Mixed Lymphocyte Reaction FAQ’s

Mixed Lymphocyte Reaction is an in-vitro cell-based assay that replicates an adaptive immune response. The MLR uses lymphocytes (T-cells and B-cells) along with antigen presenting cells (monocytes, dendritic cells, and macrophages) to produce a cell-mediated immune response.

Cell mediated responses are caused by the presentation of a foreign antigen to T-cells, which then produce an adaptive response. The antigens are presented to T-cells by “antigen presenting cells” (APCs).

The MLR induces an adaptive immune response by one of two mechanisms:

• Indirect allorecognition (autologous): APCs and T-cells are both from the same individual. The APC will come across a foreign pathogen, ingest it, and then present a portion of that pathogen (antigen) to the T-cell.
• Direct allorecognition (allogeneic): The APCs and T-cells are from different individuals (e.g. transplant). Immune cells are highly specific, can differentiate self from foreign sources and, as such when the dissimilar APC and T-cells interact, the T-cell responds directly to the foreign APC.

An adaptive immune response is a delayed reaction as the body generates a targeted response to a pathogen; as opposed to an innate response which acts acutely. The adaptive response takes a longer time because lymphocytes (like T-cells and B-cells) must first recognize the new pathogen, and then develop a specific response by producing antibodies, coordinating other immune cells, and attacking the pathogen. By the end of this process, memory cells are formed. These cells can recognize that pathogen in the future to provide quicker and more effective responses.

MLRs have two main variables – cell type and cell source:

• Cell type can either be a mixed population (e.g. PBMCs), or an isolated population(s) (e.g. DCs and T-cells). DC/T-cell reactions produce a much stronger response than PBMC reactions; however PBMC reactions are more representative of in-vivo conditions.
• Cell source can be allogeneic (APCs and T-cells from different individuals); autologous (APCs and T-cells from same individual); or xenogeneic (APCs and T-cells from different species).

Therefore, there are several combinations that can be used to produce different effects:

• Autologous DC / T-cell: T-cell response is due only to foreign antigen presentation by DC (mimics normal body response).
• Allogeneic/Xenogeneic DC / T-cell: response is due to dissimilar cell source (mimics transplantation)
• Autologous PMBCs: response is due only to foreign antigen or co-culture material (mimics normal body response)
• Allogeneic/Xenogeneic PBMC: response is due to dissimilar cell source (mimics transplantation)

Yes, in addition cell to source and cell types, there are also cell treatments. When performing an allogeneic PBMC reaction using PBMCs from two dissimilar individuals, both PBMCs populations have the ability to react. One of the populations is treated in order to arrest its response (proliferation), therefore it can only function as a stimulator for the other PBMC population.

Allogeneic MLR options:

• One-way MLR: Cells from two different individuals are used, one is treated and rendered non-proliferative so that only the other population is allowed to react. Used to measures individual responses (e.g. screening against multiple donors).
• Two-way MLR: Cells from two different individuals are used, both are allowed to respond. This is used to produce quicker and stronger responses.
• Pooled: Cells from multiple different individuals are used. These can be used in both one-way and two-way MLRs to produce even greater responses.

Understanding how a therapeutic product (drug, device, tissue, therapy, etc.) effects our adaptive immune response is critically important for the safety of the potential patients. Because the MLR is used to produce in-vitro cell-mediated adaptive immune responses, it is a useful tool for assessing a products safety profile, understanding its mechanism of action, or measuring its efficacy.

MLRs can be used in the following scenarios:

• Immunogenicity assessments (pre-clinical, pre-IND, manufacture/lot release)
• Proof of concept studies: Immunological profile, efficacy, and mechanism of action (pre-clinical and pre-IND)
• Potency Tests (manufacture/lot-release)
• Transplant Tolerance (clinical)

Because the MLR is a tool used to measure in-vitro cell-mediated adaptive immune responses, it has a wide variety of applications where biological activity of a product or therapy needs to be considered. At Xeno Diagnostics our clients span a wide range of fields including:

• Immuno-oncology: using the MLR to assess efficacy of checkpoint inhibition or immunogenicity of cellular and gene therapies (CGT).
• Transplant Immunology: using the MLR to assess immunogenicity, donor compatibility, or tolerization.
• Regenerative Medicine: using MLR to assess efficacy and immunogenicity of a variety of products including minimally manipulated human cells, tissues, and cellular- and tissue-based products (HCT/Ps).

There are three main responses to measure: viability, proliferation, and cytokine release. These can be measured in a variety of ways, from colorimetric ELISAs to Flow Cytometry.

Yes, flow cytometry is a powerful tool to measure cellular responses for the MLR. It allows for precise single cell measurements that can be performed on cell subsets (e.g. T-cells); it is also beneficial for monitoring changes in a product, if the product is a cellular therapy. Flow Cytometry can be used to measure:

• Phenotypes (T-cells, Tregs, NK, B-cells, monocytes, DCs, etc.)
• Viability and Apoptosis
• DNA replication and Cell Cycle
• Cell division and proliferation
• Activation Makers
• Intracellular cytokines
• Protein and Kinase Phosphorylation/Activation

Because there are many options for MLR format (allo, auto, 1-way, 2-way, PBMC or DC/T-cell) and a variety of readouts, the design must be carefully considered to produce the most mechanistically appropriate and relevant information for the intended use.

Xeno Diagnostics excels in designing bespoke MLRs for our clients to fit their needs. We consider the clients intended purpose, check the compatibility of their product, and factor in their regulatory needs to deliver a fit-for-purpose assay on time, in budget, all with publication quality results.

• Regulatory Compliance: Xeno Diagnostics is a high-complexity CLIA accredited and GLP compliant lab. RUO studies are conducted as “GLP-like”.
• Experience: Xeno has decades of experience performing MLRs and co-cultures, helping clients successfully navigate FDA guidelines concerning these assays.
• Customization: We offer a wide range of options for our services. We have several analytical capabilities including Flow Cytometry, Immunoassays, Luminex assays, Western Blots, PCR, and ELISpot among other options. We also offer custom assay development services to ensure that your assay meets quality expectations.
• Donor Sourcing: Xeno Diagnostics has its own IRB approved blood donation program for recallable donors. This allows us to maintain a donor bank on-site and ensure that the donors used for our studies are available for long term use. Each of our donors are HLA typed and pre-screened with an MLR for reactive donor pairs.
• Quality: Clients receive a detailed report for each study including analysis, statistics, publication quality figures and graphics, and interpretation by our experts.
• Confidence: Our staff takes every opportunity to communicate with our clients to ensure that the assays are designed to their specifications. We perform regular progress updates and timelines, and we take the time to discuss results with our clients to help interpret and plan next steps in their product development cycle.
• Ease-of-use: Xeno Diagnostics is quick and reliable when it comes to turn-around times and meeting client budgets.