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Effective Combination Adjuvants Engage Both TLR and Inflammasome Pathways To Promote Potent Adaptive Immune Responses

Emilie Seydoux, Hong Liang, Natasha Dubois Cauwelaert, Michelle Archer, Nicholas D. Rintala, Ryan Kramer, Darrick Carter, Christopher B. Fox and Mark T. Orr
J Immunol July 1, 2018, 201 (1) 98-112; DOI: https://doi.org/10.4049/jimmunol.1701604
Emilie Seydoux
*Infectious Disease Research Institute, Seattle, WA 98102; and
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Hong Liang
*Infectious Disease Research Institute, Seattle, WA 98102; and
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Natasha Dubois Cauwelaert
*Infectious Disease Research Institute, Seattle, WA 98102; and
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Michelle Archer
*Infectious Disease Research Institute, Seattle, WA 98102; and
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Nicholas D. Rintala
*Infectious Disease Research Institute, Seattle, WA 98102; and
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Ryan Kramer
*Infectious Disease Research Institute, Seattle, WA 98102; and
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Darrick Carter
*Infectious Disease Research Institute, Seattle, WA 98102; and
†Department of Global Health, University of Washington, Seattle, WA 98195
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Christopher B. Fox
*Infectious Disease Research Institute, Seattle, WA 98102; and
†Department of Global Health, University of Washington, Seattle, WA 98195
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Mark T. Orr
*Infectious Disease Research Institute, Seattle, WA 98102; and
†Department of Global Health, University of Washington, Seattle, WA 98195
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Abstract

The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain–containing adapter inducing IFN-β are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1β are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC−/− animals. Importantly, the early proliferation of Ag-specific CD4+ T cells was completely ablated after immunization in ASC−/− animals. Moreover, numbers of Ag-specific CD4+ T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC−/−, NLRP3−/−, and IL-1R−/− mice compared with wild-type mice and were completely ablated in TLR4−/− animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4+ T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.

Introduction

Vaccines against infectious diseases have proven to be effective against a number of pathogens, and their development and improvement were one of the major public health advances of the past century. Early vaccines consisted of attenuated or inactivated pathogens, which are strongly immunogenic but may be reactogenic owing to an inclusion of pathogen components. Modern vaccines use purified or synthetic subunit Ags, but these are often poorly immunogenic on their own, as they generally do not effectively stimulate innate immunity (1). Indeed, attenuated or inactivated vaccines contain pathogen-associated molecular patterns (PAMPs) and trigger the release of danger-associated molecular patterns (DAMPs). These are recognized by innate pattern recognition receptors (PRRs) such as the TLRs, an event that is critical to mount an effective immune response (2). Adjuvants, which contain formulated ligands for PRRs and thus mimic particular aspects of the normal response to pathogens, are used in vaccine formulations to enhance immunogenicity and modulate the type of immune response to defined Ags.

The first clinically approved combination adjuvants, AS01 and AS04, which contain the TLR4 agonist monophosphoryl lipid A (MPL) and either the saponin QS-21 or alum, respectively, were developed empirically based on an augmented adaptive immune response (3). It has subsequently been found that both aluminum salts and QS-21 can activate the inflammasome pathway (4, 5). Also, the FDA-approved squalene oil-in-water emulsion (SE) adjuvants MF59 and AS03 have been reported to enhance adaptive immunity by driving the production of DAMP molecules, which activate the inflammasome cascade to produce the inflammatory cytokines IL-18 and IL-1β (1, 6, 7). Thus, the inflammasome has emerged as a central node in the mechanism of action of all adjuvants that have been advanced to clinical usage.

The inflammasome is a multiprotein complex implicated in host defense against pathogens and is composed of specific sensor proteins, the adaptor protein ASC, and the inflammatory protease procaspase 1 (8, 9). Several sensor proteins have been described to assemble inflammasomes, but the leucine-rich repeat-containing protein (NLR) family member NLRP3 is one of the most well characterized and studied (10). The canonical pathway of NLRP3 inflammasome assembly is based on two signals (11): the first signal is triggered by the binding of ligands to TLR and induces the synthesis of pro–IL-1β and NLRP3 through NF-κB signaling. The second signal may be triggered by a variety of different compounds, ranging from exogenous chemicals such as nanoparticles, silica, and asbestos (12, 13); the saponin-containing adjuvant QS-21 (4); or aluminum salts (5) to self-components released upon damage that accumulate in nonsteady state locations, (e.g., host lipids, metabolites [ATP, uric acid], RNA, and DNA) (7, 14, 15). The assembly of the inflammasome proteins triggers the activation of the protease caspase-1, which in turn results in maturation and release of active IL-1β and IL-18 and induces pyroptosis, a specialized form of inflammatory cell death.

