Mixed Lymphocyte Reaction FAQ’s

The Mixed Lymphocyte Reaction (MLR) is a functional assay which measures the proliferative response of lymphocytes from one individual (the responder) to lymphocytes from another individual (the stimulator). In the ‘one-way’ MLR, stimulator lymphocytes are treated to render them non-proliferative so that measurement of the responding cells is not obscured. The MLR can be performed on lymphocytes from the same individual (AUTO-MLR), from different individuals of the same species (ALLO-MLR), or individuals from different species (XENO-MLR).

Lymphocytes from both the graft donor and the recipient are isolated using a mechanical cell separation or Ficoll-Paque density gradient centrifugation. Stimulator cells are treated with mitomycin C which binds to DNA rendering the cells non proliferative. Responder cells are left untreated and therefore able to proliferate when stimulated. Equal numbers of viable stimulator and responder cells are added to test wells and incubated for 5 days. On day 6, bromodeoxyuridine (BrdU) is added to the wells to be incorporated in place of thymidine in the DNA of proliferating cells. After 24 hours, the cells are analyzed for BrdU incorporation by an ELISA-like method. Comparisons of the stimulated test cells with control, non-stimulated responder cells yields a stimulation index which can be used to compare proliferation in the various cell combinations.

There are four different types of observed responses as follows:

• AUTO – Autologous (negative control) is a measure of an individual’s cellular response to “self”.
• ALLO – Allogeneic is a measure of an individual’s response to cells from another individual of the same species.
• XENO – Xenogeneic is a measure of an individual’s response to cells from an individual of a different species.
• MITO – Mitogeneic (positive control) is a measure of a cell’s response to a known chemical (mitogen) which causes cells to proliferate.

A good positive control is the Mitogenic response which proves that the responder cells are viable and responsive to stimulation. A good negative control is obtained by preparing wells containing only mitomycin C- treated responder cells. These cells should remain non-proliferative compared to non-treated responder cells and/or mitogenic test cells.

The Mixed Lymphocyte Reaction is used to evaluate the potential for host versus graft or graft versus host reaction in transplantation which might be caused by mismatched tissue types or other antigenic differences between individuals.

Repeated measurements will determine if the recipient is becoming sensitized to the donor over a period of time.

The following should all be taken into consideration when setting up the experimental design of a Mixed Lymphocyte Reaction:

• Volume of blood needed to provide the requisite number of lymphocytes.
• Duration of stimulation to elicit a measurable proliferative response (usually 4-7 days).
• Whether the MLR is the test of choice for your experiment.
• Choose appropriate controls, assure sterility and the absence of endotoxin.
• The method of lymphocyte separation must also provide acceptable cell viability.

NEW! – We have added another page to discuss this question, as we have received numerous questions about experimental design and the optimization of Mixed Lymphocyte Reactions.

You may also want to consider in advance the regulatory requirements for submission.

We suggest the following:

• Cytotoxic responses that are mediated by antibodies and complement.
• IgM levels for acute antibody response in the host.
• IgG which develops after the initial IgM response.
• Knowledge of both antibody classes will assist in evaluation of the adaptive immune system response of the recipient.

Learn more about our contract research services and other capabilities here.