• Mixed Lymphocyte Reaction (MLR) Bioassays at Marin Biologic Laboratories


    Posted on Dec 12, 2019

    MLR bioassays are requested by the US FDA to monitor immune responses with new agents, as tested with in vitro cultured human T-lymphocytes obtained from multiple blood donors via MarinBio’s phlebotomists. In a TWO-WAY MLR, the client sample is co-cultured with Peripheral Blood Mononuclear Cell (PBMC) derived T-cells from two genetically distinct normal individuals resulting in cell activation, cell blast transformation, cell DNA synthesis and T-cell proliferation. For a ONE-WAY MLR only one blood donor’s T-cells are incubated with client sample(s). MarinBio’s MRL bioassays reproducibly determine whether client test drugs or biologic agents stimulate, inhibit or have no significant effect on human T-cell-mediated cellular immune responses. MarinBio has significant experience and is highly expert in successfully adapting and completing MLR bioassays for clients and their regulatory agencies.

    MarinBio provides robust, rapid, efficient, cost effective and reproduceable assays for small- or large-scale MLR bioassays, including automated high-throughput screening bioassays by MLR. MarinBio performs blood draws from human volunteer donors on site and routinely performs MLR assays for clients. Formats to measure T-cell proliferation include state-of-the-art flow Attune NxT flow cytometry with 4 lasers and capable of monitoring up to 16 colors and automated sampling of MLR bioassays performed in multi-well plates. High-throughput and multiplexing is available.

    Our flexible MLR assay design can be customized to client specifications. Cells from multiple human allogeneic donors enables MarinBio clients and their regulatory agencies to determine, with high accuracy and sensitivity, any significant human T-cell-mediated immunomodulatory activities of the client test agent samples (e.g. checkpoint inhibitor therapeutic antibodies). Thus, using normal human allogeneic donor cell populations and multiparameter, state-of-the-art flow cytometry, MarinBio routinely measures the effects of client test agents on human T-cell-activation/proliferation, -cytokine release, -cell surface marker expression. For example, Marin Bio’s MLR measures individual or multiple cell surface markers (e.g. CD137, CD25, CD71, etc.) and/or intracellular markers (e.g. Ki-67, IFN- γ).

    Shown below in the top two panels are flow cytometric analyses of proliferating human T-cells. The top left panel shows proliferating T-cells in MLR from one human donor alone, as compared to the top right panel which shows the proliferating cells from both donors mixed together. Increasing amounts of CellTrace™ Violet (CTV) staining of individual cells is shown along the y-axis and increasing cell size, as measured by forward cell scatter, is shown along the x-axis. CTV stained T-cell flow cytometric monitoring of approximately ten or more generations of proliferating cells (before the signal is overtaken by intrinsic cellular auto-fluorescence) provides consistent homogenous CTV staining, so that distinct cell replication generations are discernable, where proliferating/responding T-cells have a successive 50% reduction in CTV following each T-cell division/replication. The photomicroscope image of the cultured cells from one normal blood donor alone is shown in the bottom, left panel and the photomicroscope image obtained from culturing the two normal blood donors (a TWO-WAY MLR) showing aggregates of proliferating T-cells is shown in the bottom, right panel.

    Increasing Cell Size In Forward Cell Scatte