T-lymphocyte hybridomas

6.9 T-lymphocyte hybridomas

T-cell hybridomas have been used for studies on the T-cell receptor and as a source of T-cell- derived cytokines and other regulatory effector molecules, particularly suppressor factors. They are also used for evaluating cell recognition reactions. The methods used to prepare T-cell hybridomas are essentially the same as for B-cell hybrids: an activated population of T cells primed to respond to a particular antigen, or activated with mitogen (phytohaemagglutinin a PHA, or concanavalin A aCon A) is fused with a tumour-cell line of T-cell origin, usually the murine AKR thymoma BW-5147. Fusion products are assessed for phenotype and karyotype (to determine that they are true T-lymphocyte–tumour-cell hybrids) and finally cloned and functionally selected depending upon the activity (or activities) desired.

MATERIALS AND EQUIPMENT Activated murine lymphocytes (see Technical notes)

8-azaguanine-resistant variant of the murine AKR thymoma BW-5147 (but see also Technical notes) Tissue culture medium (Appendix B.6), alone or with 10% fetal bovine serum (FBS) Polyethylene glycol solution PEG-1500 (prepared as in Section 2.5) Concentrated HAT medium (see Section 2.5.4) 96-well microtitre plates

24- and 48-well tissue culture plates Irradiated (25 Gy) spleen cells and thymocytes, syngeneic with the lymph-node donor

METHOD

1 Mix 2 × 10 7 washed BW-5147 cells in serum-free medium with 1 × 10 8 washed stimulated murine lymphocytes in a 50-ml conical tube and centrifuge (150 g for 15 min at room temperature).

2 Carefully remove the supernatant by aspiration and tap the tube to resuspend the cells.

3 Place the tube in a water bath at 37°C and add 1 ml of a 1 : 1 mix of PEG-1500 in serum- free medium; add dropwise over 45 s.

4 Allow the tube to stand for 45 s and then over 5 min add 50 ml of serum-free medium prewarmed to 37°C with gentle mixing.

5 Leave the cell suspension at 37°C for 5 min and then centrifuge to pellet the cells (150 g for

15 min at room temperature).

6 Remove and discard the supernatant before resuspending the cells in serum-free medium. Centrifuge and resuspend in tissue culture medium containing 10% FBS.

7 Dispense in 100 µl aliquots into 96-well microtitre plates.

8 Incubate at 37°C for 24 h, and then add 50 µl of threefold concentrated HAT medium to each well.

9 Every 5 days, remove half of the supernatant and replace with fresh single-strength HAT medium, for at least 3 weeks.

First hybrids will begin to appear after about 10 days and new ones will continue to appear for the next 3 weeks. Growing hybrids should be karyotyped, phenotyped and screened for antigen

6.9T-LYMPHOCYTE HYBRIDOMAS

been confirmed. Cloning may be facilitated by the addition of 1 × 10 5 per well irradiated syn- geneic spleen cells. Feeder cells also act as antigen-presenting cells if required by an antigen- specific response. The cells should be freshly prepared and irradiated immediately before they are required. Samples of each clone of interest should be cryopreserved after screening, in case of not only accidental loss, but also population drift resulting in loss of function.

TECHNICAL NOTES • Activate T-lymphocytes by:

(a) priming in vivo by injection of antigen in adjuvant subcutaneously at the base of the tail, restimulation in vitro and expansion in IL-2-containing medium to achieve the required cell numbers; or

(b) stimulation with mitogen (PHA or Con A); stimulation in vitro followed by expansion in IL-2- containing medium. Hybridomas derived from these cell populations would not be expected to show antigen specificity.

• The BW-5417 line should be cultured every 2–3 months in 2 × 10 5M 6-thioguanine to maintain sensitivity to aminopterin in HAT. • Other murine tumour-cell lines such as FS6-14.13 can also be used for fusion to murine T-cells to produce T-cell hybridomas. Human T-lymphocyte hybridomas can be produced using appropriate human T-cell tumour lines, e.g. MOLT-4.

