To identify membrane proteins involved in cytotoxic T-cell (CTL) stimulation, we screened our proprietary Membrane Proteome Array (MPA) for activation of CTLs. The MPA comprises 5,300 different human membrane proteins, each overexpressed in live HEK-293 cells. CTLs were layered on the MPA, incubated overnight, stained for an activation marker, and quantified for expression by high-throughput flow cytometry. We identified 38 putative membrane proteins involved in CTL activation. Eight of these “hits” are known activators of CTLs, such as CD86. Other hits include membrane proteins that are involved in multiple functional activities. Here, we describe the functional validation of two of these previously unreported stimulatory proteins. For Hit A, we converted the extracellular domain to an Fc-fusion protein and used the construct to probe the MPA for binding partners. We identified two proteins that interact with Hit A: one protein found on neurons and one protein found on T cells. Hit B was a commercially available molecular agonist. Using the agonist in our assay, we were able to modulate the extent to which this target stimulated CTLs. In addition to identifying factors of CTL activation, we are currently using the MPA to identify novel viral receptors and therapeutic targets for neurodegenerative diseases, as well as deorphaning and testing the specificity of antibodies.

Citation Format: Jonathan T. Sullivan, Duncan Huston-Paterson, Charles Azuelos, Benjamin J. Doranz. Identification of novel costimulatory pathways in T cells with functional assays on the Membrane Proteome Array (MPA) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A89.