Matrix-Based Specific T-Cell Activation Reporter for Nearly-Digital Mapping of Antigen-Specific T Cells via Heterotypic Cell Micro-Communities and Digital Microfluidics

Antigen-specific T cells constitute a low percentage of peripheral blood mononuclear cells (PBMCs) and require antigen presentation by autologous antigen-presenting cells for activation and cytokine secretion. Conventional interferon-γ release assay (IGRA) averages rare activation events beyond cellular resolution, while single-cell compartmentalization disrupts the intracellular synapses for T cell immunity. Here, we developed mSTAR (matrix-based Specific T-cell Activation Reporter), an active-matrix digital microfluidic platform that partitions microliters of PBMCs into a nanoliter-scale matrix of 16 cell microcommunities (CMCs), each retaining tens to hundreds of PBMCs in their native heterotypic composition to preserve the natural physiological immune response. In a proof of concept, mSTAR executes parallel IGRA for a tuberculosis disease model, including CMC stimulation and magnetic-bead-based immunofluorescence assay. The IGRA of the mSTAR system achieves a limit of detection of 3.70 pg/mL, yielding 16 interferon-γ quantification readouts and ratios between CMCs incubated with different stimulants (negative, positive, or specific antigen). This enables the mapping and reporting of activated antigen-specific T cells in the matrix, thereby achieving functional phenotyping of each CMC. Poisson-based modeling of the droplet matrix converts rare T-cell activation events into a nearly digital estimation, and the positive CMCs encapsulating target cells can be retrieved for further characterization without loss. By bridging functional phenotyping and molecular features without prior cell sorting or engineering, mSTAR provides a programmable immunoanalytical infrastructure for broad downstream applications such as antigen-specific immunotherapy development or T-cell receptor discovery.