Liver-targeted delivery of bioengineered induced tregs via nanoparticles prevents and treats acute gvhd

Abstract Induced regulatory T cell (iTregs) therapy holds promise for graft-versus-host disease (GvHD), but clinical translation has been limited by phenotypic instability and insufficient tissue localization to inflamed organs. Recognizing the liver as both a major site of acute GvHD (aGvHD) pathology and a critical immunologic interface where alloantigen presentation and systemic tolerance are orchestrated, we hypothesized that localizing iTregs to the liver could establish a local immune regulatory hub, enabling sustained immunomodulation and thereby altering the systemic trajectory of aGvHD. To enable tissue-specific targeting, we developed a bioorthogonal click chemistry platform using 4-aminophenyl β-D-galactopyranoside-conjugated, lipid-coated nanoparticles loaded with Ac4ManNAz (ALPA NPs). These NPs are selectively taken up by hepatocytes via the asialoglycoprotein receptor (ASGPR), enabling durable azide tagging through metabolic glycoengineering. iTregs were surface-modified with dibenzocyclooctyne (DBCO) generating DBCO-iTregs that retained full suppressive function in vitro compared to unmodified iTregs. Upon adoptive transfer, DBCO-iTregs covalently linked to azide-expressing hepatocytes, leading to significantly enhanced hepatic localization compared to unmodified iTregs (p < 0.0001). We next evaluated therapeutic efficacy of our approach in both parent→F1 and fully MHC-mismatched murine bone marrow transplantation (BMT) models. B6D2 or BALB/c mice were lethally irradiated and transplanted with B6 donor T cells and marrow. For prevention experiments, ALPA NPs were administered on days -3 and -2 (17.5 mg/kg) followed by iTreg or DBCO-iTreg and conventional T cell infusion on day 0. For treatment approaches, ALPA NPs were given on days 4 and 5 followed by iTreg or DBCO-iTreg infusion on day 7 after bone marrow transplant (BMT). In co-transplantation models, DBCO-iTregs + ALPA NPs significantly reduced clinical GvHD scores, serum AST/ALT levels, and improved survival compared to unmodified iTregs (p < 0.05) or DBCO-iTregs delivered with free Ac4ManNAz (p < 0.01). In treatment models initiated after GvHD onset, liver-targeted DBCO-iTregs conferred durable improvements in survival, weight maintenance, and histologic protection of hepatic and intestinal tissues compared to controls. These effects were dependent on NP-mediated targeting, as DBCO-iTregs or free Ac4ManNAz alone showed no benefit. Mechanistically, flow cytometry revealed that liver-targeted DBCO-iTregs reduced the proportion of IFN-γ+ donor iTregs (CD45.1+) in the liver compared to unmodified iTregs (17.3% ± 3.7 vs. 33.2% ± 3.3, p < 0.01), suggesting enhanced phenotypic stability. Furthermore, conventional donor CD4+ and CD8+ T cells (CD45.2+) in the liver and spleen of recipients treated with DBCO-iTregs had significantly reduced percentage of cells expressing IFN-γ compared with BM + T control recipients (CD4+ liver: 32.1 ± 8.0% vs. 48.5 ± 8.4%, p < 0.05; CD8+ liver: 5.9 ± 1.0% vs. 18.7 ± 3.7%, p < 0.001; spleen: 1.9 ± 0.5% vs. 16.7 ± 8.1%, p < 0.0001), highlighting a broad impact on effector T cell responses. Correspondingly, serum and liver IFN-γ levels were significantly reduced in DBCO-iTreg + ALPA NPs-treated mice. Notably, when co-transplanted, DBCO-iTregs isolated from the liver had greater expression of FoxP3 compared to unmodified iTregs (p < 0.05) suggesting improved in vivo stabilization of FoxP3. Finally, liver-targeting DBCO-iTregs + ALPA NPs preserved graft-versus-leukemia (GvL) activity while preventing aGvHD, suggesting potential for combinatorial efficacy. In summary, our findings demonstrate that liver-specific targeting of iTregs via nanoparticle-based glycoengineering enables potent local immunoregulation, preserves iTreg stability, suppresses effector T cell responses, and improves systemic control of aGvHD without compromising GvL activity. This platform offers a novel and translatable strategy for organ-specific immune modulation in allogeneic transplantation and potentially autoimmunity.