In Vivo Modulation of Dendritic Cells by Engineered Materials: Towards New Cancer Vaccines

Abstract

Therapeutic cancer vaccines are emerging as novel and potent approaches to treat cancer. These vaccines enhance the body's immune response to cancerous cells, and dendritic cells (DCs), an initiator of adaptive immunity, are a key cell type targeted by these strategies. Current DC-based cancer vaccines are based on ex vivo manipulation of the cells following their isolation from the patient, followed by reintroduction to the patient, but this approach has many limitations in practical cancer treatment. However, recent progress in materials science has allowed the design and fabrication of physically and chemically functionalized materials platforms that can specifically target DCs in the body. These materials, through their in vivo modulation of DCs, have tremendous potentials as new cancer therapies. Nanoparticles, which are several orders of magnitude smaller than DCs, can efficiently deliver antigen and danger signals to these cells through passive or active targeting. Three-dimensional biomaterials, with sizes several orders of magnitude larger than DCs, create microenvironments that allow the effective recruitment and programming of these cells, and can be used as local depots of nanoparticles targeting resident DCs. Both material strategies have shown potential in promoting antigen-specific T cell responses of magnitudes relevant to treating cancer.

Figure 1

DC biology relevant to creating a cascading cytotoxic T lymphocyte (CTL) response upon infection. Immature DCs (iDC) in peripheral tissues (e.g., skin) encounter and eliminate pathogens by phagocytosis. DCs digest the antigens into small fragments of peptides, present those on their surface coupled to the major histocompatibility complex (MHC), and become mature DCs (mDC). Mature DCs also express receptors (e.g., CCR7) that allow them to migrate to lymph nodes (LNs) in response to gradients of chemokine (e.g., CCL19/21) secreted from LNs. In LNs, DCs transfer the antigenic information to naïve CD8+ T cells via interactions between their surface receptors, including both MHC/antigen-T cell receptor (TCR) binding and CD80/86-CD28 interactions. CTLs are particularly driven by recognition of fragments presented from MHC class I molecules (one particular type of MHC). The activated CTLs leave the LNs to kill infected cells or pathogens. Inset: fluorescent microscope image of dendritic cells [5].

Figure 2

DC-based cancer vaccine via ex vivo manipulation of DCs. A patient’s own peripheral blood mononuclear cells (PBMCs) are taken from the blood, followed by isolation of monocytes from PBMCs. Monocytes are cultured in the presence of cytokine cocktails (GM-CSF and IL-4) to generate autologous DCs. The immature DCs are pulsed with tumor antigens and TLR agonists to generate mature DCs presenting tumor antigen on their surface. Finally the mature DCs are injected back to patient’s body, where it is expected they will migrate to LNs to invoke tumor-specific CTL responses.

Figure 3

Nanoparticles loaded with cancer antigens and danger signals can target DCs in peripheral tissue via passive phagocytosis or active DC targeting with specific antibodies. Following nanoparticle uptake and processing, DCs become mature, present antigens on their surface, and migrate to LNs to activate T cells.

Figure 4

Immunization with antigen-loaded liposome enhances antigen-specific lysis by CTL in compared to immunization with soluble antigen. (a) Schematic presentation of antigen (OVA)-loaded liposome and soluble OVA used in immunization. (b) OVA-specific in vitro CTL activity using T cells isolated from mouse spleen [42].

Figure 5

Co-encapsulation of antigen (OVA) and TLR ligand (CpG) leads to efficient CTL response. (a) Schematic presentation of PLGA particles encapsulating OVA and CpG in a single particle, and PLGA particles loaded separately with OVA and CpG, respectively. (b) Representative flow cytometry plots of splenocytes from mice 6 days after subcutaneous vaccination with single PLGA formulation containing coencapsulated OVA and CpG oligonucleotide or physical mixture of OVA- and CpG-loaded PLGA particles [53]. SIINFEKL tetramer is specific peptide sequence for OVA, and the number in the box represents the percentage of T cells specific for this OVA antigen with the two vaccinations approaches. (c) Quantitative representation of in vivo cytolysis of injected tumor cells [53].

Figure 6

Targeting DCs with antigen-containing liposome. (a) A schematic presentation of antigen-bearing liposome coupled with DC-specific antibody or non-specific protein. (b) FACS data for lymph node cells of mice injected with fluorescein-labeled liposome coupled with non-specific peptide and anti-DEC-205 [44]. The upper right quadrant of each plot represents the percentage of DCs containing fluorescein-labeled liposome. (c) Representative images of the metastasis of melanoma to lungs in mice when the mice were vaccinated with liposome containing IFN-γ and engrafted with non-specific peptide or anti-DEC-205 (metastasis are black spots) [44].

