The MLR has been a mainstay for immunological drug discovery for decades, with a wide range of read-out assays such as cell proliferation and cytokine ELISA. The use of flow cytometry as a technique for measuring MLR responses opens up a wide range of possibilities.

What is MLR?

MLR measures incompatibility between two individuals (1,2).  It was the required method for choosing donors of transplantation grafts until tissue typing for human leukocyte antigen (HLA) was instituted (3).  MLR is still used to demonstrate safety of stem cell and other cell or device therapy (1).  Typically, an equal number of donor 1 and donor 2 blood mononuclear cells, mostly lymphocytes, are co-cultured for about 5 days.  Due to HLA incompatibility, an immune reaction is initiated: cells proliferate, display activity markers, and produce cytokines (4). The main stimulators in the reaction are the small number of “dendritic” cells in the population (5). The responder cells are T-cells.

Two-Way versus One-Way MLR

Two-way MLR is a coculture of two donor cells untreated; usually cells from both donors proliferate. In one-way MLR, proliferation of one of the two donor cells is inhibited, by irradiation, e.g. 60Co, or by mitomycin-c treatment. These cells are called Stimulators, and the reacting cells are called Responders. One use of a one-way MLR is to determine if a primary tumor cell or cell line stimulates in the MLR (2).  Such tumor cells are naturally proliferating and thus blocking this activity allows their immune stimulating properties to be measured.

How is MLR Measured?

Flow cytometry is used to measure proliferation and activity markers (1).  Production of cytokines into the supernatant is measured by Enzyme Linked ImmunoSorbent Assay (ELISA) or other specific protein methods (4).

What is Flow Cytometry?

Flow cytometers are powerful laser-based instruments used to rapidly analyze individual cells and particles (6).  Single cells flow through the cytometer analyzer in a liquid bubble, where cell size and internal complexity are determined by refraction and reflection signals, respectively, in the visual spectrum.  Other characteristics can be measured simultaneously, such as viability, cell surface and intracellular protein expression, DNA content and ion flux changes. This technology allows for rapid multi-parametric analysis of single cells, typically examining more than 20,000 cells per second.

 

Flow cytometry can measure various cellular properties, including:

  • Cell size and volume
  • Cell viability
  • Cell cycle stage
  • Protein expression
  • DNA content
  • Surface and intracellular markers

 

In the figure below, human blood lymphocytes, monocytes and granulocytes were analyzed by flow cytometry. The forward scatter of visible light (FSC-A, x-axis) defines cell size and side scatter (SSC-A, y-axis) defines intracellular complexity. Each dot represents a single cell. The figure shows lymphocytes (including B and T Lymphocytes subsets), a small set of larger and more complex monocytes, and a heterogeneous group of granulocytes which are polymorphonuclear and contain intracellular granules.

 

Human peripheral blood mononuclear cell representation in a flow cytometry density plot chart. Side (SSC-A) and Forward (FSC-A) Scatters based on cell areas are represented.

Fig. 1: Human peripheral blood mononuclear cell representation in a flow cytometry density plot chart. Side (SSC-A) and Forward (FSC-A) Scatters based on cell areas are represented.

 

Marin Biologic Laboratories offers exceptional scientific services with our cutting-edge Attune NxT Acoustic Focusing flow cytometers.  The GMP validated instruments utilize four lasers allowing up to 16 fluorescent color combinations as well as automatic sampling of a 96 well plate.

Flow Cytometry for MLR

Cell proliferation

Flow cytometry can accurately measure the number of T cell divisions in an MLR. The process is as follows. One of the two blood donor cells is prelabeled with dye. If the cells do not proliferate, the dye stays in individual cells at the same concentration as originally.  If the cells proliferate, the dye concentration gets successively halved at each cell division and this is measured by flow. This is assessed in flow cytometry by gating on single lymphocytes (live cells, forward and side scatter), and then selecting CD3 surface positive cells, a marker of T lymphocytes.  Both the overall change in fluorescence intensity and the number of divisions can be measured.

Marin Biologic Laboratories Case Study-1 Demonstrating MLR proliferation

Human peripheral blood mononuclear cells (PBMCs) are gated for size and complexity, live marker and the plotted based on their cell proliferation dye (CPD) intensity. When single donor PBMCs are incubated by themselves, not many cells lose CPD staining indicating low proliferation rate of the cells (empty box, first plot). When two histocompatibility mis-matched donor PBMCs are incubated together, the PBMC population that is stained with CPD staining loses an important amount of intensity in their CPD staining signal. This indicates that the unstained PBMCs induce CPD-stained PBMC proliferation (empty box second plot).

