The Adjuvant Combination of dmLT and Monophosphoryl Lipid A Activates the Canonical, Nonpyroptotic NLRP3 Inflammasome in Dendritic Cells and Significantly Interacts to Expand Antigen-Specific CD4 T Cells

Abstract

Adjuvants are often essential additions to vaccines that enhance the activation of innate immune cells, leading to more potent and protective T and B cell responses. Only a few vaccine adjuvants are currently used in approved vaccine formulations in the United States. Combinations of one or more adjuvants have the potential to increase the efficacy of existing and next-generation vaccines. In this study, we investigated how the nontoxic double mutant Escherichia coli heat-labile toxin R192G/L211A (dmLT), when combined with the TLR4 agonist monophosphoryl lipid A (MPL-A), impacted innate and adaptive immune responses to vaccination in mice. We found that the combination of dmLT and MPL-A induced an expansion of Ag-specific, multifaceted Th1/2/17 CD4 T cells higher than that explained by adding responses to either adjuvant alone. Furthermore, we observed more robust activation of primary mouse bone marrow-derived dendritic cells in the combination adjuvant-treated group via engagement of the canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex. This was marked by a multiplicative increase in the secretion of active IL-1β that was independent of classical gasdermin D-mediated pyroptosis. Moreover, the combination adjuvant increased the production of the secondary messengers cAMP and PGE2 in dendritic cells. These results demonstrate how certain adjuvant combinations could be used to potentiate better vaccine responses to combat a variety of pathogens.

Figure 1.. The combination adjuvant increases the expansion and activation of antigen-specific CD4 T cells during vaccination.

Mice were immunized intradermally with a single dose of the model antigen 2W1S with and without dmLT, MPL-A, or the combination of the two. 10 days later, the cervical lymph nodes were harvested and antigen-specific CD4 T cell responses were assessed by flow cytometry using MHC-II tetramers. (A, C) Representative flow plots from the CLN and total number of expanded (B) CD44⁺ I-A^b:2W1S⁺ CD4 T cells and (D) activated CD25⁺ antigen-specific CD4 T cells were determined. Treatments were compared using One-Way ANOVA with Tukey’s post hoc test for multiple comparisons (n=6 per group *, p<0.05; **, p<0.01; ***, p<0.001; ****p<0.0001). Data shows a representation of 4 independent experiments. Numbers above gates represent the (A) percent of antigen specific CD4 T cells out of CD4 T cells and the (C) percent of antigen specific CD4 T cells expressing CD25.

Figure 2.. The combination adjuvant imparts a mixed Th phenotype in antigen specific CD4 T cells following vaccination.

Mice were immunized intradermally in the ear with the model antigen 2W1S with and without dmLT, MPL-A, or the combination of the two. 10 days later, the cervical lymph nodes were harvested, and antigen-specific CD4 T cells were enriched using MHC-II tetramers and column-based magnetic cell isolation. Enriched 2W1S cells were counted, plated in a 96-well plate at 1 × 10⁶ cells/well, and stimulated with 2W1S antigen for 72 hours at 37C, 5% CO2. Cell supernatants were harvested, and secreted cytokines were assessed. Treatments were compared using One-Way ANOVA with Tukey’s post hoc test for multiple comparisons (n=3 per group *, p<0.05; **, p<0.01; ***, p<0.001; ****p<0.0001). Data show a representation of 2 independent experiments.

Figure 3.. The combination adjuvant distinctly alters the transcription profile in DCs with an enrichment in canonical inflammasome signaling.

