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:
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 MLR 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 MLR:
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: