Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles

Abstract

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8⁺ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8⁺ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8⁺ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens.

Keywords: CD4; CD8+; CTL; Caspase-1; Caspase-11; GSDMD; IL-1; PLGA; T cells; Th1; adjuvant; cell-mediated immunity; non-canonical inflammasome; polymeric nanoparticles; vaccine.

Graphical abstract

Figure 1

Particle size dictates the induction of T CD8⁺ and IFN-γ responses Effector CD8⁺ T cells specific to the H-2 Kb OVA epitope SIINFEKL (C-I Tmer) were quantified tetramers 7 days after booster. (A) Representative dot plots for SIINFEKL-specific CD8⁺ and CD44⁺. (B and C) Percentage of CD44⁺, Tmer⁺ cells over total CD3⁺, CD8⁺, and total numbers in spleens. (D) IFN-γ in splenocytes supernatants after ex vivo stimulation with OVA for 72 h. (E) Total OVA-specific IgG titers in serum 7 days after booster. Statistical differences were calculated per multiple comparison ANOVA and Tukey-Kramer post-hoc test to determine significant differences between all groups on Log10 data. Representative of two experiments, n = 4/group.

Figure 2

50-nm PS nanoparticles drive potent antigen-specific anti-tumor immunity (A) Immunization scheme and s.c. challenge with 3.5 × 10⁵ B16-OVA cells. Tumors were measured daily. (B) Spider plots show early development of tumors. Lines represent individual mice. (C) Kaplan-Meier survival graphs up to 25 days after challenge. (D) Extended survival analysis up to day 100. (A–C) n = 19 in PBS and OVA groups, n = 17 in 50-nm particle + OVA group. (D) n = 14 for PBS and OVA, n = 12 for 50-nm particle + OVA. Statistical significance for survival analyses determined by Mantel-Cox test. (E) CD8α T cell depletion efficacy in peripheral blood by fluorescence-activated cell sorting over total CD3⁺T cells on day 31 and 35 post priming displayed as mean frequency ± SEM. An unpaired T test was used to determine statistical significance where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Kaplan-Meier survival analysis of challenged mice; a Mantel-Cox test was used to determine statistical significance between 50-nm NP OVA immunized mice with CD8⁺T cell depletion and non-depleted 50-nm NP OVA immunized mice given isotype control where ∗p < 0.05. n = 7 mice per group.

Figure 3

Induction of cell-mediated immunity by PLGA adjuvants depends on particle size (A) An increase in percentage and total numbers of H-2Kb SIINFEKL-specific CD8⁺ T cells is observed on day 21 after vaccination with 50-nm PS and 60-nm PLGA particles (graphs and dot plots). (B) 100- and 500-nm PLGA particles fail to induce CD8⁺ responses in the spleen after vaccination. (C–E) IFN-γ in splenocytes supernatants after 72 h of ag-restimulation. Data are displayed as mean of triplicate samples ± SEM. (D–F) OVA-specific IgG titers in serum 7 days after booster. Statistical differences per multiple comparison ANOVA and Dunnett’s or Fisher LSD post-hoc test. Representative of two experiments for (A), (C), and (D) and one for (B), (E), and (F). (A, C, D) n = 5 mice per group. (B, E, F) n = 4 mice per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; asterisks (∗) represent differences to antigen alone, dots (⋅) represent comparisons between groups with adjuvant; ns: not significant.

Figure 4

Particle size influences antibody class switching without affecting antibody neutralizing activity Wild-type (WT) or Ifng^−/− mice received i.m. OVA (A-G) or s.c. ClfA (H–L) alone or in combination with PS particles. PBS was used as vehicle control and alum as positive control. Serum antibodies were measured by ELISA on day 21. (A–C) OVA-specific IgG1, IG2b, and IgG2b after i.m. vaccination. (D–G) Total antibodies and isotypes in WT and Ifng^−/−. (H) Total ClfA-specific antibodies after s.c. vaccination. (I–K) ClfA-specific titers for IgG1, IG2b, and IgG2b after s.c. vaccination. (L) Neutralizing activity as percent inhibition of S. aureus binding to fibrinogen, with 100% being bacterial adherence in the absence of serum. Results are shown as mean ± SEM; symbols represent individual animals. Statistical differences were calculated per multiple comparison ANOVA and Dunnett’s test compared with antigen alone or selected Fisher LSD for selected groups. Representative of two experiments for WT mice immunized with OVA (n = 4/group) or ClfA (n = 5/group) and one for Ifng^−/− (n = 4/group).