BMDCs were cultured from the bone marrow of C57BL/6 mice. Cells were seeded in 6 well plates at a concentration of 1 × 10⁶ cells/mL and stimulated with dmLT (D), MPL-A (M), or a combination (DM) for 24 hours. Cells were centrifuged, RNA extracted, and paired-end mRNA sequencing was performed. (A) Comparative analysis of differentially expressed genes between each treatment and (B) shows the pathway analysis of the top predicted upregulated pathways in the combination adjuvant treated BMDCs. The resulting biological functions, canonical pathways, and upstream regulators were filtered by setting a threshold of P < 0.05 using Fisher’s exact test. (C) Selected gene expression profiles show the abundance of expression relative to untreated and (D) cDNA was also generated and RT-qPCR was performed in a time course on select inflammasome genes. (E) Analysis of predicted upstream regulators from RNA sequence data was performed using IPA software. Data was analyzed using the ΔΔCT method, normalizing all samples to GAPDH and comparing relative expression levels to those of untreated cells. Treatments were compared using Two-Way ANOVA with Dunnett’s post hoc test for multiple comparisons (n=3 per treatment per timepoint *, p<0.05; **, p<0.01; ***, p<0.001; ****p<0.0001). Data show a representation of 2 independent experiments.

Figure 4.. The combination adjuvant activates the NLRP3 inflammasome and increases the production of secondary messengers.

BMDCs were cultured from the bone marrow of C57BL/6 mice. Cells were seeded in 6 well plates at a concentration of 1 × 10⁶ cells/ml and stimulated with dmLT, MPL, or in combination for 24 hours at 37C. BMDCs were lysed and assessed via western blot (A-F) to determine cytosolic NLRP3, pro and cleaved IL-1β and caspase 1, (H) COX-II, and β-actin as a standardization control and (I) supernatant was assayed for PGE₂ by ELISA. (G) After 21 hours, the phosphodiesterase (PDE) inhibitors rolipram and cilostazol were added at 50μM to prevent conversion of the secondary messenger cAMP and cell lysates and supernatant harvested at 24 hours to determine total cAMP induction. Treatments were compared using One-Way ANOVA with Tukey’s post hoc test for multiple comparisons (n=3 per treatment *, p<0.05; **, p<0.01; ***, p<0.001; ****p<0.0001). Data show a representation of 3 independent experiments.

Figure 5.. Activation of DCs is NLRP3 dependent, but Caspase 1 and GSDMD independent.

BMDCs were cultured from the bone marrow of wild type, NLRP3 KO, ASC KO, Caspase 1 KO, and GSDMD KO mice. Cells were seeded in 6 well plates at a concentration of 1 × 10⁶ cells/mL and were left untreated or stimulated with dmLT, MPL, or a combination of the two for 24 hours. Cells were analyzed for the expression of surface markers indicative of activation by flow cytometry. (A) Representative gating strategy for the expression of activation markers CD40, CD80, CD86, and MHC-II in cells differentiated from wild type animals. Data show the percentage of DCs positive for each activation marker in (B) wild type, (C) Caspase 1 KO, (D) ASC KO, (E) NLRP3 KO, and (F) GSDMD KO cells. Treatments were compared using Two-Way ANOVA with Tukey’s post hoc test for multiple comparisons (n=3 per treatment per timepoint *, p<0.05; **, p<0.01; ***, p<0.001; ****p<0.0001). Data show a representation of 3 independent experiments.

Figure 6.. IL-1β secretion, but not other Th polarizing cytokines require key components of the NLRP3 inflammasome complex but are independent of gasdermin D mediated pyroptosis.

BMDCs were cultured from the bone marrow of wild type and inflammasome KO C57BL/6 background mouse femurs and differentiated using GM-CSF. Cells were seeded in 96 well plates at a concentration of 1.5 × 10⁵ cells per well and stimulated with dmLT MPL-A, or a combination of the two for 1, 3, 6 and 24 hours. (A) heat maps IL-1β, IL-6, TNF, and IL-12p40 secretion after 24 hours is shown (B) IL-1β kinetics from single and combination adjuvant treated wild type and (C) combination adjuvant treated inflammasome KO BMDCs was determined via ELISA and expressed as pg/ml. (D) Cytotoxicity was determined after 24 hours by quantifying LDH release with LPS + ATP treatment serving as a positive control. Significance was calculated using two-way ANOVA with Dunnetts post hoc test for multiple comparisons against untreated (A), GSDMD (B), and LPS + ATP (C) (n=3–6 per group ****, p < 0.0001). Data shown is a representation of 3 independent experiments.