Antigen-presenting cells (APCs) are powerful tools to expand antigen-specific T cells ex vivo and in vivo for tumor immunotherapy, but suffer from time-consuming generation and biosafety concerns raised by live cells. Alternatively, the cell-free artificial antigen-presenting cells (aAPCs) have been rapidly developed. Nanoscale aAPCs are recently proposed owing to their superior biodistribution and reduced embolism than conventional cell-sized aAPCs, but pose the challenges: easier cellular uptake and smaller contact surface area with T cells than the cell-sized counterparts. This study aimed to fabricate a new "stealth" nano-aAPCs with microscale contact surface area to minimize cellular uptake and activate antigen-specific T cells by combination uses of ellipsoidal stretch, PEGylation, and self-marker CD47-Fc conjugation.

Biomimetic nanoparticles have been reported as immune modulators in autoimmune diseases and allograft rejections by numerous researchers. However, most of the therapeutics carrying antigens, toxins or cytokines underlay the mechanism of antigen presentation by cellular uptake of NPs through pinocytosis and phagocytosis. Few researches focus on the direct and antigen-specific modulation on T cells by NPs and combined use of multiple regulatory molecules. Here, polylactic-co-glycolic acid nanoparticles (PLGA-NPs) were fabricated as scaffold to cocoupling H-2Kb-Ig dimer, anti-Fas mAb, PD-L1-Fc, TGF-β and CD47-Fc for the generation of alloantigen-presenting and tolerance-inducing NPs, termed killer NPs and followed by i.v. injection into a single MHC-mismatched murine model of alloskin transplantation. Three infusions prolonged alloskin graft survival for 45 days; depleted most of H-2Kb alloreactive CD8+ T cells in peripheral blood, spleen and local graft, in an antigen-specific manner. The killer NPs circulated throughout vasculature into various organs and local allograft, with a retention time up to 30 h. They made contacts with CD8+ T cells to facilitate vigorous apoptosis, inhibit the activation and proliferation of alloreactive CD8+ T cells and induce regulatory T cells in secondary lymphoid organs, with the greatly minimized uptake by phagocytes. More importantly, the impairment of host overall immune function and visible organ toxicity were not found. Our results provide the first experimental evidence for the direct and on-target modulation on alloreactive T cells by the biodegradable 200-nm killer NPs via co-presentation of alloantigen and multiple regulatory molecules, thus suggest a novel antigen-specific immune modulator for allograft rejections.

Numerous nanomaterials have been reported in the treatment of multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). But most of these nanoscale therapeutics deliver myelin antigens together with toxins or cytokines and underlay the cellular uptake and induction of tolerogenic antigen-presenting cells by which they indirectly induce T cell tolerance. This study focuses on the on-target and direct modulation of myelin-autoreactive T cells and combined use of multiple regulatory molecules by generating a tolerogenic nanoparticle.