Production of human T-lymphocyte lines
6.8 Production of human T-lymphocyte lines
6.8.1 Human T-lymphocyte lines
Procedures for deriving human T-lymphocyte lines are essentially similar to those described for the mouse (see Section 6.7), but with some important differences and limitations. Human lines are usually derived using either ethical (vaccination) or fortuitous (usually an infection) immun- ization. They invariably need repeated donations from the same individual to provide both T lymphocytes (a minor requirement) and feeder cells (a massive requirement). Although it is possible to select a panel of HLA-D-matched individuals to supply feeder cells or to transfect the required major histocompatibility complex (MHC) molecules into antigen-presenting cells, this requires sophisticated laboratory back up. Attempts have been made to establish lines of Epstein–Barr virus-transformed autologous B cells from the T-lymphocyte donor and use these as antigen-presenting cells, but without uniform success.
MATERIALS AND EQUIPMENT Peripheral blood drawn from an antigen-sensitized individual (see Technical notes) Antigen Tissue culture medium Pooled normal human serum (NHS)
3 H-thymidine 96-well microculture plates, U-shaped wells
24-well culture plates Tissue culture flasks, 25 cm 2 Automated cell harvester Beta spectrometer
C H A P T E R 6: Isolation of cells
METHOD
As with the murine T cells, it is important to establish a dose–response curve for the antigen to
be used.
1 Fractionate peripheral blood mononuclear cells (PBMC) from heparinized blood by density gradient centrifugation (Section 6.2.2).
2 Wash the cells twice by centrifugation and resuspend in tissue culture medium containing 10% pooled NHS.
3 Dispense 100 µl aliquots of cells (2 × 10 6 /ml) into the wells of U-shaped well microtitre plates. Add 100 µl aliquots of antigen in doubling dilutions in 100 µl medium.
4 Incubate at 37°C in a humidified atmosphere of 5% CO 2 in air for 5 days.
5 Add 37 × 10 3 Bq of 3 H-thymidine to each well and incubate for a further 6 h.
6 Harvest the wells using an automated cell harvester and measure isotope incorporation using a b counter.
7 The results are expressed as an index of stimulation, as defined for murine lymphocytes above.
8 Plot a dose–response curve to determine the optimum antigen concentration required to give maximum T-lymphocyte proliferation.
TECHNICAL NOTES • Although many of the antigenic constituents of human vaccines or common infectious
organisms have been used to produce T-lymphocyte lines, those of the influenza virus have been particularly useful because parallel developments in molecular biology have made a wide range of defined ‘flu peptides readily available.
• Please 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
Human T-lymphocyte line production
MATERIALS AND EQUIPMENT Heparinized human peripheral blood Tissue culture medium Pooled normal human serum (NHS) Source of IL-2, either supernatant from the cell line MLA-144, or recombinant human material Antigen 96-well microculture plates, flat wells
Tissue culture flasks, 25 cm 2 growth area
24-well culture plates
METHOD
1 Prepare human peripheral blood lymphocytes from heparinized blood by density gradient centrifugation (Section 6.2.2) and resuspend in tissue culture medium containing 10% pooled NHS.
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6.8PRODUCTION OF HUMAN T-LYMPHOCYTE LINES
2 Adjust the cell concentration to 1–2 × 10 6 /ml and dispense in 10 ml aliquots into 25-cm 2 tissue culture flasks or in 2 ml aliquots in 24-well cluster plates. Add the required antigen at its optimum concentration.
3 To maintain high cell density incubate the flasks upright at 37°C in a humidified atmosphere of 5% CO 2 in air for 6–7 days.
4 Harvest the cells and separate the blasts by density gradient centrifugation (Section 6.2.2) and wash the cells in tissue culture medium by centrifugation.
The T-cell lines can be maintained in IL-2-containing medium with repeated rounds of stimula- tion with antigen and feeder cells. They are much easier to grow than their murine equivalents, to the extent that they can be cloned at or soon after primary plating.
6.8.2 Human T-lymphocyte cloning
MATERIALS AND EQUIPMENT As previous section, but including phytohaemagglutinin (PHA)
METHOD
1 To prepare autologous irradiated feeder cells, separate leucocytes from heparinized blood, wash and irradiate with 40 Gy from a high-energy source.
2 Adjust the cell concentration to 1 × 10 6 /ml in medium containing 10% pooled NHS, IL-2, antigen at the optimum concentration and a submitogenic concentration of PHA (e.g. 0.01–0.1 µg/ml ait is necessary to titrate the concentration with each new batch of PHA).
3 Resuspend fractionated T-cell blasts after in vitro stimulation as described in the previous section. Prepare suspensions at 100, 33, 10, 3.3 and 1 cell/ml in tissue culture medium containing 10% human serum. Prepare sufficient for 48 microcultures of each of the three higher concentrations and 96 cultures for each of the two lower concentrations.
4 Plate out 100 µl aliquots of the T-lymphocyte suspensions and incubate at 37°C in a humidified atmosphere of 5% CO 2 in air for 7 days.
5 Examine the plates under a phase-contrast inverted microscope for growth. The clones tend to grow in clumps and so are very easy to identify.
6 Positive wells can be expanded into 24-well cluster plates containing 1 × 10 6 irradiated autologous feeder cells per well in medium supplemented with serum, IL-2, antigen and PHA.
7 After a further 7 days’ incubation the T-cell blasts can be further expanded on an irradiated feeder layer grown in 25-cm 2 culture flasks with antigen stimulation.
To ensure monoclonality, repeat steps 1–6. T-cell clones can be maintained for short periods in IL-2-containing medium but will need stimulation with antigen and feeder cells every 6–14 days.
C H A P T E R 6: Isolation of cells