Mixed-lymphocyte reaction (MLR) and cell-mediated cytolysis (CMC)
9.8 Mixed-lymphocyte reaction (MLR) and cell-mediated cytolysis (CMC)
MATERIALS AND EQUIPMENT CBA or C3H and DBA/2 mice P815Y mastocytoma cells Sodium 5l chromate X-ray machine or g source Gamma spectrometer
9.8.1 Mixed-lymphocyte reaction
METHOD
1 Prepare spleen-cell suspensions from C3H and DBA/2 mice.
2 Irradiate DBA/2 cells (30 Gy); these will be used as MLR-stimulator cells. Irradiate immediately before putting into culture. The stimulatory capacity of irradiated cells falls within a few hours if they are allowed to stand at 4°C.
3 Prepare MLR cultures using irradiated DBA/2 and C3H cells. Mix 10 6 of each cell type and culture in 3 ml of medium in 5 ml Falcon plastic tubes as in Protocol A.
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9.8MIXED-LYMPHOCYTE REACTION (MLR) AND CELL-MEDIATED CYTOLYSIS (CMC)
Prepare sufficient replicates of each tube to provide cells for the CMC assay on the 4th day of MLR culture (see Protocol B) (viability of MLR cultures varies athis must be standardized for each laboratory) and, in addition, prepare three replicates of tubes 1–3 for the assay of DNA synthesis in the MLR culture.
4 On the 4th day of the MLR culture collect cells for the CMC assay (Protocol B).
5 On the 5th day of the MLR culture add 3 H-thymidine to three replicates of tubes 1–3 to assay for DNA synthesis. We have given absolute numbers of MLR cells rather than the usual lymphocyte : target ratio. In fact, the efficiency of target-cell killing is not ratio dependent over a wide range.
A MLR protocol.
Tube number
X-irradiated cells
10 6 C3H or CBA C3H- or CBA-responder cells
2 × 10 6 DBA/2
10 6 DBA/2
0 10 6 10 6 * This is a better control than unirradiated cells alone as irradiated cells might exert a slight inhibitory activity
upon the generation of possible CMC cells.
B CMC protocol. Tube number (three replicates)
10 5 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ MLR lymphocytes from tube number:
51 Cr-labelled mastocytoma cells
9.8.2 Cell-mediated cytolysis
METHOD
1 Label mastocytoma cells with 51 CrO 4 .
2 Count number of viable lymphocytes recovered from MLR (Protocol A).
3 Prepare cell mixtures in 2 ml of medium as shown in Protocol B.
4 Culture for 6 h at 37°C in a CO 2 incubator.
5 Resuspend the cells after culture and centrifuge (150 g for 10 min at 4°C).
6 Remove 1 ml of the supernatant from each tube for g counting.
C H A P T E R 9: Lymphocyte function
9.8.3 Calculation of isotope release (equivalent to target-cell destruction)
METHOD
1 Lyse an aliquot of 10 5 original 5l Cr-labelled mastocytoma cells either by freezing and thawing (three times at 37°C and –20°C) or with 10% w/v saponin.
2 Spin down insoluble material from the lysate and count radioactivity in the supernatant. Use this value as the maximum (100%) isotope release.
3 Calculate spontaneous release from the labelled mastocytoma (tube 6 in triplicate) as a percentage of the total counts released by saponin. The mean of these three determinations will be used to correct the release observed in lymphocyte–target mixtures (tubes 1–5).
4 Calculate experimental release for each lymphocyte–target mixture as a percentage of the total counts released by saponin (tubes 1–5, in triplicate).
5 Calculate specific release as follows:
% specific release = 10 [ R e − R s ] 100 − R s
where R e is mean percentage experimental release and R s is mean percentage spontaneous release.
6 Plot a graph of percentage specific release for each group against the number of MLR- derived cells used to lyse the mastocytoma cells. Calculate also the standard deviation of each group.
TECHNICAL NOTE In experimental determinations of CMC it is advisable to assay at 4, 6 and 8 h to determine the optimum under your conditions, rather than at the single time point as suggested here.
9.8.4 CMC with PHA blasts
The applicability of CMC may be extended to any system of alloantigens using 51 Cr-labelled PHA blasts as target cells. PHA blasts may be produced en masse as follows.
MATERIALS AND EQUIPMENT As Section 9.5 (mitogenic response), but in addition: Inbred mice Tissue culture medium containing fetal bovine serum and antibiotics Plastic tissue culture flasks
METHOD
1 Prepare cell suspension from mouse lymph nodes.
2 Count cells and adjust to 3–5 × 10 6 /ml.
3 Add optimal concentration of PHA (see methods in Section 9.5).
4 Add 20 ml of cell suspension to each bottle and gas for 60 s with 5% CO 2 in air.
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9.8MIXED-LYMPHOCYTE REACTION (MLR) AND CELL-MEDIATED CYTOLYSIS (CMC)
5 Place bottles on their sides in a 37°C incubator. The kinetics of the response are essentially similar to those seen in Section 9.5.
6 After 72 h, pool cells, wash three times in tissue culture medium and label with 51 CrO 4 .
7 Use for CMC as above in Section 9.8.2.
TECHNICAL NOTES • The protocols above are technically less demanding when carried out with human peripheral
blood lymphocytes (PBL). In general, PBL, even from the mouse, are much easier cells to culture and give a very low spontaneous background.
• The ability to expand T lymphocytes to form large clonal populations using antigen stimulation and the cytokine interleukin 2 (IL-2) means that cytotoxic effector cells may be generated from even a single progenitor cell grown in limiting dilution culture. Although the implications of this powerful cell technology are enormous, such refinements are natural developments of the techniques described here.