Figure 7

Targeting of nanoparticles to lymph node. (a) Schematic presentation of 100- and 25-nm fluorescently labeled PPS NPs (left) and corresponding fluorescence lymphangiography after injection of NPs into mouse-tail skin (right) [80]. 25-nm NPs enter the dermal lymphatic capillary network much more efficiently than 100-nm NPs. Scale bar, 1 mm. (b) The 25-nm, but not the 100-nm, nanoparticles are visible in mouse lymph node sections 24 h after injection [80]. Cell nuclei shown in blue (DAPI); scale bar, 200 mm. (c) Quantification of FACS data for uptake of NPs by MHC II + cells and CD11c+ cells (DCs) in lymph node [79]. (d) A restimulation assay data to determine CD8 T-cell memory by IFN-γ production, showing CD8 T-cell memory after treatment with OH-functionalized 25 nm OVA-NPs but not CH₃O-functionalized NPs [80].

Figure 8

Accumulation of nanoparticles to lymph node according to their size (diameter). (a) Immunofluorescence microscopy on frozen sections of popliteal LN isolated from mice injected with 20- or 1000-nm green fluorescent nanoparticles into footpad [81]. Red fluorescence depicts B220 staining specific for B cell follicles. Scale bar, 100 μm. (b) Trafficking of particles in vivo. Left and right footpads of DC-depleted (left) and control (right) mice were injected with 20- or 500-nm red fluorescent nanoparticles, respectively [81]. Popliteal LN that have acquired nanoparticles are shown with arrowheads and injection sites with arrows.

Figure 9

Schematic presentation of 3D polymer scaffold cancer vaccine. 3D porous polymer scaffold can load various signals, including chemokines, antigens, and danger signals, to manipulate dendritic cells in situ in the body. Immature DCs migrate to 3D polymer scaffold in response to the gradient of chemokine released, mature, and proliferate in the 3D microenvironment. The mature DCs then leave the scaffolds and migrate to the lymph nodes for activation of naïve T cell.

Figure 10

(a) Accumulation of MHC II+ cells (green) around BSA rods (left) and MIP-3β rods (right) [82]. Scale bar, 100 μm. (b) Kinetics of LN-homing DCs from BSA rods (◦) and MIP-3β rods (●)[82]. (c) CTL activities after co-implantation of OVA rods and MIP-3β rods against E.G7-OVA cells as target [82]. Prophylactic (d) and therapeutic (e) immunity through immunization of MIP-3b rods + antigen rods (●), BSA rod + antigen rods (◦), MIP-3b rods + BSA rods (■), or BSA rods + BSA rods (□)[82]. Prophylactic model received two immunization on day −14 and −7 along with hapten (DNFB) application, followed by 3LL tumor inoculation on day 0 [82]. Therapeutic model received 3LL tumor inoculation on day 0 and four immunization on days 2, 9, 16, 23 long with hapten application.

Figure 11

(a) SEM image of macroporous PLG scaffold. Scale bar, 1000 μm. (b) H&E staining of sectioned PLG scaffolds explanted from subcutaneous pockets in the backs of C57BL/6J mice after 14 days: blank scaffolds (BLANK) and GM-CSF (3000 ng)-loaded scaffolds (GM-CSF) [86]. Scale bar, 500 μm. (c) Number of CD11c+ DCs isolated from PLG scaffolds at day 14 after implantation in response to doses of 0, 1000, 3000, and 7000 ng of GM-CSF [86]. (d) The number of CD11c+CCR7+ host DCs isolated from matrices loaded with PEI-ODN control, 10 μg PEI-CpG-ODN, 400 and 3,000 ng GM-CSF and 400 and 3,000 ng GM-CSF in combination with 10 μg PEI-CpG-ODN at day 7 after implantation [86]. Inset: photographs of inguinal lymph nodes from control mice (right) and after 10 days implantation of matrices incorporating 10 μg CpG-ODN + 3,000 ng GM-CSF. A comparison of the survival time in mice for (e) prophylactic [86] and (f) therapeutic [87] cancer vaccination. (g) Ratio of CD8a+ T cells versus FoxP3+ Treg cells residing within PLG scaffolds loaded with GM-CSF and lysates (GM+Lys) alone or in combination with CpG-ODN (GM+Lys+CpG) at day 12 after implantation [87].