Investigating the Role of Different Co-receptors on T-cell Activation Through IFN-γ Secretion Using Spatially Controlled Cell Monitoring Platform

Conventional methods for studying T-cell activation typically assess receptor engagement and downstream functional outputs in separate experimental formats, thereby obscuring the spatial correlation between initial receptor arrangement and localized functional outputs. This makes it difficult to examine the individual and combined effects of receptors and co-receptors engagement within the immunological synapse within the same controlled cellular microenvironment. Building upon the recently published CellStudio platform, previously validated for monitoring growth factor interactions in adherent cell models like mesenchymal stem cells and HeLa cells, we adapted this modular system to investigate non-adherent immune cells. This platform integrates Printing and Vacuum Lithography (PnVlitho) with bead-based immunoassays to generate defined activation patterns surrounded by cytokine capture antibodies, establishing a "present-and-measure" framework for localized biosensing. Jurkat T-cells were patterned on fibronectin alone or in combination with anti-CD3 and/or anti-CD4 antibodies, enabling precise engagement and activation of the co-receptors. Cell capture, basal contact morphology, and IFN-{gamma} secretion were evaluated for each condition. While CD4 engagement alone had a minimal effect, presenting anti-CD3 and anti-CD4 together caused the cells to spread into wide, circular contact zones and trigger the strongest local IFN-{gamma} signals. These findings show that first adaptation of CellStudio for suspension cells enables the standardized comparison of receptor-dependent differences in Jurkat-cell capture, contact morphology, and local cytokine-associated signals within a spatially defined assay, emerging as a powerful tool for understanding co-receptor cooperation in T-cell function and immunotherapy.