Immune cells do not live in isolation but interact to coordinate their many actions. One of the chief routes they foster communication is through direct physical interactions that enables them to read and interpret signals mediated at membrane interfaces. Despite the critical importance of these direct interactions in determining crucial developmental and functional immunological responses, their dynamic nature together with vast heterogeneity and polyfunctionality of individual immune cells have presented technical challenges for their systematic investigation. In particular, only limited tools are available that can exert control over the individual cells and their microenvironments to be able to precisely define interactions and deeply profile their outcomes at the individual cell level to resolve emerging immune responses within each single-cell.

To fill this critical void, this thesis presents the development and implementation of novel microfluidic technologies for single-cell analysis of direct cell-cell interactions in immunology. By combining carefully designed weir-based hydrodynamic traps with a multistep cell loading procedure, the microfluidic devices capture and controllably pair hundreds of cells in parallel. This approach provides requisite control over interactions with one-to-one interacting partners, welldefined and synchronous initiation of interactions, and enduring contacts. It also provides full control over the soluble microenvironment by solution exchange without losing cell registration. Accordingly, these features enable monitoring and assaying lymphocyte interactions longitudinally from the beginning with multiparametric single-cell measurements. These capabilities in turn allow probing into complete immune cell activation window from the very onset for direct correlation analyses within hundreds of individual cells in a single experiment.

We apply these new 'microfluidic cell pairing' technologies to quantitative investigation of lymphocyte interactions to elucidate lymphocyte activation dynamics and their relation to diverse functional behaviors at the single-cell level. These studies help resolve qualitatively and quantitatively distinct calcium signaling patterns in single CD8 T cells based on varying T cell receptor affinities which correlate with differential cytokine output. Similar studies with natural killer (NK) cells identify a previously unreported inverse correlation between the strength of early calcium signaling and cytokine production, and further indicate a calcium-dependent mechanism for selective regulation of cytotoxicity and cytokine production in NK cells. Collectively, these findings provide essential insight into the regulation and evolution of immune responses within individual immune cells, and establish the potential of these new microfluidic technologies to address important questions on many aspects of cell-cell interactions across biology in general and in immunology in particular.