We have developed a synthetic TLR4 agonist called glucopyranosyl lipid adjuvant (GLA), which, when combined with an SE, induces potent Th1 and T follicular helper cell (TFH) responses, increases Ag-specific B cells, increases Ab titers, and stimulates isotype switching (16–21). GLA-SE adjuvanticity relies on the engagement of both the MyD88 and TIR domain–containing adapter inducing IFN-β signaling pathways (17), which are upstream of NF-κB. Moreover, potent Th1 responses are dependent on type I and II IFNs, IL-12, IL-18, and IL-18R–driven innate IFN-γ production (20, 21). Also, GLA-SE is predominantly captured in the draining lymph nodes (dLNs) by subcapsular macrophages, which are important for IL-18 secretion, generation of Th1 responses, and strong B cell responses characterized by the production of increased germinal center (GC) B cells as well as isotype-switched preplasmablasts (PPB) producing IgG2c (22).

To further elucidate the mechanisms of action of the combination adjuvant GLA-SE, we hypothesize that ATP is one of the necessary DAMPs that are released upon immunization with GLA-SE and that it results in the subsequent activation of the NLRP3 inflammasome. We show in this article that the SE triggers the infiltration of innate cells into the lymph nodes (LNs) and their secretion of the active form of IL-1β. Activation of the inflammasome cascade and release of IL-1β were necessary to the adjuvanticity of GLA-SE, as deletion of inflammasome components greatly ablated the infiltration of innate cells and both adaptive Th1 and B cell responses. In addition, we show that extracellular ATP, a known activator of NLRP3 inflammasome, augments the adjuvanticity of GLA-SE. The findings of this study allow a deeper understanding of the molecular mechanisms that drive the potent adjuvant activity of the clinical stage adjuvant GLA-SE and have implications for the development of future combination adjuvants based on a paradigm of combined TLR and inflammasome activation.

Materials and Methods

Methods were performed in accordance with relevant regulations and guidelines.

Mice, immunizations, and tissue harvesting

Female wild-type (WT) C57BL/6, IL-1R−/−, NLRP3−/−, Caspase 11−/−, Caspase 1-11−/−, P2XR7−/−, TLR4−/−, and OT-II [B6.Cg-Tg(TcraTcrb)425Cbn/J] mice aged 6−10 wk were purchased from the Jackson Laboratory. ASC−/− mice were a kind gift of A. Hise (Case Western Reserve University). All strains were on the C57BL/6 background, maintained in specific pathogen-free conditions, and kept in the facility for a minimum of 7 d prior to usage. All animal experiments and protocols used in this study were approved by the Infectious Disease Research Institute’s Institutional Animal Care and Use Committee.

Mice were immunized via an i.m. injection in the calf muscles of hind limb with 2.5 μg of ID97 recombinant protein (23) and 1 μg of PE (ProZyme, Hayward, CA) formulated in 5 μg of GLA in SE (2% oil) (19, 24, 25). In experiments to assess proliferation, mice were immunized with 5 μg of EndoGrade endotoxin-free OVA (Biovest International) formulated in 5 μg of GLA in SE (2% oil). In experiments with apyrase, high-activity apyrase (10 U/leg, A2230-100UN; Sigma-Aldrich) was included in the immunization mix. In experiments with oxidized ATP (oxATP) (A6779; Sigma-Aldrich), mice were injected twice i.m. with 100 μl of 6 mM oxATP on days −2 and −1 prior to immunization with GLA-SE.

For adaptive immune responses, spleens and inguinal dLNs were collected in RPMI 1640 7 d postimmunization. Cell suspensions were obtained by manual disruption. RBCs contained in spleens were lysed using the RBC Lysis Buffer (eBioscience). For innate responses, dLNs were harvested 1, 6, and 24 h postimmunization in PBS + 0.5% BSA + EDTA-free protease inhibitor mixture (1:100; Sigma-Aldrich) + 10 μg/ml Brefeldin A (GolgiPlug; BD Biosciences). For muscle experiments, calf muscles of hind limbs were collected 6 h postimmunization in HBSS, chopped into small pieces, and digested in an enzymatic solution containing Liberase TM (70 μg/ml working solution; Roche Applied Science) using a gentleMACS Dissociator (Miltenyi Biotec). Total viable cells were counted (Guava Technologies; EMD Millipore) and plated at 2 × 106 cells per well in round-bottom 96-well plates.

