Microculture technique and response to non-recall antigens (primary immune response in vitro)

9.6 Microculture technique and response to non-recall antigens (primary immune response in vitro)

This technique uses a maximum of only 10 5 responding cells per culture. The reduced cell num- ber allows a greater number of variables to be tested per experiment. The introduction of semi- automated procedures has greatly reduced the time required for plating out and harvesting.

MATERIALS AND EQUIPMENT Blood, containing heparin (10 IU/ml). The heparin must be preservative free Tissue culture medium Lymphoprep

3 H-thymidine ( 3 H-TdR) ause at 37 × 10 4 Bq/ml in tissue culture medium

Scintillation fluid Microculture trays, 96 wells, flat bases Eppendorf multidispenser Cell-harvesting machine Beta scintillation counter

9.6MICROCULTURE TECHNIQUE AND RESPONSE TO NON-RECALL ANTIGENS

METHOD

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

2 Carefully layer 6 ml of diluted blood onto 3 ml Lymphoprep or similar separation medium to obtain PBMC (will contain lymphocytes and antigen-presenting cells).

3 Centrifuge at 400 g (interface force) for 20 min at room temperature. A misty layer of lymphocytes will be visible at the plasma–density gradient interface.

4 Remove lymphocytes using a Pasteur pipette and mix with an equal volume of tissue culture medium.

5 Centrifuge at 250 g for 15 min at room temperature and remove the supernatant.

6 Wash twice in tissue culture medium by centrifugation (150 g for l0 min at room temperature).

7 Remove an aliquot of cells and determine the number of viable lymphocytes/ml. Adjust to

2 × 10 6 lymphocytes/ml.

8 Prepare cultures in microwells according to the following Protocol.

Protocol. Control wells

Stimulated wells

100 µl tissue culture medium

50 µl tissue culture medium

50 µl stimulant*

50 µl lymphocyte suspension

50 µl lymphocyte suspension

50 µl autologous plasma 200 µl total volume

50 µl autologous plasma

200 µl total volume

* Mitogen, antigen or allogeneic cells, at optimum concentration.

9 Set up three to five replicate cultures of each treatment using an Eppendorf multidispensing pipette.

10 Replace the lid and place the culture tray in a humidified incubator gassed with 5% CO 2 in air.

11 The magnitude of the mitotic response is determined by the addition of 50 µl of 3 H-TdR to each well before harvesting. As an approximate guide:

(a) For PHA cultures add 3 H-TdR 40–48 h after the initiation of culture; incubate for 4 h at 37°C before harvesting. (b) For mixed lymphocyte or antigen-stimulated (e.g. Candida or PPD) cultures add 3 H-TdR

5 days after the initiation of culture; incubate for 6 or 18 h at 37°C before harvesting, depending on the sensitivity of the assays in your hands. Once the assay is highly reproducible, you will need fewer counts to detect a significant difference between experimental groups so a shorter ‘pulse’ time will be possible.

(c) Primary responses may be studied in this microculture system with harvesting

around day 6–7 rather than the 4–5 days that are optimal for recall antigens (Young et al. 1995).

Continued

C H A P T E R 9: Lymphocyte function

12 Harvest the cultures using a semi-automatic cell-harvesting procedure.

13 Dry filter strips from harvesting machine at 37°C for at least 3 h.

14 Remove discs from the filter strips and place each disc in a counting vial containing scintillation fluid.

15 Count b emissions in a scintillation counter; assess the results as in Section 9.5.1.

TECHNICAL NOTES • The volume of PBMC may be usefully increased to 200 ml for non-recall antigens to increase the

chances of responding cells being present. • Autologous plasma gives low backgrounds but for convenience this can be replaced with human AB serum. Make up the medium to 5% serum. • Addition of dendritic cells has been reported to increase the sensitivity for detecting responses to non-recall antigens (Macatonia et al. 1989).

9.6.1 Dye-monitored lymphocyte proliferation

Lymphocyte proliferation monitored by the incorporation of 3 H-thymidine is very sensitive but its measurement involves the lysis of the proliferating cells and their harvesting, so no further characterization is possible of the same cell population. Alamar Blue dye changes its colour from blue to red in the presence of proliferating cells. This was originally thought to be brought about by the release of metabolites into the medium that then acted upon the dye. It is now known that the colour change is dependent on the presence of activated cells interacting directly with the dye. The dye is added to cell cultures, in microtitre plates, 24–48 h before measurement. Plates are then monitored in an ELISA reader at 600 nm (oxidized dye) and 570 nm (reduced dye). This is subtracted from the reading at 570 nm to give a measure of prolif- eration (Zhi-Jun et al. 1997). As there is no interference with the cells, plates may be read again at various time points. The cells may also be washed and further studied in other systems, for instance in the fluorescence-activated cell sorter after labelling with appropriate antibodies.