• The activated lymphocyte population may be enriched for T cells by nylon wool column frac- tionation (Section 6.6.3) under sterile conditions prior to fusion. • To facilitate phenotypic analysis (Section 6.9.3), it is convenient to use a T-lymphocyte donor that expresses the Thy-1.2 gene product such as C3HeB/Fe and many other strains, in contrast to the Thy-1.1 expressed by the AKR-derived BW-5147 line.

6.9.1 Screening for functional T hybrids

The techniques used to analyse the T hybridomas will depend upon the effector function rescued and whether the cells are antigen-specific. In general, the techniques cited in Section 9.4 may be adapted for use with T-hybrid lines. T-cell hybrids tend to be unstable, often exhibiting very high ploidy numbers immediately after fusion, but rapidly losing chromosomes thereafter. Clones of interest should be cryopreserved and growing clones checked and recloned to maintain their antigen-specific effector function.

6.9.2 Karyotype analysis

MATERIALS AND EQUIPMENT Actively growing T hybrids

Colchicine (usually supplied as Colcemid) 0.1% w/v solution Trypsin, 1.0% w/v solution Potassium chloride, 0.5% solution Buffered Giemsa stain, freshly made prior to use (see Section 11.7)

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

METHOD

1 Transfer about 10 7 actively growing and dividing T-hybridoma cells into a tissue culture grade conical centrifuge tube. Add 0.1 ml of 0.1% colchicine and incubate at 37°C for 2 h. This arrests the cells in mitosis.

2 Centrifuge to pellet the cells and remove the supernatant.

3 Resuspend the cells vigorously, using a vortex mixer, add 10 ml of 0.5% potassium chloride and incubate for 10 min to allow the lymphocytes to swell and burst.

4 Centrifuge to pellet the contents of the tube, remove the supernatant and slowly resuspend the pellet in 1 : 3 solution of glacial acetic acid and methanol to fix the nuclei.

5 Repeat the centrifugation and fixing twice and finally resuspend the pellet in about 10 drops of fixative.

6 Wash a clean microscope slide with a few drops of fixative and wipe dry with a clean tissue to completely degrease.

7 Breathe onto the slide to warm and moisten it. Using a Pasteur pipette deposit a single drop of the cell suspension onto the slide from a height of 15–30 cm. Tilt the slide to drain the fixative away and allow the slide to dry. This procedure ruptures the nuclei and allows the chromosomes to spread. It requires a certain amount of practice to achieve good separation of the chromosomes.

8 Dip the slide in the trypsin for 20 s and wash with tap water.

9 Stain with buffered Giemsa (Section 11.7).

10 Wash with tap water.

11 Observe under a transmitted light microscope: at × 40 magnification to select a suitable nuclear spread, and then to × 100 magnification for counting.

6.9.3 Phenotypic analysis

Karyotypic analysis (Section 6.9.2) provides the definitive demonstration of hybrid status, but in the initial stages it is convenient to select on the basis of a hybrid cell-surface phenotype. Most strains of mice express the Thy-1.2 gene product, whereas AKR mice, from which the tumour-cell line BW-5147 was derived, express Thy-1.1. Thus hybrids are HAT resistant, express the Thy-1.2 antigen and sometimes coexpress Thy-1.1. Lines selected after HAT treatment which express only Thy-1.1 should be discarded as they are probably HAT-resistant revertants of the tumour-cell line.

MATERIALS AND EQUIPMENT Anti-Thy-1.1 and anti-Thy-1.2 monoclonal antibodies (for direct or indirect immunofluorescent

staining; Section 4.2.5) Phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) UV microscope or continuous-flow cytofluorimeter

METHOD

1 Wash 6 × 10 6 of the hybrids by centrifugation and divide into 3 aliquots.

2 Process for direct or indirect immunofluorescence using anti-Thy-1.1, anti-Thy-1.2 and irrelevant (control) antibodies (Section 8.2.1).

3 Examine by fluorescence microscopy or by flow cytometry (Section 8.5).

6.9T-LYMPHOCYTE HYBRIDOMAS