Flow cytometry

For intracellular cytokine staining, cells were stimulated for 2 h with media (RPMI 1640 + 10% FCS) or ID97 (10 μg/ml) at 37°C and subsequently incubated with Brefeldin A for an additional 8 h at 37°C. Cells were surface stained with CD4 BV650 (clone RM4-5; BioLegend), CD8 BV510 (clone 53-6.7; BioLegend), B220 BV510 (clone RA3-6B2; BioLegend), CD11b BV510 (clone M1/70, BD Biosciences), and CD44 allophycocyanin-eF780 (clone IM7; eBioscience) together with Fc receptor block (anti-CD16/32 Ab) followed by permeabilization with Cytofix/Cytoperm (BD Biosciences) and intracellular staining with CD154 PerCP-eF710 (clone MR1; eBioscience), TNF eF450 (clone MP6-XT22; eBioscience), IL-2 allophycocyanin (clone JES6-5H4; eBioscience), and IFN-γ PE-Cy7 (clone XMG1.2; eBioscience). Cells were gated as singlets > lymphocytes > CD4+ CD8− B220− CD11b− > CD44+ > cytokine+.

For peptide MHC class II (pMHC-II) tetramer staining, cells were incubated with the Ag85B (Rv1886) p25 tetramer (National Institutes of Health Tetramer Core Facility at Emory University, Atlanta, GA) and Fc receptor block for 1 h at 37°C. Cells were then surface stained with CXCR5 PerCP-eF710 (SPRCL5; eBioscience), CD8 BV510, B220 BV510, CD11b BV510, PD-1 (CD279) BV605 (clone 29F.1A12; BioLegend), CD4 BV650, and CD44 allophycocyanin-Cy7. Cells were subsequently permeabilized in Foxp3/Transcription factor Fix and Perm buffer (eBioscience) for 1 h at room temperature and then intracellularly stained with Foxp3 Alexa Fluor 488 (clone FJK-16s; eBioscience) and T-bet BV421 (clone 4B10; BioLegend). Cells were gated as singlets > lymphocytes > CD4+ CD8− B220− CD11b− > Tetramer+ CD44+ > CXCR5+ PD-1+ (TFH), CXCR5− PD1− T-bet+ (Th1), or CXCR5− PD1− Foxp3 (T regulatory cells [Treg]).

For B cell staining, cells were stained with Fc receptor block and IgM PerCP-eF710 (clone II-41; eBioscience), CD95 BV421 (clone Jo2; BD Biosciences), CD138 BV605 (clone 281-2; BD Biosciences), B220 BV785 (clone RA3-6B2; BioLegend), GL7 eF660 (clone GL-7; eBioscience), CD38 AF700 (clone 90; eBioscience), IgD allophycocyanin-Cy7 (clone 11-26c.2a; BioLegend), IgG1 FITC (clone RMG1-1; BioLegend), and IgG2a BV510 (clone R19-15; BD Biosciences) followed by permeabilization and intracellular staining with IgG1 FITC and IgG2a BV510. PE-specific B cells were identified by staining cells with 1 μg/ml (surface staining) and 0.2 μg/ml (intracellular staining) PE. Cells were gated as singlets > lymphocytes > B220 B cells > PE+ > CD95+ GL7+ (GC), CD95− GL7− CD138+ (PPB), or CD95− GL7− CD138− CD38+ IgD+ IgM+ (memory) > IgG1+ or IgG2a+.

