Multimodal probing of T cell sensitivity with hexapod heterostructures

Studies employing antigen-presenting systems at the ensemble or single cell level can provide complementary insights into T-cell activation and signaling. However, synthetic material toolkits that probe T cells at both levels are lacking, although they would be essential in advancing basic immunology and immunotherapy. Here, we develop a biomimetic antigen-presenting system (bAPS) using hexapod heterostructures (i.e., a sub-micrometer hematite core, and nanostructured silica branches with diverse surface modifications) for single-cell, single-molecule stimulation and ensemble modulation of T-cell signaling. We report with single molecule resolution of: T-cell sensing/activation by a single agonist peptide-major histocompatibility complex; distinct T-cell receptor (TCR) responses to discriminate structurally similar peptides that differ by just one amino acid; and the superior sensitivity of TCR antigen recognition compared to chimeric antigen receptors (CARs). We also show that magnetic field-induced piconewton×micrometer-level torques on the hexapods amplify immune responses in suspension T and CAR-T cells. Furthermore, by using our bAPS, we have developed a reliable and high-throughput new method for identifying neoantigen-specific TCRs at the single-cell level. The multimodal hexapod bAPS presents an unexplored, nanotechnology-based biointerface tool for investigating recognition, signaling, and the biochemical/mechanical dual sensitivity of T cells and beyond. Examination of paired single-cell RNA-seq and single-cell TCR-seq in CD4 and CD8 T cells obtained from in vitro hexapod-stimulatation of mixed 1% target and 99% wildtype cells and sorting for TNF-alhpa or IL2 positive populations