Sachiko Hirosue

VEGF-C Promotes Immune Tolerance in B16 Melanomas and Cross-Presentation of Tumor Antigen by Lymph Node Lymphatics

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.

Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: Cross-presentation and T cell activation

Vaccines aiming to activate cytotoxic T cells require cross-presentation of exogenous antigen by antigen-presenting cells (APCs). We recently developed a synthetic nanoparticle vaccine platform that targets lymph node-resident dendritic cells (DCs), capable of mounting an immune response to conjugated antigen. Here, we explore routes of processing and the efficiency of MHC I cross-presentation of OVA peptides conjugated using both reducible and non-reducible linkages, exploring the hypothesis that reduction-sensitive conjugation will lead to better antigen cross-presentation. Both clathrin and macropinocytic pathways were implicated in nanoparticle uptake by colocalization and inhibitor studies. Cross-presentation by DCs was demonstrated by direct antibody staining and in vitro stimulation of CD8(+) T cells from OT-I mice and was indeed most efficient with the reduction-sensitive conjugation. Similarly, we observed IFN-γ production by CD4(+) T cells from OT-II mice. Finally, immunization with the OVA peptide-bearing nanoparticles resulted in in vivo proliferation and IFN-γ production by adoptively transferred CD8(+) OT-I T cells and was also most efficient with reduction-sensitive linking of the peptide antigen. These results demonstrate the relevance of the poly(propylene sulfide) nanoparticle vaccine platform and antigen conjugation scheme for activating both cytotoxic and helper T cell responses.

Engineering Approaches to Immunotherapy

The rapidly evolving field of immunoengineering will bring new design strategies to clinical immunology. As the science of immunology grows increasingly mechanistic, motivation for developing quantitative, design-based engineering approaches has also evolved, both for therapeutic interventions and for elucidating immunological pathways in human disease. This has seeded the nascent field of “immunoengineering,” which seeks to apply engineering analyses and design approaches to problems in translational immunology. For example, cell engineers are creating ways to tailor and use immune cells as living therapeutics; protein engineers are devising new methods of rapid antibody discovery; biomaterials scientists are guiding vaccine delivery and immune-cell activation with novel constructs; and systems immunologists are deciphering the evolution and maintenance of T and B cell receptor repertoires, which could help guide vaccine design. The field is multidisciplinary and collaborative, with engineers and immunologists working together to better understand and treat disease. We discuss the scientific progress in this young, yet rapidly evolving research area, which has yielded numerous start-up companies that are betting on impact in clinical and commercial translation in the near future.

Peripherally Administered Nanoparticles Target Monocytic Myeloid Cells, Secondary Lymphoid Organs and Tumors in Mice

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.

Steady-State Antigen Scavenging, Cross-Presentation, and CD8+ T Cell Priming: A New Role for Lymphatic Endothelial Cells

Until recently, the known roles of lymphatic endothelial cells (LECs) in immune modulation were limited to directing immune cell trafficking and passively transporting peripheral Ags to lymph nodes. Recent studies demonstrated that LECs can directly suppress dendritic cell maturation and present peripheral tissue and tumor Ags for autoreactive T cell deletion. We asked whether LECs play a constitutive role in T cell deletion under homeostatic conditions. In this study, we demonstrate that murine LECs under noninflamed conditions actively scavenge and cross-present foreign exogenous Ags to cognate CD8+ T cells. This cross-presentation was sensitive to inhibitors of lysosomal acidification and endoplasmic reticulum–golgi transport and was TAP1 dependent. Furthermore, LECs upregulated MHC class I and the PD-1 ligand PD-L1, but not the costimulatory molecules CD40, CD80, or CD86, upon Ag-specific interactions with CD8+ T cells. Finally, Ag-specific CD8+ T cells that were activated by LECs underwent proliferation, with early-generation apoptosis and dysfunctionally activated phenotypes that could not be reversed by exogenous IL-2. These findings help to establish LECs as APCs that are capable of scavenging and cross-presenting exogenous Ags, in turn causing dysfunctional activation of CD8+ T cells under homeostatic conditions. Thus, we suggest that steady-state lymphatic drainage may contribute to peripheral tolerance by delivering self-Ags to lymph node–resident leukocytes, as well as by providing constant exposure of draining peripheral Ags to LECs, which maintain tolerogenic cross-presentation of such Ags.