The assay correlates very closely with the 3 H-thymidine incorporation system. See also the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) described in Sections

10.2.2 and 10.3.

9.6.2 Mixed-lymphocyte reaction

The mitotic response obtained when cells taken from two inbred strains or from two outbred individuals of any species are mixed in in vitro culture is known as the mixed-lymphocyte reac- tion (MLR). It is an in vitro counterpart of the host versus graft (HvG) or graft versus host (GvH) reactions examined later. Like the GvH reaction: (i) the majority of the responsive (as opposed to responding) cells are T lymphocytes; (ii) it has not been possible to demonstrate unequivocally an effect of previous immunization on the magnitude of the response between strains with a ‘strong’ H-2 difference; and (iii) it is possible to increase the magnitude of the response by previous sens- itization across ‘weak’ H-2 differences.

9.6 MICROCULTURE TECHNIQUE AND RESPONSE TO NON-RECALL ANTIGENS

It is important to distinguish between responsive and responding cells because of the phe- nomenon known as back stimulation; it was found that F 1 cells gave a mitotic response when mixed with X-irradiated or mitomycin-treated parental cells. According to MLR genetics the F l should not recognize the parent cells as being foreign. The mechanism proposed to explain this

back stimulation was that the blocked parental cells recognize the F 1 cells as foreign and produce ‘mitogenic factors’ (previously cytokines) which non-specifically induce proliferation in the immunologically unresponsive F l cells. MLR cultures may be performed using culture con-

ditions similar to those described for PHA but mixing 10 6 cells from each of two donors to yield the total of 2 × 10 6 per culture. A two-way MLR will result, i.e. donor A will recognize B and vice versa. In many situations it is an advantage to have a unidirectional response and so parent and F 1 mixtures can be used, or, more simply, the proliferation of either cell type may be blocked with X- irradiation or mitomycin C treatment.

A suggested experimental protocol is given below; these cultures are then used as a source of cytotoxic effector cells. Activation and expansion of T-lymphocyte populations on a clonal basis by antigenic stimulation and culture in interleukin 2 (see Section 6.7) has provided a much more sensitive and precise method of quantification of antigen-reactive T lymphocytes by limiting dilution analysis (see below).

9.6.3 Whole blood assay for T-cell responses

Purification of lymphocytes is (i) time consuming and expensive and involves collecting reason- ably large volumes of blood, and (ii) difficult when working with patients where large samples are not available, or where such demands would discourage participation in experiments. A tech- nique for assaying responses in whole blood has been developed which gives comparable results to the conventional technique with separated cells.

MATERIALS AND EQUIPMENT Blood awith preservative-free heparin as anticoagulant Microtitre, 96 U-shaped well plates Antigen or mitogen

Methyl 3 H-thymidine Tissue culture medium Cell-harvesting apparatus Beta scintillation or plate counter

METHOD

1 Dilute blood 1 : 10 with tissue culture medium.

2 Place 200 µl samples in triplicate, in multiwell plate.

3 Add antigen (see Technical notes).

4 Incubate at 37°C in humidified incubator with 5% CO 2 .

5 On day 6 (or follow time course) add 37 × 10 3 Bq 3 H-thymidine to each well.

6 Incubate for 18 h at 37°C.

7 Harvest cells, e.g. onto glass-fibre filter paper strips (as above).

8 Count in scintillation counter.

C H A P T E R 9: Lymphocyte function

TECHNICAL NOTES • The response to mycobacterial antigens was found to be optimal at around 1–10 µg/ml, but

this will need to be retested for different antigens. • Sensitive estimates of T-cell responsiveness can be obtained by determining cytokine secre-

tion following antigen stimulation. Supernatant (100 µl) samples can be taken on day 6 and estimated for interferon-g concentration by ELISA.

• It is necessary to eliminate interference in the scintillation count with haemoglobin by removal of red blood cells at the harvesting stage (see also Sewell et al. 1997).