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Mixed Lymphocyte Reactions

A Simple and Powerful Assay for Understanding T Cell Activation

Mixed Lymphocyte reactions (MLR) assays can be used to understand the effects of biologics or small molecules on T cell activation, an important response in the tumour microenvironment (TME). T cells play a significant role in the immune system. Once activated, they express cytokines that help regulate and facilitate T cell mediated immune responses. Therefore, understanding a compound’s effect on T cell activation is potentially a key indicator for determining a drug candidate’s mechanism of action (MOA).

MLR assays rely on a proportion of the “responder” T cells bearing TCRs that will be triggered by allogenic HLA molecules displayed by “stimulator” APCs. These APCs have the capacity to provide costimulatory and coinhibitory signals in the assay.

Aquila offer a range of customisable MLR protocols, and we will work with you to advise on the most suitable MLR for your research question, tailoring the assay accordingly to ensure a strong data readout.

The simplest MLR protocol combines PBMCs from two donors as either “two-way” cultures, in which each PBMC population can act as stimulator and responder, or a “one-way” MLR where one PBMC population is first irradiated so that it can function as a stimulator but not a responder.

A more defined MLR system uses monocyte-derived dendritic cells (Mo-DC), which can be immature or matured under varying conditions, depending on the target of interest. Responder T cells are isolated from HLA-mismatched donors, providing a source of alloreactive T cells.

Figure 1. Allogeneic MLR

Readouts for T cell function include cytokine production e.g. IL-2, IFN-γ (ELISA, multiplex assays). Flow cytometry can be added to assess proliferation (CFSE dilution), cell cycle (Ki-67) and activation status (e.g. CD25, PD-1, LAG-3, TIM-3 etc). Cytokine production, transcription factor expression and signalling pathways (e.g. phospho-STATs) can be studied by intracellular staining. Activation status of DCs (e.g. HLA, CD80, CD83, CD86) can also be assessed by flow cytometry.

Figure 2. Readouts for T cell function. IL-2 expression is measured after 2 days and IFN-γ expression is measured after 5 days.

Aquila can help you understand the immunomodulatory effects of your drug candidates within the tumour microenvironment and/or in combination with other immune modulators. We offer a tailored program of work and data you can trust, giving you greater confidence in your lead compound selection. Aquila have extensive in vitro immunology, pre-clinical immuno-oncology and specialist multiplex histology experience. Our suite of immuno-oncology services and assays can be specifically customised to your project, efficiently progressing your route to clinic.

Interested to hear more about how Aquila’s customisable MLR assays? Get in touch so we can support your best route to clinic.


Mixed lymphocyte reaction (MLR) assays can be used to understand the effects of biologics or small molecules on T cell activation, an important immune response...


We can help you understand your target's and/or compound's mechanism of action to progress your drug discovery pipeline.


Immuno-oncology is an exciting and progressing area of research that uses manipulation of the immune system to successfully target cancer.

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