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Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocytebinding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4 + and CD8 + T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4 +

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6chi Ly6g− monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6clo Ly6g+ granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6chi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8+ T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1702-8) contains supplementary material, which is available to authorized users.

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Lymphatic vessels drain fluid, antigens, and immune cells from the periphery to the lymph nodes (LNs). In addition to transporting activated dendritic cells to mount adaptive immune responses in the LN, lymphatic drainage is brings soluble antigens from the periphery to LN-resident immature dendritic cells and B cells. It is also the most common site for cancer metastasis. Despite its importance, the role of tumor-associated lymphatic vessels and their drainage to the LN in regulating host immune responses to the tumor is poorly understood. We show that tumor expression of VEGF-C, the most potent lymphatic growth factor, promotes pro-tumor immune tolerance by several mechanisms. For one, it enhances drainage to the draining LN, where tumor antigens along with suppressive cytokines bathe the LN and could affect B and T cell education there. Second, tumor VEGF-C upregulates CCL21 in the tumor stroma and surrounding lymphatic vessels, which itself promotes T cell infiltration and suppression. Third, VEGF-C drives peritumoral and LN lymphangiogenesis, which can modulate the myeloid cell repertoire towards more suppressive phenotypes. These changes resulted in increased infiltrations of regulatory T cells and myeloid-derived suppressor cells, and increased levels of regulatory cytokines. Interestingly, VEGF-C-expressing tumors were impervious to prior immunization against tumor antigen, unlike control-transfected tumors, which were hindered by the vaccine-induced immune response. Together, these findings suggest that lymphatic drainage serves to maintain peripheral tolerance and that tumors may hijack such mechanisms to escape host immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 298. doi:1538-7445.AM2012-298