Immune Function & Modulation Assays
Mixed Lymphocyte Reaction (MLR) Assay
Does your compound modulate dendritic-cell function, alter T-cell activation, or influence the interaction between the two?
Our allogeneic MLR assay combines primary human monocyte-derived dendritic cells (moDCs) and CD4+ T cells from unrelated donors to assess compound effects on dendritic-cell function and the resulting T-cell response. IFN-γ provides a primary functional readout, complemented where needed by cytokine profiling and immunophenotyping, while independent donor pairs enable assessment of response variability and consistency across donor backgrounds.
MLR assay format and readouts
- Format
- 96-well plate
- Model
- Allogeneic co-culture of primary CD4+ T cells (responder) and monocyte-derived dendritic cells (moDCs; stimulator)
- Primary readout
- IFN-γ release as a surrogate of T-cell activation
- DC phenotype
- flow cytometry immunophenotyping, including CD86, CD80, CD83, CD206, CD209 and CD1a, where included
- On request
- IL-2, IL-8, IL-10 and IL-12, extended cytokine profiling, proteomics and transcriptomics
- Reference controls
- Checkpoint inhibitors including anti-PD-1 nivolumab and anti-PD-L1 atezolizumab, and adenosine-pathway modulators, selected to investigate suppression or relief of suppression in the model.
MLR responses in allogeneic human moDC/CD4+ T-cell co-cultures vary across independent donor pairs and are modulated by PD-1 blockade and adenosine. Peripheral blood monocyte-derived mature dendritic cells are co-cultured with allogeneic CD4+ T cells, and IFN-γ and IL-2 released into the supernatants are quantified. The allogeneic co-culture response increases after PD-1 blockade with nivolumab and is reduced under adenosine treatment.
Imane Nafia, PhD
Chief Scientific Officer, Explicyte
Assay principle
How the MLR assay works
1
Establish the allogeneic co-culture
Monocyte-derived dendritic cells from one donor (stimulator) are co-cultured with CD4+ T cells from a second, unrelated donor (responder) in 96-well plates, eliciting an allogeneic T-cell response.
2
Introduce the test compound
3
Quantify the immune response
4
Characterize the biological context
Illustrative MLR data
MLR assay for cancer immunotherapies: example results
PD-1 blockade enhances the allogeneic MLR response
Mature monocyte-derived dendritic cells are co-cultured with allogeneic CD4+ T cells, and IL-2 (A) and IFN-γ (B) are quantified in the supernatants. Nivolumab increases the MLR response, as reflected by higher IL-2 and IFN-γ levels. These data illustrate the assay's ability to detect changes in allogeneic T-cell responses following checkpoint inhibition.
Adenosine modulates dendritic-cell phenotype and suppresses the MLR response
Monocyte-derived DCs were exposed to adenosine, with or without an adenosine-receptor antagonist. Flow cytometry was used to profile DC differentiation and maturation (A), while IL-10 and IFN-γ were quantified in DC and co-culture supernatants (B).
Adenosine is associated with a CD1a-low/CD14+ DC phenotype, increased IL-10 and reduced IFN-γ in the MLR. Adenosine-receptor antagonism reverses these changes, supporting a role for adenosine signaling in the modulation of the allogeneic DC/T-cell response.
Making a difference as a preclinical CRO in oncology
Why work with Explicyte
Deep immuno-oncology experience
More than 100 in vitro campaigns over ten years, supporting 30+ peer-reviewed publications in immuno-oncology.
A dedicated PhD study director
A single PhD-level study director oversees your program from experimental planning through final report discussion.
Independent donor pairs
Characterized donors and controlled differentiation, with studies run across independent responder–stimulator pairs to assess the consistency of compound effects across distinct donor backgrounds.
From functional response to biological insight
Cytokine profiling, flow cytometry immunophenotyping, proteomics and transcriptomics can be integrated to characterize compound-associated changes in dendritic-cell biology and the ensuing T-cell response.
View our platform →
Paul Marteau, PharmD (study director), Imane Nafia, PhD (CSO), Loïc Cerf, MSc (COO), Alban Bessède, PhD (founder, CEO), Jean-Philippe Guégan, PhD (CTO)
Contact our team
Discuss your MLR assay
Whether you're testing an immune checkpoint inhibitor, an agonist antibody, or a small molecule acting on an immunosuppressive pathway, we'll build an MLR study around your mechanism and endpoints — from single-agent screening to combination activity profiling.
Frequently asked questions
Frequently asked questions about mixed lymphocyte reaction (MLR) assays
What does a mixed lymphocyte reaction (MLR) assay measure?
A mixed lymphocyte reaction assay evaluates how a test compound modulates the interaction between allogeneic dendritic cells and T cells. In Explicyte’s model, IFN-γ provides a primary functional readout of the co-culture response, complemented where needed by dendritic-cell phenotyping and additional cytokine or molecular analyses.
What cells are used in Explicyte's MLR assay?
The assay combines primary human monocyte-derived dendritic cells (moDCs) from one donor with CD4+ T cells from a second, unrelated donor. This donor mismatch elicits an allogeneic T-cell response that can be modulated by the test compound.
Which functional and phenotypic readouts are available?
IFN-γ release is used as the primary functional readout and as a surrogate of T-cell activation. Additional cytokines, including IL-2, IL-8, IL-10 and IL-12, can be measured according to the study design. Dendritic-cell phenotype can also be characterized by flow cytometry using markers such as CD86, CD80, CD83, CD206, CD209 and CD1a.
Which reference controls can be included?
Reference controls may include checkpoint inhibitors such as the anti-PD-1 antibody nivolumab and the anti-PD-L1 antibody atezolizumab, as well as adenosine-pathway modulators. Appropriate controls are selected according to the study design and biological question.
Can compounds be tested at different stages of the MLR assay?
Yes. Test compounds can be introduced during dendritic-cell differentiation, maturation, at the co-culture stage, or across selected experimental windows. This flexibility allows compound effects to be investigated at different stages of the dendritic-cell/T-cell response.
How can the assay help distinguish effects on dendritic cells from effects on the T-cell response?
By controlling the timing of compound exposure and combining functional readouts with dendritic-cell phenotyping, the assay can help distinguish effects arising during dendritic-cell differentiation or maturation from effects occurring during the subsequent allogeneic T-cell response. Additional molecular profiling can be incorporated when deeper characterization is required.
How is donor-to-donor variability addressed?
Studies can be performed across independent responder–stimulator donor pairs to determine whether compound-associated effects are consistent across distinct donor backgrounds rather than being restricted to a single donor combination.
Which therapeutic modalities can be tested in an MLR assay?
The assay can be used to evaluate both biologics and small molecules, as single agents or in combination. Examples include immune checkpoint inhibitors, agonist antibodies and compounds targeting immunoregulatory pathways such as the adenosine axis.
What additional analyses can be integrated?
MLR studies can be extended with broader cytokine profiling, flow cytometry immunophenotyping, proteomics and transcriptomics to characterize cellular and molecular changes associated with compound response.
