The T-cell:Dendritic cell co-culture is a quick way for multi-compound testing in an in-vitro immune microenvironment. Arrayed compound screening in a semi-automated platform can provide robust data to facilitate decision-making in a fast-paced industry.
Conceived in the mid-'60s to assess the host-graft reactivity research in transplantation1, the mixed lymphocyte reaction (MLR) assay continues to be part of regulatory bodies' standard procedure for medical clearance to clinical trials. Here are revised aspects of the assay and how Revvity's services can help you nurture your development.
What is a MLR assay?
In drug discovery, the MLR is a technique used to determine the immunogenicity of a compound, commonly used to evaluate the potential impact of a small therapeutic molecule or biologic in the immune system. To assess the immunomodulatory effect of compounds in-vitro, lymphocyte proliferation and cytokine production are observed in an allogeneic reaction, meaning there are mixed cells from different donors together in culture to model an immune microenvironment. Based on the lymphocyte cell population to be stimulated, two types of MLR are commonly used: one- and two-way assays.
The most common is a unidirectional MLR, where only one lymphocyte population can respond or proliferate. In bidirectional MLR, both cell populations can increase and should be considered in specific experimental designs. For data acquisition, the cell culture workflow for MLR can include high throughput flow cytometry and associated validated tools for cytokine release measurements.
How does the allogenic MoDC:T Cell assay work?
There are many ways to perform MLR. The most common is a T cell-based MLR, which uses two different types of cells: antigen-presenting cells and peripheral blood lymphocytes. Monocytes obtained from the peripheral blood of healthy donors are differentiated in vitro to become a dendritic cell, a professional antigen-presenting cell (Mo-DC). Then, purified CD3+ T lymphocytes isolated from an unrelated individual are co-cultured with the Mo-DC, the T cells proliferate, responding to the allogeneic major histocompatibility complex (MHC) molecules present in the surface of the dendritic cells. Drugs can modulate the effects of the interaction, which is a powerful tool to assess the effectiveness of immunomodulatory capabilities of a new compound.
The MLR assay assesses multiple donors to reflect better how a client test molecule will behave across the population. We build confidence in the data by quantifying the dividing lymphocytes and measuring their response by cytokine release detection (see reference 2).
Why should I consider a MLR for my project?
The MLR constitutes a reactivity test that measures the cell microenvironment reaction to a drug, indicating how the compound would interact with the body. Drug developers also use this assay to test the efficacy of cancer immunotherapies, vaccines, and other therapeutics that potentially increase, decrease, or repolarize the interaction between T cells and the antigen-presenting cells. Apart from the widely known immune-oncology applications, the applications for MLR can also span from autoimmunity3, inflammation4, and reproductive immunology5 or be used as a model for immune synapse in a COVID-19 complication scenario6.
The immunogenicity assessment obtained by the MLR can be crucial for the preclinical stages of the drug development pipeline because the compound created could become immunogenic and trigger a non-desired immune response. If this occurs, the individual could develop an allergic reaction or suffer anaphylactic shock. Furthermore, it can reduce the effectiveness of the treatment and lead to undesired autoimmunity. Therefore, demonstrating the absence of secondary immune effects in the early stages of the development pipeline is crucial for the subsequent regulatory approvals of EMA and FDA.
Revvity's MLR assay
We can help as a biopharmaceutical service partner to analyze immunogenicity. We offer a MLR assay platform for testing therapeutics that will modify the interaction between the dendritic cell and T cells to either increase, decrease, or repolarize the T cell response to get answers to the immunogenicity of your compound concisely.
Key Advantages:
- Concise, reproducible, and robust quantitative data
- Streamlined process to simplify your workflow
- Quickly remove obstacles to progress in as little as four weeks
- Up to 16 compounds in a single assay starting with eight reading points each
- Multiple donors to better reflect how a test molecule will behave across the population
Read-outs:
- Cytokine Analysis – HTRF™ , LEGENDplex™, or AlphaLISA™ analytes available
- Proliferation Analysis – flow cytometry
References:
- Bain, B. & Lowenstein, L. Genetic studies on the mixed leukocyte reaction. Science (80-. ). (1964) doi:10.1126/science.145.3638.1315.
- Tourkova, I. L., Yurkovetsky, Z. R., Shurin, M. R. & Shurin, G. V. Mechanisms of dendritic cell-induced T cell proliferation in the primary MLR assay. Immunol. Lett. (2001) doi:10.1016/S0165-2478(01)00235-8.
- Lee, Y. E. et al. The synergistic local immunosuppressive effects of neural stem cells expressing indoleamine 2,3-dioxygenase (IDO) in an experimental autoimmune encephalomyelitis (EAE) animal model. PLoS One (2015) doi:10.1371/journal.pone.0144298.
- Matsui, F. et al. Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function. J. Gastroenterol. (2018) doi:10.1007/s00535-018-1447-1.
- Talwadekar, M. D., Kale, V. P. & Limaye, L. S. Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study. Sci. Rep. (2015) doi:10.1038/srep15784.
- Izrael, M. et al. Astrocytes Down regulate Inflammation in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome: Applicability to COVID-19. Front. Med. 8, 740071 (2021).
