Mixed Lymphocyte Culture / Reaction (MLC / MLR)

Principle

T cells from one individual are mixed and co-cultured with lymphocytes from a second
individual. If the cells of one individual recognized other’s MHC molecules as foreign,
they proliferate and divide. The proliferative response is usually measured with 3H-
labeled thymidine uptake assay.
There are two variants of the test: “two-way” – cells of both individuals are free to
proliferate, and “one-way” – cells of one of the individuals are irradiated or treated with
mitomycin so they can’t proliferate. Thus, cells of only one of the individuals can
proliferate.

Objective

- To detect alloreactive T cells and histoincompatibility

- Used for selection of donors for bone marrow transplants - lowest possible
alloreactive response is essential (limitation: no accurate quantitation of
alloreactive T cells)
- Selection of donors for living-related renal allotransplantation
- Typing of HLA-D antigens
- Used in diagnosis of immunodeficiency diseases.

Procedure (“one-way” test)

Isolation of lymphocytes from peripheral blood from two individuals

Irradiation/treatment with
mytomicin C of lymphocytes from
one of the individuals (yellow)
 Irradiated cells act as stimulator
cells but cannot proliferate
Cells from the two individuals
are mixed
3 to 7 days later: T-cell
proliferation assay (e.g. 3H-
thymidine uptake) (if the
unirradiated lymphocytes contain
alloreactive T cells, they will
proliferate and differentiate to
effector cells as a result of
recognition of foreign MHC
molecules).

Molecular Biology of the Cell 4th ed. Alberts, Bruce Et al.

Example

Taken from: Goutarou Katsuno et al. The immunosuppressive effects of ciprofloxacin

during human mixed lymphocyte reaction. Clinical Immunology (2006) 119, 110—119

The goal of this experiment was to check the effects of ciprofloxacin on intracellular
expression of IFN-γ in monocytes and T-cells during MLR. Cells from two unrelated
individuals were mixed and incubated under different conditions for 24 hours. All the
reagents were added at the beginning of MLR. Next, expression of IFN-γ was determined
with flow cytometry

Results:
 CIP at 100 µg/ml had no effect on the expression of intracellular IFN-γ in both
monocytes and T-cells
IL-18 at 100 µg/ml upregulated the expression of intracellular IFN-γ both in
monocytes and T-cells
CIP suppressed the IL-18-enhanced expression of intracellular IFN-γ both in
monocytes and T cells
CIP also suppressed the LPS enhanced intracellular IFN-γ expression in T-cells during
MLR

References:
 http://www.mercksource.com/pp/us/cns/cns_hl_dorlands.jspzQzpgzEzzSzppdocs
zSzuszSzcommonzSzdorlandszSzdorlandzSzdmd_c_66zPzhtm#12270597
 Goutarou Katsuno et al. The immunosuppressive effects of ciprofloxacin during
human mixed lymphocyte reaction. Clinical Immunology (2006) 119, 110—119
 Molecular Biology of the Cell 4th ed. Alberts, Bruce et al.