Development of a Mixed Lymphocyte Reaction (MLR) assay for evaluating the allogenicity of cell therapy

Cell therapies are emerging as a powerful class of treatments capable of addressing complex and previously incurable diseases, but their development remains challenging. In particular, allogeneic cell therapies offer scalable, “off-the-shelf” solutions, yet they carry risks related to immune compatibility and potential adverse reactions such as graft-versus-host disease.

The Mixed Lymphocyte Reaction (MLR) assay is a well-established in vitro method used to evaluate allogeneic immune responses by measuring T cell activation and proliferation.

This application note describes the development and optimisation of a non-radioactive MLR assay combining flow cytometry-based proliferation readouts and cytokine quantification, providing a comprehensive strategy to assess allogeneic responses.

Assay performance overview

• Development of a non-radioactive MLR assay combining CellTrace staining, EdU incorporation, and cytokine quantification for assessing allogeneic responses
• Optimised CellTrace-based proliferation assay enabling detection of multiple cell divisions and clear discrimination between responding and stimulating cell populations
• Demonstration of assay sensitivity and specificity to distinguish autologous versus allogeneic responses across multiple donors

How this supports your cell therapy development programme?

This MLR assay provides a comprehensive framework for evaluating immunological compatibility and immune activation in cell therapy development.

• Improves interpretation through combined proliferation and cytokine readouts
• Offers flexibility with multiple analytical approaches (Flow cytometry with CellTrace or EdU, Gyrolab, Luminex)
• Allows early detection of immune activation through cytokine secretion profiling
• Enhances robustness through optimised assay conditions and controlled experimental design

This approach provides a robust and adaptable solution for immunogenicity assessment in advanced therapy development.

Application areas

• Evaluation of allogeneic immune responses between PBMCs from unrelated donors
• Assessment of immunogenicity risks in allogeneic cell therapy products
• Characterisation of T cell proliferation and activation mechanisms
• Measurement of cytokine release (IL-6, IFNγ) as indicators of immune activation
• Proof-of-concept evaluation of cell therapy products in co-culture systems