Activation Induced Marker Assay to Detect and Monitor T-cells Using Image Cytometry 3078

Abstract Description Activation-induced marker (AIM) assays are a well-established, and accessible way to measure the antigen specific response in immune cells. In this work, an AIM assay was developed and demonstrated a multiplex detection method to monitor T-cell activation in patients with Rheumatoid Arthritis using a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca® PLX Image Cytometer. A dosage titration of PHA-P was performed on PBMCs to simulate T-cell activation and calculate a half maximal effective concentration (EC50) of Phytohaemagglutinin P (PHA-P) for further AIM assay work. Once stimulated, CD3 positive T-cells were evaluated for the activation markers, CD69 and CD25, and exhaustion molecule CD279 for disease specific response. After 24- hours, the level of surface marker expression median fluorescent intensity (MFI) values was graphed to evaluate patient response to stimulus. Orthogonal cytokine detection assay was performed using to confirm activation of T-cells using interleukin 2 (IL-2) as a reference. Results show stunted expression of surface markers and cytokine levels in those diagnosed with rheumatoid arthritis compared to healthy. The AIM assay showed to be reproducible and has the potential to be automation compatible. The proposed image cytometric AIM assay may prove to be an efficient tool to rapidly screen clinical patient samples for T-cell activation in the setting of disease treatment or progression. Funding Sources Allen Institute for Immunolgy Topic Categories Basic Autoimmunity (BA)