Polymer micelles with pyridyl disulfide-coupled antigen travel through lymphatics and show enhanced cellular responses following immunization

Poly(ethylene glycol)-stabilized poly(propylene sulfide) core (PEG-PPS) nanoparticles (NPs) smaller than 50 nm efficiently travel to draining lymph nodes and interact with antigen-presenting cells (APCs) to induce potent immune responses following intradermal immunization. To determine if a similar system could be developed that could be more easily and reproducibly prepared and eliminated faster in vivo, we created block copolymers of PEG-bl-PPS capable of self-assembling into 25-35 nm micelles (MCs). Biodistribution studies showed that these MCs were able to travel to draining lymph nodes, where they preferentially interacted with APCs. To couple cysteine-containing antigens to the surface of the MCs, a new polymer was synthesized with a terminal pyridyl disulfide (PDS), forming PDS-PEG-bl-PPS-benzyl. When mice were immunized in conjunction with free CpG as an adjuvant, ovalbumin-conjugated MCs (MC-Ova) generated more (2.4-fold) Ova-specific CD8(+) T cells in the blood and higher (1.7-fold) interferon-gamma levels from splenocytes upon restimulation than in mice immunized with free Ova and CpG. When comparing this MC platform to our PEG-PPS NPs with disulfide-linked Ova, no significant differences were found in the measured responses. These results indicate that PDS-functionalized MCs are efficient antigen delivery vehicles that enhance immune responses compared to immunization with free protein.

Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses

Background Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age‐specific vaccine formulations to overcome suboptimal immunization. Objective Small‐molecule imidazoquinoline Toll‐like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age‐ and species‐specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs. Methods Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist–encapsulating polymersome formulations were then synthesized. Novel 96‐well in vitro assays using neonatal human monocyte‐derived DCs and humanized TLR8 mouse bone marrow–derived DCs enabled benchmarking of the TLR8 agonist–encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25–loaded BCG‐mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice. Results Although alum‐adjuvanted vaccines induced modest costimulatory molecule expression, limited TH‐polarizing cytokine production, and significant cell death, BCG induced a robust adult‐like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL‐12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist–adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B‐specific CD4+ T‐cell numbers. Conclusion TLR8 agonist–encapsulating polymersomes hold substantial potential for early‐life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early‐life vaccines. Graphical abstract Figure. No Caption available.

Primary Human and Rat Beta Cells Release the Intracellular Autoantigens GAD65, IA-2 and Proinsulin in Exosomes Together with Cytokine-Induced Enhancers of Immunity.

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D.

Vaccine nanocarriers: Coupling intracellular pathways and cellular biodistribution to control CD4 vs CD8 T cell responses

Nanoparticle delivery systems are known to enhance the immune response to soluble antigens (Ags) and are thus a promising tool for the development of new vaccines. Our laboratory has engineered two different nanoparticulate systems in which Ag is either encapsulated within the core of polymersomes (PSs) or decorated onto the surface of nanoparticles (NPs). Previous studies showed that PSs are better at enhancing CD4 T cells and antibody titers, while NPs preferentially augment cytotoxic CD8 T cells. Herein, we demonstrate that the differential activation of T cell immunity reflects differences in the modes of intracellular trafficking and distinct biodistribution of the Ag in lymphoid organs, which are both driven by the properties of each nanocarrier. Furthermore, we found that Ags within PSs promoted better CD4 T cell activation and induced a higher frequency of CD4 T follicular helper (Tfh) cells. These differences correlated with changes in the frequency of germinal center B cells and plasma cell formation, which reflects the previously observed antibody titers. Our results show that PSs are a promising vector for the delivery of Ags for B cell vaccine development. This study demonstrates that nanocarrier design has a large impact on the quality of the induced adaptive immune response.

Modes of antigen presentation by lymph node stromal cells and their immunological implications

Antigen presentation is no longer the exclusive domain of cells of hematopoietic origin. Recent works have demonstrated that lymph node stromal cell (LNSC) populations, such as fibroblastic reticular cells, lymphatic and blood endothelial cells, not only provide a scaffold for lymphocyte interactions but also exhibit active immunomodulatory roles that are critical to mounting and resolving effective immune responses. Importantly, LNSCs possess the ability to present antigens and establish antigen-specific interactions with T cells. One example is the expression of peripheral tissue antigens, which are presented on major histocompatibility complex (MHC)-I molecules with tolerogenic consequences on T cells. Additionally, exogenous antigens, including self and tumor antigens, can be processed and presented on MHC-I complexes, which result in dysfunctional activation of antigen-specific CD8+ T cells. While MHC-I is widely expressed on cells of both hematopoietic and non-hematopoietic origins, antigen presentation via MHC-II is more precisely regulated. Nevertheless, LNSCs are capable of endogenously expressing, or alternatively, acquiring MHC-II molecules. Transfer of antigen between LNSC and dendritic cells in both directions has been recently suggested to promote tolerogenic roles of LNSCs on the CD4+ T cell compartment. Thus, antigen presentation by LNSCs is thought to be a mechanism that promotes the maintenance of peripheral tolerance as well as generates a pool of diverse antigen-experienced T cells for protective immunity. This review aims to integrate the current and emerging literature to highlight the importance of LNSCs in immune responses, and emphasize their role in antigen trafficking, retention, and presentation.