Flow cytometry based functional assays essential for characterizing biological significance

The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. Flow-cytometry-based functional assays can be essential for elucidating the mechanism of a drug. While standard flow cytometry can identify immune subsets and their relative abundance within a sample, functional assays allow for the characterization of the biological significance of these populations including suppressive capability or activation status of a target population.

Functional assays usually involve ex vivo stimulation of cells with drugs, antibodies, and/or cytokines, followed by staining for markers in order to quantify a biological response. We have validated functional assays that allow effective characterization of immune subsets, including:

• Mixed Lymphocyte Reactions (MLRs): Splenocytes from two allogenic strains of mice, a stimulator population and a responder population, are co-cultured ex vivo. Proliferation of the responder population is measured with CFSE staining, which is an amine-reactive dye that allows tracking of cellular division for up to seven cycles. Splenocytes from tumor-naïve stimulator animals trigger proliferation, while splenocytes from animals bearing certain types of tumors will suppress proliferation. This assay can be used to measure alterations in immune suppression resulting from drug treatment. The below data represent the suppression of proliferation in the MLR when spenocytes from A20-tumor-bearing mice are used as stimulator cells.

• NK Cell Stimulation: Splenocytes or dissociated tumors are cultured with IL-12 and IL-18, treated to prevent excretion of produced cytokines. Expression of markers including IFN-γ are quantified to identify activated NK cells within the total NK cell population.

• B-cell Stimulation: Splenocytes or dissociated tumors are cultured with IgM and expression of markers including CD69 are quantified to identify activated B cells within the total B cell population.
• T-cell Stimulation: Splenocytes can be cultured with IL-2, anti-CD28, and anti-CD3e or a drug of interest after CFSE staining. After stimulation, differential rates of proliferation and survival can be measured. The below data represent proliferation in culture with (red) and without (blue) antibody stimulation.

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