Figure 5

IL-1 and IL-18 differentially regulate nanoparticle-induced IFN-γ and CD8⁺ T cell responses (A–D) Ag-specific isotypes in serum 7 days after i.m. booster. (E and F) Percentage and number of OVA H-2Kb CD8⁺ T cells in spleens 7 days after booster. (G and H) IFN-γ and IL-10 secretion in splenocytes on day 21, after 72h ex vivo ag-stimulation. Results are presented as mean ± SEM. Statistical differences are per one-way ANOVA and Fisher LSD (for Tmer⁺ cell quantification) or Dunnett’s (for cytokine quantification) post-hoc tests. IFN-γ and IL-10 data are shown as mean ± SEM; ∗ denotes differences between selected groups, ⋅ denotes difference compared with control (OVA alone), ns: not significant. Representative of three experiments for Il1r1^−/− mice and two for Il18^−/− mice (n = 4/group for antibodies and 8/group for Tmer and cytokines).

Figure 6

Caspase-11 and size-dependent induction of GSDMD are required for antigen-specific CD8⁺ responses (A) WT mice were vaccinated i.m. with 50-nm particles and OVA, with or without the caspase-1 inhibitor Ac-YVAD-fmk (YVAD) on days 7 and 14. Ag-specific responses were quantified in splenocytes on day 21 using H-2Kb/OVA (SIINFEKL) MHC Tetramers. PBS or OVA alone were used as vehicle or antigen alone controls. Percentage of OVA-specific CD8⁺ over total CD8⁺ and CD44⁺ T cells. (B) Total number of OVA-specific CD8⁺ T cells in the spleen. (C) Quantification of IFN-γ in culture supernatants 72 h after OVA stimulation. (D and E) Analysis of H-2Kb/OVA (SIINFEKL) specific CD8⁺ lymphocytes in vaccinated Casp11^−/− and WT mice. (F) Quantification of IFN-γ in culture supernatants 72 h after OVA stimulation. (G) Induction of GSDMD is size dependent. BM-DCs were primed with LPS or left unprimed and were then stimulated with 50-nm or 1-μm particles in combination with LPS for 18 h. Cell lysates were probed for full-length and cleaved GSDMD protein, and β-actin was used as a loading control. LPS + Nigericin-treated BM-DCs were used as positive control for GSDMD cleavage. Data are representative of two independent experiments. (H and J) WT mice were vaccinated as before. Some groups received the GSDMD inhibitor NSA i.p. 1 h before vaccination or i.m. at the time of vaccination. (G–H) Percentage and total number of OVA-specific CD8⁺ T cells in the spleen. (I) IFN-γ in culture supernatants 72 h after OVA stimulation. Statistical differences are per one-way ANOVA and Tukey-Kramer post-hoc test. Symbols: ∗ denotes differences between treatment groups and ⋅ to OVA alone. Representative for one experiment for (A)–(C) and (H)–(J) and two for (D)–(F) and (I). Each symbol represents one mouse.

Figure 7

Size-dependent production of ROS controls the induction of CD8⁺ responses (A–D) BM-DCs were stimulated with increasing concentrations of 50-nm (red), 200-nm (orange), or 1-μm (green) particles for 6 or 18 h or were left untreated (gray histograms). ROS production was measured by fluorescence-activated cell sorting on gated live cells using Cell-ROX gated on single live cells. Percentages in histograms indicate proportion of live cells after each treatment. (D) Mean fold change in Cell-ROX MFI compared with unstimulated bone marrow-derived dendritic cells controls is displayed ± SD. Data are representative of three biological/experimental repeats. (E) Numbers of CD44^hi, Tmer⁺, and CD8⁺ T cells in spleens of WT mice treated with the ROS scavenger TEMPOL administered i.m. or i.p. during prime/boost. Statistical differences are per one-way ANOVA and Dunn’s post-hoc test. Data are representative of one experiment. Each symbol represents one mouse.