Development of a T-cell activation panel using image cytometry in rheumatoid arthritis patient samples

Abstract

Abstract Rheumatoid arthritis (RA) is an autoimmune disease that results in cartilage damage and bone loss due to chronic inflammation of joints. Treatments for RA include disease-modifying antirheumatic drugs and nonsteroidal anti-inflammatory drugs, as well as the inhibition of activated T-cells. Consequently, there is a need to screen RA patient samples rapidly and effectively to monitor treatment efficacy, disease progression, immune response, and predictive markers. In this work, we developed and demonstrated a multiplex detection method to monitor T-cell activation in a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca™ PLX Image Cytometer (Revvity). First, a dosage titration of PHA-P was performed on PBMCs to stimulate T-cell activation. Once stimulated, CD3 positive T-cells were evaluated for CD69, CD25, and the checkpoint molecule CD279. After 24 hours, the percent surface marker positive T-cells and the level of marker expression (median fluorescent intensity values) were used to determine optimal PHA-P dosage. Results showed concentration dependent increases in the expression levels with a calculated EC50 value for PHA-P ranging from 20.16-30.78 µg/mL with a 95% CV based on the CD279 titration curve. The proposed image cytometric surface marker detection method may prove to be an efficient tool to rapidly screen RA patient samples for T-cell activation in the setting of clinical disease treatment or progression.