Specific Stimulation and Multiplex Secretion Analysis of T Cells Using a Suspendable Hydrogel Platform Enables Immunophenotyping at the Single-Cell Scale

The ability to characterize and separate cells based on their surface marker expression profiles using flow cytometry revolutionized our understanding of the immune system at the level of single cells. However, the use of surface protein quantification to functionally evaluate T cells following stimulation fails to capture important outcomes of T cell activation, most prominently, cytokine secretion. This key limitation hinders elucidation of the mechanistic correlates of T cell function, which in turn limits our ability to design effective cell therapies for diseases such as cancer and autoimmune disorders. Here, we employ a single-cell microparticle-based technology (termed nanovials) to assess both the cell surface expression profiles and the protein secretion behavior of stimulated T cells. Using protein-conjugated nanovials, we demonstrated the capacity to stimulate ovalbumin-specific primary mouse CD8⁺ T cells, both nonspecifically by coating anti-CD3 and anti-CD28 antibodies on the particle, and specifically by coating ovalbumin peptide-loaded major histocompatibility complexes and anti-CD28 antibody on the particle. We further showed that nanovials could simultaneously capture secretions of multiple cytokines from single cells and that viable T cells contained within the nanovials could be sorted based on cytokine secretion levels. We applied this platform to characterize the phenotypic profile of resting T cells that would go on to secrete the interferon gamma (IFNγ) cytokine following activation on nanovials by staining cells for surface marker expression prior to on-particle stimulation. Overall, the nanovial platform and assays developed herein help elucidate the functional properties of T cells, which will inform the engineering of future cell therapies.