Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display

Abstract

Cancer vaccine immunotherapy facilitates the immune system's recognition of tumor-associated antigens, and the biomolecular design of these vaccines using nanoparticles is one important approach towards obtaining strong anti-tumor responses. Following activation of dendritic cells (DCs), a robust CD8+ T cell-mediated adaptive immune response is critical for tumor elimination. While the role of efficient antigen-presenting myeloid DCs (mDCs) is conventionally attributed towards vaccine efficacy, participation by highly cytokine-producing plasmacytoid DCs (pDCs) is less understood and is often overlooked. We examined vaccines based on the E2 protein nanoparticle platform that delivered encapsulated TLR9 agonist bacterial-like DNA (CpG1826 or CpG1018) or TLR7 agonist viral ssRNA to determine their efficacy over free agonists in activating both mDCs and pDCs for antigen presentation. Although mDCs were only activated by nanoparticle-encapsulated TLR9 agonists, pDCs were activated by all the individually tested constructs, and CpG1826 was shown to induce pDC cytokine production. Transfer of secreted factors from pDCs that were stimulated with a vaccine formulation comprising peptide antigen and CpG1826 enhanced mDC display of the antigen, particularly when delivered in nanoparticles. Only when treated with nanoparticle-conjugated vaccine could pDCs secrete factors to induce antigen display on naïve mDCs. These results reveal that pDCs can aid mDCs, highlighting the importance of activating both pDCs and mDCs in designing effective cancer vaccines, and demonstrate the advantage of using nanoparticle-based vaccine delivery.

Figure 1.. Proposed responses of dendritic cells to protein nanoparticle cancer vaccines.

A) E2 nanoparticle vaccines, which are represented in panel B as red circles. E2 structure (gray) was generated in ChimeraX using Protein Data Bank ID code 1b5s (E2). Conjugated onto E2 are antigen (red) on the particle surface and adjuvant (green) in the interior of the particle. E2 nanoparticles are ~27 nm in diameter. B) Schematic of proposed mDC and pDC responses to nanoparticle cancer vaccines. In response to vaccine, mDCs and to a lesser extent pDCs may directly activate T cells, which can recognize and eliminate tumor cells. Plasmacytoid DCs may aid mDCs in this process through the transfer of cytokines and antigens. Represented are mDCs (orange cells), pDCs (purple cells), vaccine (red circles), cytokines (blue dots), antigens (red dots), T cells (turquoise cells), and lysed tumor cells (pink).

Figure 2.. Adjuvant- and antigen-conjugated nanoparticles.

A) Reaction scheme for the conjugation of CpG1826, CpG1018, or ssRNA adjuvants to the interior cysteines of E2 (D381C) nanoparticles. B) SDS-PAGE of nanoparticles encapsulating TLR agonist. Lanes: (1,6) molecular weight ladder; (2) E2; (3) CpG1826-E2; (4) CpG1018-E2; (5) ssRNA-E2. Construct bands at ~35 kDa confirm conjugation. C) Representative hydrodynamic diameter measurements of E2 encapsulating TLR agonists and E2 control. D) Representative TEM images of CpG1826-E2, CpG1018-E2, and ssRNA-E2. Scale bars = 50 nm. E) Reaction scheme for the conjugation of SIINFEKL to the exterior lysines of E2 nanoparticles. F) SDS-PAGE of complete E2 vaccine formulation or reaction intermediates. Lanes: (1,5) molecular weight ladder; (2) E2; (3) CpG-E2, (4) CpG-S-E2. G) Representative hydrodynamic diameter measurements of E2, CpG-E2, or CpG-S-E2. CpG: CpG1826. S: SIINFEKL.

Figure 3.. Evaluation of mDCs and pDCs.

Gating scheme for bone-marrow-derived A) mDCs and B) pDCs. BMDCs were first gated based on size (left panel), and then for CD11c and B220 (right panel). Subsets are phenotypically defined as CD11c^high B220⁻ mDCs and CD11c^low B220⁺ pDCs SSC: side scatter. FSC: forward scatter.

Figure 4.. MHC II response of mDCs and pDCs to encapsulated TLR agonists.

Myeloid DC (A-C) or plasmacytoid DC (D-F) MHC II+ MFI fold increase versus PBS control. For each condition or formulation, corresponding concentrations of E2 and adjuvant are displayed. White bars are PBS- and E2-only controls. Red bars indicate 100 ng/mL LPS (mDC positive control) or 200 ng/mL LPS (pDC positive control). Green, blue, and orange bars correspond to free (light bars) or encapsulated (dark bars) CpG1826, CpG1018, or ssRNA, respectively. A) MHC II expression of mDCs incubated with PBS, LPS, E2, and CpG1826 controls or CpG1826-E2. B) MHC II expression of mDCs incubated with CpG1018-E2 or control. C) MHC II expression of mDCs incubated with ssRNA-E2 or control. D) MHC II expression of pDCs incubated with CpG1826-E2 or control. E) MHC II expression of pDCs incubated with CpG1018-E2 or control. F) MHC II expression of pDCs incubated with ssRNA-E2 or control. Mean ± SEM. Statistics: 1-way ANOVA, Bonferroni’s test. ≥3 independent biological replicates, 2-3 technical replicates per independent biological replicate. Adj: adjuvant. *p ≤ 0.05, **p ≤ 0.01. Experimental procedure is outlined in Figure SI-1.

Figure 5.. Plasmacytoid DCs secrete an array of cytokines in response to incubation with vaccine.

Displayed are concentrations of cytokines (top graph of each set) and fold increase in cytokine concentration relative to PBS negative control (bottom graph of each set), secreted from pDCs after incubation with vaccine or control. For 24 hr or 48 hr incubation periods, pDC secretions of A) IFN-β, B) TNF-α, C) IL-6, D) IL-10, and E) IL-12p70 were determined by LegendPlex^™ assay. Incubation conditions included PBS, 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL (CpG + S), or CpG-S-E2 containing 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL. Mean ± SEM. Statistics: 1-way ANOVA, Bonferroni’s test. 3 independent biological replicates, 2 technical replicates per biological replicate. CpG: CpG1826. S: SIINFEKL. *p ≤ 0.05.

Figure 6.. Plasmacytoid DCs aid mDCs to display antigen.

A) Schematic for pDC supernatant transfer studies. For incubation steps, mDCs or pDCs were given 1 μg/mL CpG-S-E2 vaccine or controls (PBS negative control, 2 μg/mL SIINFEKL positive control, or 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL). B) Myeloid DC SIINFEKL display with or without media transfer from pDCs that were incubated with PBS for 24 hr starting on Day 8. (C-E) Myeloid DC SIINFEKL display following supernatant transfer protocol for mDCs or pDCs incubated with C) PBS, D) 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL, and E) 1 μg/mL CpG-S-E2 (containing 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL) for 24 hr starting on Day 9 (mDCs) or Day 8 (pDCs). Shown is display fold increase in antigen display versus that of mDCs incubated with PBS. Mean ± SEM. Statistics: 1-way ANOVA, Bonferroni’s test. 3 independent biological replicates, 2-3 technical replicates per biological replicate. CpG: CpG1826. S: SIINFEKL. *p ≤ 0.05.