A spontaneous multifunctional hydrogel vaccine amplifies the innate immune response to launch a powerful antitumor adaptive immune response

Abstract

Substantial progress has been made with cancer immunotherapeutic strategies in recent years, most of which mainly rely on enhancing the T cell response. However, sufficient tumor antigen information often cannot be presented to T cells, resulting in a failed effector T cell response. The innate immune system can effectively recognize tumor antigens and then initiate an adaptive immune response. Here, we developed a spontaneous multifunctional hydrogel (NOCC-CpG/OX-M, Ncom Gel) vaccine to amplify the innate immune response and harness innate immunity to launch and maintain a powerful adaptive immune response. Methods: Ncom Gel was formed by a Schiff base reaction between CpG-modified carboxymethyl chitosan (NOCC-CpG) and partially oxidized mannan (OX-M). The effects of the Ncom Gel vaccine on DCs and macrophages in vitro and antigen-specific humoral immunity and cellular immunity in vivo were studied. Furthermore, the antitumor immune response of the Ncom Gel vaccine and its effect on the tumor microenvironment were evaluated. Results: The Ncom Gel vaccine enhanced antigen presentation to T cells by facilitating DC uptake and maturation and inducing macrophages to a proinflammatory subtype, further leading to a T cell-mediated adaptive immune response. Moreover, the innate immune response could be amplified via the promotion of antigen-specific antibody production. The Ncom Gel vaccine reversed the tumor immune microenvironment to an inflamed phenotype and showed a significant antitumor response in a melanoma model. Conclusions: Our research implies the potential application of injectable hydrogels as a platform for tumor immunotherapy. The strategy also opens up a new avenue for multilayered cancer immunotherapy.

Keywords: Ncom Gel vaccine; adaptive immune response; cancer immunotherapy; innate immunity; spontaneous multifunctional hydrogel.

Scheme 1

A spontaneous multifunctional NOCC-CpG/OX-M hydrogel (Ncom Gel) vaccine was designed to harness innate immunity against tumors. The innate immune response was amplified through the activation of DCs and macrophages after Ncom Gel vaccine administration. In addition, innate immune cells launched and maintained a powerful adaptive immune response through the function of the Ncom Gel vaccine.

Figure 1

Design and characterization of the Ncom Gel. (A) Schematic showing NOCC-CpG/OX-M hydrogel formation. (B) Rheological analysis of the NOCC-CpG/OX-M hydrogel. The curves of the energy storage modulus (G') and loss modulus (G") during the crosslinking of OX-M (20 mg/mL) and CpG-b-NOCC (20 mg/mL) to hydrogels were obtained. (C) Representative SEM image of the hydrogel. Scale bar, 200 µm. (D) Morphological observation of the hydrogels (1) NOCC-CpG, (2) OX-M, and (3) Ncom Gel.

Figure 2

The Ncom Gel vaccine enhances the uptake of antigen by DCs. (A) Schematic illustration of DC 2.4 cellular uptake. (B) DC 2.4 cells were cultured with free OVA-RBITC or OVA-RBITC/Ncom Gel and determined by flow cytometry (n = 3, mean ± s.d., * P < 0.05, **P < 0.01, *** P< 0.001 and **** P< 0.0001). (C-D) The histograms of DCs 2.4 analyzed using flow cytometry at 6 h and 8 h. (D) DC 2.4 cells were coincubated with soluble OVA-RBITC or OVA-RBITC/Ncom Gel at 37°C for 6 h, stained with Lysol-Tracker Green and DAPI and recorded by laser scanning confocal microscopy (Scale bar= 20 µm).

Figure 3

Evaluation of APC activation by the Ncom Gel vaccine in vitro. (A-C) Flow cytometry analysis of the expression of CD80, CD83 and CD86 on BMDCs (n=3, mean ± s.d., **P < 0.01, *** P< 0.001, *** P< 0.0001). (D-E) The secretion of IL-6 and TNF-α in BMDC suspensions detected by ELISA kits (n = 3, mean ± s.d., * P < 0.05, **P < 0.01, *** P< 0.001 and **** P< 0.0001). (F) Schematic illustration of DC activation through multiple stimuli. (G-I) The percentages of CD80⁺ Raw264.7 (A), CD40⁺ Raw264.7 (B) and CD86⁺ Raw264.7 (C) cells in various groups (n=3, mean ± s.d., * P < 0.05, **P < 0.01, *** P< 0.001 and **** P< 0.0001).

Figure 4

The Ncom Gel vaccine dramatically enhanced the production of antigen-specific antibodies in vivo. (A) Schematic illustration of immunization and analysis schedule. (B) The titer of anti-OVA IgG antibodies in plasma was detected by ELISA. (C-D) The titers of anti-OVA IgG1 antibodies and anti-OVA IgG2b antibodies in plasma on days 21, 28 and 35 were detected by ELISA (n=3, mean ± SD, * P < 0.05, **P < 0.01, *** P< 0.001 and **** P< 0.0001).

Figure 5

The Ncom Gel vaccine has a significant effect on the adaptive immune response in vivo. (A-D) Antigen-specific T cell analysis in the spleen after immunization with different formulations (A, C, CD4⁺ IFN-γ⁺ T cell analysis; B, D, CD8⁺ IFN-γ⁺ T cell analysis). (E) Detection of IFN-γ secreted by T cells by ELISA after restimulation with antigen (n=3, mean ± s.d., * P< 0.05, *** P< 0.001).

Figure 6

The Ncom Gel vaccine dramatically inhibits melanoma growth in C57BL/6 mice. (A) Treatment scheme for C57BL/6 mice with B16F10-OVA tumors. (B) Average tumor growth curves of B16F10-OVA tumor-bearing mice were recorded after treatment with PBS, OVA/CpG, OVA/OX-M, OVA/NOCC-CpG, OVA/Ncom Gel, OVA/alum or NcomGel (n=6; data are represented as the means ± SEM and analyzed with two-way ANOVA with Bonferroni correction tests. * P < 0.05, **P < 0.01, *** P< 0.001 and **** P< 0.0001.) (C) Survival curves of mice after the mentioned treatments. (D) Body weight curves were recorded during the treatment. (E) Individual mouse tumor growth curves in different treatment groups.

Figure 7

The Ncom Gel vaccine changes the tumor immune microenvironment through a strong systemic immune response. (A-B) Representative flow cytometry dot plot of tumor infiltrating CD8⁺ T cells and CD4⁺ T cells (A) and Tregs (B) 2 days following the last treatment. (C) Quantitative data of CD4⁺ T cells and CD8⁺ T cells were analyzed (n=3 biologically independent samples). (D) The ratios of CD8⁺ T cells to CD4⁺ T cells in the tumor immune microenvironment (n=3 biologically independent samples). (E) Quantitative data of Tregs were examined (n=3 biologically independent samples). (F-H) The frequencies of MDSCs and DCs in tumors (F) and representative flow cytometry dot plots were analyzed following the last treatment (n=3 biologically independent samples). (I-J) Representative flow cytometry dot histograms of M1 TAMs (CD11b⁺ F4/80⁺ CD86⁺) and M2 TAMs (CD11b⁺ F4/80⁺ CD206⁺) in tumors are shown. (K-L) The frequencies of M1 TAMs and M2 TAMs in tumors examined 2 days after the last treatment (n=3 biologically independent samples). Mice were divided into the following groups: (1) NS, (2) OVA/CpG, (3) OVA/Ncom Gel, and (4) Ncom Gel. All data are represented as means ± s.d. and analyzed with one-way ANOVA with Tukey test. * P < 0.05, **P < 0.01, *** P< 0.001 and **** P< 0.0001.