from T-lymphocyte lines (MLA-144 (gibbon line) for use with human cells or EL4 for use with murine cells) or from normal lymphocytes (e.g. rat spleen) stimulated in bulk with concanavalin A (mitogenic stimulation as for phytohaemagglutinin aPHA). IL-2 is constitutively produced by the cell lines; however, its production may be maximized by growing the cells to their plateau density, washing them into serum-free medium and incubating overnight at 37°C in a humidified atmosphere containing 5%

CO 2 in air. Interleukin-2 from any of these sources will maintain murine T-lymphocytes, but human T- lymphocytes need primate IL-2.