Purpose: 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. Methods: The spherical polylactic-co-glycolic acid nanoparticles were fabricated using a double-emulsion method, and then stretched twofold using film-stretching procedure followed by PEGylation and co-coupling with CD47-Fc, H-2K b /TRP2 180-188-Ig dimers, and anti-CD28. The resulting PEGylated and CD47-conjugated nanoellipsoidal aAPCs (EaAPC PEG/CD47) were co-cultured with macrophages or spleen lymphocytes and also infused into melanoma-bearing mice. The in vitro and in vivo effects were evaluated and compared with the nanospherical aAPCs (SaAPC), nanoellipsoidal aAPCs (EaAPC), or PEGylated nanoellipsoidal aAPC (EaAPC PEG). Results: EaAPC PEG/CD47 markedly reduced cellular uptake in vitro and in vivo, as compared with EaAPC PEG , EaAPC, SaAPC, and Blank-NPs and expanded naïve TRP2 180-188-specific CD8 + T cells in the co-cultures with spleen lymphocytes. After three infusions, the EaAPC PEG/CD47 showed much stronger effects on facilitating TRP2 180-188-specific CD8 + T-cell proliferation, local infiltration, and tumor necrosis in the melanoma-bearing mice and on inhibiting tumor growth than the control aAPCs. Conclusion: The superimposed or synergistic effects of ellipsoidal stretch, PEGylation, and CD47-Fc conjugation minimized cellular uptake of nano-aAPCs and enhanced their functionality to expand antigen-specific T cells and inhibit tumor growth, thus suggesting a more valuable strategy to design "stealth" nanoscale aAPCs suitable for tumor active immunotherapy.
PurposeNumerous 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.Materials and methodsPoly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) were fabricated by co-coupling MOG40–54/H-2Db-Ig dimer, MOG35–55/I-Ab multimer, anti-Fas, PD-L1-Fc and CD47-Fc and encapsulating transforming growth factor-β1. The resulting 217 nm tolerogenic nanoparticles (tNPs) were administered intravenously into MOG35–55 peptide-induced EAE mice, which was followed by the investigation of therapeutic outcomes and the in vivo mechanism.ResultsFour infusions of the tNPs durably ameliorated EAE with a marked reduction of clinical score, neuroinflammation and demyelination. They were distributed in secondary lymphoid tissues, various organs and brain after intravenous injection, with retention over 36 h, and made contacts with CD4+ and CD8+ T cells. Two injections of the tNPs markedly decreased the MOG35–55-reactive Th1 and Th17 cells and MOG40–55-reactive Tc1 and Tc17 cells, increased regulatory T cells, inhibited T cell proliferation and elevated T cell apoptosis in spleen. Transforming growth factor-β1 and interleukin-10 were upregulated in the homogenates of central nervous system and supernatant of spleen cells.ConclusionOur data suggest a novel therapeutic nanoparticle to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine in the antigen-specific combination immunotherapy for T cell-mediated autoimmune diseases.
Antigen-presenting cells expand antigen-specific T cells ex vivo and in vivo for tumor immunotherapy, but are time-consuming to generate and, as live cells, raise biosafety concerns. An alternative is found in cell-free artificial antigen-presenting cells (aAPC), but these only present two or three kinds of immune molecules. Here, we describe a multipotent artificial antigen-presenting cell (MaAPC) that delivered 11 kinds of immune moleclues. This MaAPC simulated natural APCs through the concurent coupling of target antigens (H-2Kb/TRP2180–188-Ig dimers and H-2Db/gp10025–33-Ig dimers), costimulatory molecules (anti-CD28, anti–4-1BB, and anti-CD2), and “self-marker” CD47-Fc onto surface-modified polylactic-co-glycolic acid microparticles (PLGA-MP). These PLGA-MPs also encapsulated cytokines (IL2 and IL15), a chemokine (CCL21), and checkpoint inhibitors (anti–CTLA-4 and anti–PD-1). Culture of MaAPCs with naïve T cells for 1 week elevated the frequencies of TRP2180–188–specific and gp10025–33–specific CTLs to 51.0% and 43.3%, respectively, with enhanced cytotoxicity. Three infusions of MaAPCs inhibited subcutaneous melanoma growth in a mouse model and expanded TRP2180–188 and gp10025–33–specific CTLs 59–86-fold in peripheral blood, 76–77-fold in spleen, and 205–212-fold in tumor tissue, in an antigen-specific manner. Compared with conventional aAPCs carrying two or three immune molecules, the 11-signal MaAPCs exerted greater impact on T cells, including activation, proliferation, cytotoxicity, differentiation to memory CTLs or regulatory T cells and cytokines profiles, without detected side effects. Such MaAPCs could be used to individualize tumor immunotherapy.
In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4 and CD8 T cells in the myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG/H-2D-Ig dimer, MOG/I-A multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and "self-marker" CD47-Fc and encapsulate inhibitory cytokine (TGF-β1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65-79% of MOG-specific CD4 and 46-62% of MOG-specific CD8 T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule-dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide-reactive T cells; reduced MOG peptide-reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-β1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell-mediated autoimmune diseases.
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