Human PBMCs stained with CPD show no proliferation when one donor is cultured and high proliferation when unmatched cells are mixed.

Figure 2. Human PBMCs stained with CPD show no proliferation when one donor is cultured and high proliferation when unmatched cells are mixed.

 

Activation markers

Activation of T cells (For example an agonist CD3 antibody) upregulate specific surface receptors, such as CD25 (the IL-2 receptor), CD71 (the transferrin receptor), and co-stimulatory molecules (CD26, CD27, CD28, CD30, CD154 or CD40L, and CD134).  These can be assessed in flow cytometry by adding staining. At 5 day coculture, cells receive a different fluorescently stained antibody for each marker of interest. Then gate on T lymphocytes as above and measure activation by fluorescent signals in that channel (9).

Intracellular cytokine detection

Activated lymphocytes, both surface-bearing CD4 “helper” T cells, and CD8 positive “killer” T cells, produce cytokines. Typical cytokines that show activation induced upregulation include Interleukin 2 (IL2) and Interferon gamma (IFN gamma) (4).  This typical targets are secreted by the cells and can be measured in cell supernatants. However, some other cell activation markers are not expressed in the cell surface but intracellularly (like granzyme in CD8+ cells or FoxP3 in regulatory T cells), making the process more complex to measure them.  Flow cytometry can measure Intracellular proteins first by fixing and permeabilizing cells and then detecting specific proteins using a specific fluorescein-labeled antibody.  Since blood lymphocytes are usually quiescent, single donor, unstimulated cells will be negative for intracellular cytokine production while positive MLR cocultures will have high production.  A significant advantage in this approach is that T cell subsets can be characterized in combination with cytokine expression analysis. (Th subtypes, Tregs, etc.).

Immunosuppression by Stem Cells in the MLR

Embryonic Stem Cells (ESC) and other types of stem cells (placenta, dedifferentiated skin, etc.) do not stimulate in MLR even if the same donor’s blood cells are positive.  These cells are usually immunosuppressing (3). There is no reaction of normal “Responder” cells when cocultured with inactivated stem cells.  To measure the immunosuppressive strength of stem cells, a two-way MLR is set up using two normal blood cells. They will give a positive MLR.  In parallel, inactivated stem cells are added (three cell types).  The stem cells will suppress the normal MLR response.  To measure the strength of immunosuppression, stem cells are cocultured at equal numbers with the normal donor blood cells, and at successive 1:2 dilutions.  Usually, the immunosuppressive effect disappears at the 1:8 ratio of stem cell to normal cell.

 

Marin Biologic Laboratories Case Study-2 Demonstrating Inhibition of MLR by Stem Cells

Human PBMCs stained with CPD show proliferation when unmatched cells are mixed, but when cells are also incubated with stem cells (with inhibitory effects) cell proliferation is reduced.
Figure 3. Human PBMCs stained with CPD show proliferation when unmatched cells are mixed, but when cells are also incubated with stem cells (with inhibitory effects) cell proliferation is reduced.

 

References

1. Mangelick et al. Characterization of CD4+ and CD8+ T cells responses in the mixed lymphocyte reaction by flow cytometry and single cell RNA sequencing. Front Imunol 2024.  DOI: 10.3389/fimmu.2023.1320481
2. Nicotra et al. Mesenchymal stem/stromal cell quality control: Validation of mixed lymphocyte reaction assay using flow cytometry according to ICH Q2(R1). Stem Cell Res & Ther. 11:426,2020
3. Baxter-Lowe, L.A. The changing landscape of HLA typing: Understanding how and when HLA typing data can be used with confidence from bench to bedside. Hum Immunol 82:466, 2021
4. Bishara, et al. Cytokine production in human mixed leukocyte reactions performed in serum-free media. J Immunol Meth 215:187, 1998.
5.Tourkova, I.L. et al. Mechanisms of dendritic cell-induced T cell proliferation in the primary MLR assay.  Immunol Lett 78:75, 2001.
6. McKinnon, K.M.  Flow Cytometry: An Overview. Curr Protoc Immunol  2018. doi: 10.1002/cpim.40
7. Davis et al. Modified cell trace violet proliferation assay preserves lymphocyte viability and allows spectral flow cytometry analysis. Cytometry Part A 2024. https://doi.org/10.1002/cyto.a.24830
8. Herzig et al. A streamlined proliferation assay using mixed lymphocytes for evaluation of human mesanchymal stem cell immunomodulation activity. J Immunol Meth 2021. DOI: 10.1016/j.jim.2020.112915
9.Poloni et al. T‐cell activation–induced marker assays in health and disease. Immunol Cell Biol 101:491, 2023

 

 

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