Sartorius logoT cells play a critical role in adaptive immune responses, including pathogen elimination, and tumor immunosurveillance. The binding of the T-cell receptor (TCR) to peptides complexed with major histocompatibility complex on antigen-presenting cells, along with engagement of co-receptors such as CD4 or CD8 and co-stimulatory molecules, triggers an intricate signaling mechanism.

TCR signal initiation is mediated by cytosolic tyrosine kinases leading to signal amplification through a network of serine-threonine kinases. Activation of the TCR pathway in naive and effector T cells leads to T-cell activation, proliferation, and cytokine production.

Modulating TCR engagement and the TCR signaling pathway using biologics, small molecules, or genetic engineering is highly relevant to many therapeutic areas, including cancer immunotherapy, adoptive cell therapy, and vaccine development. Genetic defects, mutations, and other mechanisms resulting in increased T-cell kinase activity also are involved in many autoimmune diseases making them attractive targets for the direct inhibition of T-cell activation.

The discovery and development of small molecules and antibodies targeting T-cell function, as well as T cell–based cell therapies and cell manufacturing, require assays to rapidly and reliably profile T-cell activation and cell health. To address the need for rapid monitoring, we developed an optimized, high-content multiplexed assay using high-throughput flow cytometry to measure T-cell activation.

TCA Assay

The Intellicyt® Human T Cell Activation Cell and Cytokine Profiling Kit (TCA Kit) collapses the traditional workflow by evaluating cell
phenotype, T-cell activation markers, cell proliferation, and cell viability, and quantitates secreted cytokines in a single assay using a 96- or 384-well plate format. The TCA Kit is applicable to many functional assay workflows, for example, during the development of checkpoint inhibitors and, cell therapies and during cell manufacturing.

The TCA assay discriminates between live and dead cells by using a membrane integrity dye, which stains only dead cells by DNA intercalation. Viable T-cell subsets are identified using CD3, CD4, and CD8 antibodies, and cell surface activation markers measure early activation (CD69+), late activation (CD25+) and even later activation (HLA-DR+) in the different T-cell subpopulations. A bead-based assay quantitates levels of secreted IFN-γ and TNF-α in the same sample well. For long-term studies, cells are stained using a proliferation dye prior to culture to quantitate cell division.

The panel design of the TCA assay dictates a violet, blue, and red (VBR) laser configuration. The acquisition protocol and data analysis, including event gates and gating strategy, activation metrics, heat maps, standard curves, and IC50/EC50 curves, are autogenerated using the TCA template and integrated ForeCyt® software.

Screening Kinase Inhibitors

In recent years, there has been renewed interest in using phenotypic screens for drug discovery. This screening methodology does not require a specific drug target or knowledge of its role in the disease pathology; it uses a specific, relevant biological model or signaling pathway to identify appropriate hits.

Phenotypic screens are being used with novel compound libraries as well as drug repurposing and chemogenomic libraries. The use of these known libraries allows for signature and phenotypic matching where the characteristic of a drug with desirable properties can be matched to another drug with an unknown clinical profile.

To illustrate the value of the Intellicyt® iQue Screener PLUS platform for T-cell function in phenotypic screening, the TCA Kit was used to screen a 152–small molecule library of kinase inhibitors (KIs) for their ability to inhibit human primary T-cell activation in peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 beads. Samples were acquired on the Intellicyt® iQue Screener PLUS VBR platform, and the early/late activation markers CD69, CD25 and HLA-DR cell as well as proliferation were assessed in viable CD4 and CD8 lymphocytes.

To assess T-cell function, the levels of secreted IFN-γ and TNF-α were quantitated. Data were analyzed: heat maps and IC50 curves were generated, and cytokines were quantitated. Profile Map, a unique analysis tool in ForeCyt® software, was used to integrate
assay metrics with Boolean logic to quickly locate hits using defined multiplexed criteria.

Assay Biochemistry and Proof-of-Concept Study Plate Setup
Assay Biochemistry and Proof-of-Concept Study Plate Setup. The different T-cell identifiers and phenotypic and functional activation markers measured in each well are seen in the left panel. A typical assay plate setup used for proof-of-concept studies is seen in the right panel. This plate includes the standard curves for cytokine quantitation and positive and negative controls.

For proof-of-concept studies, cryopreserved PBMCs were cultured for 24 hours and then stained with the Cell Proliferation and Encoder Dye B/Green. Cells were stimulated with three different well-characterized T-cell activators (CD3/28 DynaBeads, phytohemagglutinin (PHA), or staphylococcal enterotoxin B (SEB)) using a 12-point, 2-fold serial dilution series. SEB used a 4-fold serial dilution. A 12-point, 3-fold standard curve quantitated the levels of secreted INF-γ and TNF-α.

Ten microliters of sample containing cells and supernatant were transferred from the culture plate to an assay plate on days 1, 3, and 6 after stimulation, and analyzed using the TCA Kit. The time to results for the assay, including data acquisition and analysis, was approximately 4 hours. The seven-decade dynamic range of the Intellicyt® iQue Screener PLUS allows for easy discrimination between cytokine capture beads and cells using basic forward and side scatter plots.

Due to differences in the capture bead’s intrinsic fluorescence, the single cytokine capture beads are resolved into INF-γ and TNF-α populations for quantification. For the cell gates, viable single cells are determined, T-cell subsets are identified, and activation markers are assessed. The T-cell time course activation data were analyzed and line graphs generated using the multiplate Panorama feature in the ForeCyt software.

The data showed a temporal and dose response for the various activation metrics with differences observed between the three compounds. For example, TNF-α secretion peaked at day 3 with DynaBeads and SEB treatment, and significantly decreased at day 6, whereas little temporal difference in TNF-α secretion was observed with cells cultured in PHA. Further analysis showed differences in multiple metrics in cells treated with SEB compared to other activating compounds. Collectively, these data showed how the TCA Kit can rapidly generate high-content data that can identify different mechanisms of action (MOAs).

For the initial KI library screen, PBMCs were cultured in IL-2-containing media and treated for 1 hour with 20 μM of each inhibitor; then cells were activated using anti-CD3/CD28 beads. Twenty-four hours later, the TCA assay was performed and samples acquired. A plate-level analysis and visualization tool quickly identified compounds that inhibited expression of CD69 as well as KI that drastically reduced CD4+ T-cell viability.

The TCA Kit generated 15 different parameters that were integrated using the Profile Map data tool in the ForeCyt Panorama feature. Twenty-seven different KIs were identified that inhibited expression of all T-cell activation markers and cytokine secretion. Hits and the level of inhibition were ranked with an overlay line graph, providing easy visualization for each metric.

By changing parameter thresholds, Profile Map quickly identified KIs that had unique MOAs. Shifting the threshold of CD69 expression identified two hits that did not affect CD69 expression but inhibited all other T-cell activation markers. Since CD69 is often the only marker used in many traditional T-cell activation panels, these hits would not have been identified in traditional assays.

The same strategy was used to identify three KIs that did not affect CD25 expression but inhibited all other metrics, and additional compounds that showed differential cytokine secretion. Profile Map can be used to identify treatments that have similar phenotypes, which is important when trying to compare unknown compounds with drugs that have high safety profiles in the clinic.

Using this signature mapping feature, three drugs were identified that showed the same level of inhibition of all metrics as the FDA-approved rheumatoid arthritis drug Rituximab. This data illustrated the value of the high-content data generated by the Intellicyt® TCA Kit, and suggests KIs can inhibit multiple pathways to T-cell activation, it also demonstrates the insight provided by the use of the Intellicyt® platform for phenotypic screening of small molecules affecting T-cell activation.

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