Self-adjuvanted nanovaccine for cancer immunotherapy: Role of lysosomal rupture-induced ROS in MHC class I antigen presentation

Abstract

MHC class I (MHC I) antigen presentation of exogenous antigens (so called “cross presentation”) is a central mechanism of CD8⁺ cytotoxic T lymphocyte (CTL) responses essential for successful vaccine-based cancer immunotherapy. The present study constructed amphiphilic pH-sensitive galactosyl dextran-retinal (GDR) nanogels for cancer vaccine delivery, in which dextran was conjugated with all-trans retinal (a metabolite of vitamin A) through a pH-sensitive hydrazone bond, followed by galactosylation to acquire dendritic cell (DC)-targeting ability. Our results showed that pH-sensitive GDR nanogel was a self-adjuvanted vaccine carrier that not only promoted DC maturation through activating retinoic acid receptor (RAR) signaling, but also facilitated antigen uptake and cytosolic antigen release in DCs. Furthermore, pH-sensitive GDR nanogel effectively augmented MHC I antigen presentation and evoked potent anti-cancer immune responses in vivo. More importantly, we first reported that nanoparticle-triggered lysosome rupture could directly induce ROS production in DCs, which was found to be essential for augmenting proteasome activity and downstream MHC I antigen presentation. Hence, DC-targeted pH-sensitive GDR nanogels could be a potent delivery system for cancer vaccine development. Triggering lyososomal rupture in DCs with pH-sensitive nanoparticles might be a plausible strategy to elevate intracellular ROS production for promoting antigen cross presentation, thereby improving cancer vaccine efficacy.

Introduction

Protein/peptide-based therapeutic cancer vaccines have emerged as a promising approach of cancer immunotherapy, which eradicates cancer cells by evoking anti-cancer immunity in patients [1]. For effective anti-cancer therapy, cancer vaccines should be able to trigger CD8⁺ T cell activation and anti-cancer cytotoxic T lymphocyte (CTL) responses, which require MHC class I (MHC I) antigen presentation by antigen-presenting cells, such as dendritic cells (DCs) and macrophages [1], [2]. To achieve optimal MHC I antigen presentation, protein/peptide antigens need to be degraded into small peptides, which primarily occurs in proteasomes, the multicatalytic protease complexes in the cytosol. Unfortunately, tumor-derived protein or peptide antigens are usually processed in the endo/lysosome compartment that generally favors MCH class II (MHC II) rather than MHC I antigen presentation [2], [3]. Hence, enhancing MHC class I antigen presentation of tumor antigens (so called “cross presentation”) is highly desirable to improve the anti-cancer efficacy of cancer vaccines.

Reactive oxygen species (ROS), as by-products of intracellular oxidative phosphorylation, participate in both innate and adaptive immune responses [4]. Previous studies have shown that laser- or H₂O₂-triggered ROS could act as a “danger” signal to promote DC maturation and enhance its ability to promote T cell proliferation [5]. More recent studies showed that ROS not only resulted in oxidized damages of intracellular proteins, but also enhanced the activation of proteasomes, thereby serving a key mechanism enhancing MHC I presentation in viral infected tissues [6], [7]. However, whether ROS is involved the cross presentation of soluble antigens in antigen-presenting cells remains unclear.

In the past decade, biodegradable nanoparticles have been reported as a potent vaccine delivery system to improve vaccine efficacy [8], [9]. For example, nanoparticle-based vaccine formulation can effectively increase antigen uptake by DCs through active and passive targeting [10], [11]. Nanoparticles can also directly stimulate DC maturation and activation, enhancing its antigen presenting capability [12], [13], [14]. More interestingly, biodegradable nanoparticles have been reported to effectively promote MHC class I/II antigen presentation [15]. We previously reported a bioreducible polysaccharide nanogel as potent vaccine adjuvant, which enhanced MHC I/II antigen presentation and anti-cancer CTL responses through facilitating lyososome escape and cytosolic release of tumor antigens in mouse DCs [16]. Liu Q et al. reported that pH-responsive nanoparticle enhanced rapid antigen release in lysosome and induced immune response [17]. Therefore, developing engineered biodegradable nanovectors for vaccine delivery holds a great potential for enhancing cross presentation and improving cancer vaccine efficacy.

Vitamin A, as an essential micronutrient, plays a crucial role in both innate and adaptive immunity, such as B cell and DC differentiation, CD4⁺ and CD8⁺ effector T cell activation, as well as tissue-specific lymphocyte homing [18], [19], [20]. The administration of vitamin A or its active metabolite, retinoic acid (RA), significantly enhances antigen-specific antibody production, CD8⁺ effector T cell activation and mucosal immunity [20], [21], indicating its potency as a vaccine adjuvant. The immunoregulatory effect of vitamin A is primarily mediated by DCs, in which vitamin A (retinol) can be sequentially oxidized to an active metabolite, all-trans retinoic acid (ATRA), and exert its biological function through binding to nuclear receptors retinoic acid receptor (RAR) and retinoic X receptor (RXR) [22]. In the present study, dextran was conjugated with all-trans retinal, a metabolite of vitamin A and an ATRA precusor, through a hydrazone bond, followed by galactosylation to obtain amphiphilic pH-sensitive galactosyl dextran-retinal (GDR) conjugates. Upon dissolved in water, GDR conjugates spontaneously self-assembled into pH-sensitive nanogel, which effectively encapsulated OVA antigen to form GDR/OVA nanovaccine. Since DCs are the most potent antigen present cells for initiating anti-cancer immune response, we used mouse bone marrow-derived dendritic cells (BMDCs) as a cell model to evaluate the effect of GDR on DC maturation, antigen uptake, and MHC I/II antigen presentation in vitro. The anti-cancer effect of GDR/OVA nanovaccine was further evaluated in tumor-bearing mouse model. Our results showed that GDR nanogel acted as a self-adjuvanted nanocarrier robustly augmenting vaccine-induced anti-cancer immune responses.