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Immune responses are complex events that involve the interaction of multiple cell types and signaling molecules. Researchers frequently need to identify different subsets of cells and record measurements related to cell health, cell function, and cytokine secretion to sufficiently characterize the effects of a test article. Thus, profiling the effects of immunomodulatory compounds or biologics often involves multiple screens using different assay platforms. Here we describe an immune-profiling platform that utilizes the high throughput, high content flow cytometry capabilities of the iQue Screener (IntelliCyt Corporation) to assay immunophenotype (identifying CD3 and CD8 positive T-cells), cell viability, cell proliferation, and multiple cytokine secretions (IL-17f, IL-6, and TNF).
We screened a structure activity relationship (SAR) library of potential immunomodulating compounds. The library was assembled using seven compounds with known immunomodulatory activity as templates for a SAR expansion to generate a biased library of 1438 compounds. The selection applied various substructure and similarity search methods combined with the generation of virtual templates, allowing for the thorough exploration of the chemical space around these compounds. Phytohemagglutinin (PHA) activated PBMCs were treated with the library at two doses, 16.7 µM and 83.3 µM for 72 hours. We utilized the viability of CD3 and CD8 positive cells (cytotoxic T-lymphocytes, CTLs) as a flag for artifact rejection, restricting our analysis to wells that retained greater than 70% viability for this subset of cells. Based on the viability criteria, cytotoxic compounds were eliminated and 301 compounds were further analyzed. Next, compounds that altered the extent of CTL proliferation over the course of the assay to different degrees or produced different patterns of cytokine secretion were identified. This analysis methodology enabled the generation of unique, multivariate immune-activation profiles for each compound using a combination of cell activation markers and secreted cytokine markers.
In three highlighted examples from a set of diverse compounds within the library, Compound 2 demonstrated nearly complete inhibition of proliferation, while Compounds 1 and 3 demonstrated an intermediate effect. The modulation of IL-17f, IL-6, and TNF varied for each featured compound. Compound 2, which had the strongest inhibition of proliferation, demonstrated little change in IL-17f secretion, a strong inhibition of IL-6, and an intermediate effect on TNF secretion. In contrast, Compounds 1 and 3 had mild effects on proliferation, but near complete inhibition of IL-17f and TNF. IL-6 was unaffected by these two compounds. Taken together, these data illustrate the ability to generate rich immune profiles from a single assay setup on a single assay platform capable of cell and beads-based assays. This has the potential to greatly reduce the time and cost required for immune profiling in high-throughput screening environments.