Profiling for Monocyte-Regulated T-Cell Immune Responses in Human PBMC Samples: Simultaneous Assessments of Cell Surface Markers and Cytokine Secretion

Advances in immuno-oncology have accelerated the need for higher content cell analysis tools that are capable of reducing the time needed to actionable results. The complexity of immune responses necessitates that data should yield insight into both the context of mechanism (cell activation markers) and pathways (signaling molecules) for how patients would respond to a certain treatments, or which therapies are most effective for a specific patient. Traditionally, cellular endpoints and secreted cytokines have been measured using two separate assays on two different analysis platforms: cellular endpoints measured by flow cytometry or imaging, and secreted cytokines measured by plate-based or bead-based ELISA. IntelliCyt's iQue Screener PLUS with 3 lasers and 13 fluorescent channels, was built as an immunology profiling tool and can measure cellular activation markers and secreted cytokine levels in a single assay in under 20 minutes for a 384-well plate. In a highlighted example showing monocyte regulation of T cells, we profiled the immune responses of three different patients across seven different cell activation markers, cell viability, and secretion of IFNγ, TNF, and IL-10 in response to several immunogenic compounds. PBMCs from 3 individual healthy donors were treated with the monocyte activator lipopolysaccharide (LPS) and/or with T cell activator staphylococcal enterotoxin B (SEB) in duplicate dose response. Cell and compounds were incubated for 24 hours, then analyzed for viability, surface expression of CD3, CD4, CD8, CD14, CD25, CD45 and CD127, and cytokine secretion using the QBeads reagent kits (IntelliCyt Corp). ForeCyt software was utilized for data acquisition and analysis, where sequential gates were used to individually identify cells and beads from the samples based on size. The cells were further analyzed for viability and cell marker expression, and QBeads were further segregated into the individual detection beads for each cytokine. The media concentration of each cytokine was calculated via the use of a standard curve. Utilizing the built-in plate level analytics of ForeCyt, we generated 27 unique cytokine curves from a single 384 well plate, and were able to assess the differences in cytokine secretion for each of the 3 cytokines, across the 3 treatments (LPS alone, SEB alone, LPS+SEB), for all 3 donors. The cell-based measurements yielded 54 dose-response curves detailing the percentage of regulatory T-cells, T-helper cells, cytotoxic T-cells, monocytes, lymphocytes, and cell viability for each treatment/patient. The ability to simultaneously measure cell and bead based endpoints from a single sample reduces assay-to-assay variability and conserves the amount of patient cells required for testing. Taken together, these results highlight the power of the iQue Screener PLUS platform as an immunological profiling tool with not only the speed of sampling required for large-scale combinatorial studies, but the analysis power to quickly transform raw data into clinically relevant results for individual patients.