Mixed Lymphocyte Reaction (MLR) Assay

The immunological synapse that occurs between T lymphocytes and Dendritic cells (DCs) is a key component of an effective immune response and is controlled by a combination of stimulatory and inhibitory signals – also called immune checkpoints. As to trigger a better anti-tumor immune response, interventions through a fine-tuning of these control signals within the immunological synapse represent one of the most promising cancer immunotherapy strategies.

In order to investigate novel immunotherapeutics capable of enhancing DC-mediated T cell activation, Explicyte set up and validated a robust and high throughput human allogeneic mixed lymphocyte reaction (MLR) assay, which consists in a co-culture of CD4+ T cells (responder) with monocyte-derived DCs (stimulator) and in the HTRF-based quantitation of IFNγ, a relevant surrogate of T cell activation. Our assay is a well-suited system for biologics and small molecules assessment either as single agents and/or for combination therapies, including with e.g. immune checkpoint inhibitors, and can thus be ran in various types of studies spanning from screening to potency profiling and functional characterization of candidate compounds.

Illustrative data

Molecular and phenotypic characterization of human monocytes derived dendritic cells (DCs)
Primary human monocytes were freshly isolated from healthy donors and differentiated and matured into DC lineage (mDCs) and were then further characterized by multiplexed flow cytometry for their activation markers profile (ie. CD209, CD1a, CD80, CD83 and CD86) (A). Both monocytes, immature and mature DCs were captured by phase contrast imaging and revealed differential phenotypic features (B).
 

Primary human monocytes were freshly isolated from healthy donors and differentiated and matured ...

Robust optimizable MLR response in different human allogeneic T-DC donor pairs
While displaying an expected response amplitude varying from a donor pair to another one, the MLR response is robust and is further optimized following PD1 blockade. Isolated peripheral blood monocytes-derived DC from six human donors were mixed with CD4+ T cells each originating from different donors. IFNγ levels released in the supernatants were measured after 72h using a specific HTRF-based kit.

While displaying an expected response amplitude varying from a donor pair to another one, ...

PD1/PDL1 blockade characterization in an allogeneic MLR assay through IFNg release quantification
Isolated peripheral blood monocytes-derived DC from a human donor were mixed with CD4+ T cells isolated from a second donor. IFNγ levels released in the supernatants were measured 72h after co-culture using a specific HTRF-based kit. Anti-PD1 (Nivolumab and Pembrolizumab) and anti-PDL1 (Atezolizumab) antibodies potentiated DC-mediated T cell activation as highlighted by IFNg increase in the MLR assay.  

Isolated peripheral blood monocytes-derived DC from a human donor were mixed with CD4+ T cells ...

Assay principle

Mixed lymphocyte reaction











  • Human allogeneic MLR assay, based on a co-culture of CD4+ T cells (responder) with monocyte-derived DCs (stimulator) thus addressing donor-to-donor variability
  • HTRF-based quantification of key cytokines released, e.g. IFNγ, one of the  most representative surrogates of T cell activation
  • ​Robust functional assay, microplate format tailored for screening campaigns
  • Complementary analysis can be implemented to decipher MOA (eg. activation marker, signaling pathway, etc)