Principle of MLR Assay

• Responder T cells recognize alloantigens presented by stimulator antigen-presenting cells (APCs).

• Stimulator cells express foreign MHC molecules, which act as alloantigens.

• Responder T cells become activated upon recognition of foreign MHC molecules.

• Activated T cells proliferate and upregulate activation markers (e.g., CD69, CD25).

• T cell response is measured using various readouts:

• Proliferation assays

  • Cytokine secretion (IFN-γ, IL-2) using ELISA or multiplex assays.

  • Flow cytometry for activation marker expression and proliferation assessment.

Model Description

Allogeneic stimulator cells (PBMC, DC etc) are co-cultured with responder T cells, resulting in T cell activation and proliferation.

Readouts

T cell activation by flow cytometry and T cell response by cytokine secretion (IFN-γ, IL-2, and TNF-α release).

Add-On Services

Evaluation of cytokine release, proteomics, and transcriptomics.

Key Components of MLR Assay

A. Responder Cells (T Cells):

• Peripheral blood mononuclear cells (PBMCs) or purified CD4⁺/CD8⁺ T cells.

• Recognize foreign MHC molecules and initiate an immune response.

B. Stimulator Cells (Antigen-Presenting Cells, APCs):

• Can be PBMCs, dendritic cells, or B cells from a genetically different donor.

• Provide alloantigens (foreign MHC molecules) for T cell activation.

• In one-way MLR, stimulator cells are inactivated (irradiated or mitomycin C-treated)

   to prevent their proliferation.

C. Readouts for T Cell Activation and Proliferation:

• T cell activation markers:

• CD69 and CD25 expression measured by flow cytometry.

• Cytokine secretion assays:

• ELISA or multiplex assays to measure IFN-γ, IL-2, and TNF-α levels.