For innate staining (dLNs and muscles), cells were stained with Ly6G FITC (clone 1AB; BioLegend), Ly6C PerCP-Cy5.5 (clone HK1.4; eBioscience), MHC class II (MHC-II) eF450 (clone M5/114.15.2; BioLegend), CD169 (Siglec-1) BV605 (clone 3D6.112; BioLegend), CD8 BV785 or BV510 or PE-Cy7 (clone 53-6.7; BioLegend), CD4 BV785 or BV650 (clone RM4-5; BioLegend), B220 BV785 or BV510 (clone RA3-6B2; BioLegend), CD11b-AF700 (clone M1/70; BioLegend), CD11c-allophycocyanin-Cy7 (clone N418; eBioscience), NK1.1 PE or allophycocyanin (clone PKI136; BioLegend), and CD69 PE-Cy7 (clone H1.2F3; eBioscience). After permeabilization, cells were stained with pro–IL-1β allophycocyanin (clone NJTEN3; eBioscience). Appropriate isotype controls were used. Cells were gated as singlets > lymphocytes > CD4+ T cells, CD8+ T cells, B220+ B cells, Ly6G+ Ly6C+ neutrophils, Ly6C+ CD11b+ inflammatory monocytes, CD169+ CD11b+ subcapsular macrophages, NK1.1+ NK cells, MHC-II+ CD11c+ CD11b+ dendritic cells (DCs), or MHC-II+ CD11c+ CD11b− DCs > pro–IL-1β+ or CD69+.

All data were collected on Fortessa or LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo (Tree Star).

Serum endpoint titer ELISA

Mouse serum was collected by terminal cardiac bleed and subsequent centrifugation at 10,000 rpm for 5 min. Serum titers against ID97 and PE were then evaluated by Ab capture ELISA. Briefly, Corning high-binding 384-well plates (VWR International) were coated overnight at 4°C with 1 μg/ml PE or 2 μg/ml ID97 in coating buffer (eBioscience). Then, plates were blocked with 1% BSA–PBS, and serum samples were serially diluted. Detection Abs were anti-mouse IgG2c or Ig(H+L) HRP (SouthernBiotech). Plates were analyzed at 450 nm (ELx808; BioTek Instruments), and endpoints were set as the minimum dilution at which values were lesser or equal to an OD of 0.2.

IL-1β, IL-1α, and IFN-γ ELISA

At indicated times following immunization, dLNs were collected in PBS + 0.5% BSA + EDTA-free protease inhibitor mixture (1:100; Sigma-Aldrich) + 10 μg/ml Brefeldin A (GolgiPlug; BD Biosciences) and manually homogenized. Supernatants were collected, and total production of IL-1β, IL-1α, or IFN-γ was assessed using the corresponding Mouse Ready-Set-Go! ELISA kit (Affymetrix) according to the manufacturer’s instructions.

CFSE-labeled, OVA-specific CD4+ T cell proliferation assay

LNs and spleens from OVA-restricted OT-II [B6.Cg-Tg(TcraTcrb)425Cbn/J] mice were collected, and CD4+ T cells were isolated using a CD4-negative isolation kit (Miltenyi Biotec). Cells were labeled with CFSE (Fisher Scientific), and 1 × 107 stained CD4+ T cells in 300 μl of PBS were i.v. injected per mouse. Two days later, mice were immunized on the left side (ipsilateral) with a mixture of OVA + PBS or OVA + GLA-SE. Their right side (contralateral) was left unimmunized as an internal control. Three days later, dLNs (inguinal, iliac, and axillary nodes of the ipsilateral side) and non-dLNs (inguinal, iliac, brachial, and axillary nodes of the contralateral side) were collected and labeled for lineage (CD8-BV510, CD11b-BV510, B220-BV510), CD4-BV650, CD69-PE-Cy7, and CD44-allophycocyanin-Cy7 to determine by flow cytometry CFSE dilution profiles as a measure of Ag-specific T cell proliferation. Data were analyzed using the proliferation tool of FlowJo software, and proliferation is indicated as expansion index (EI) (26).

Test of apyrase protease activity

Three different types of apyrase (normal [A6410-500UN], high-activity [A2230-100UN], and recombinant [A6237-100UN]; all from Sigma-Aldrich) were reconstituted in 1× PBS (pH 7.2) to equivalent activity unit concentrations. The apyrases were then mixed with ID97 and GLA-SE and assessed for protease activity after 1 h incubation on ice. ID97 integrity was assessed by a silver-stained reducing SDS-PAGE. Briefly, samples were reduced using 1.25% 2-ME (63689; Sigma-Aldrich) in 1× lithium dodecyl sulfate buffer (NP0007; Thermo Fisher Scientific) at 90°C heat for 15 min, electrophoresed through a 4–20% Tris Glycine gel (EC60255; Life Technologies), and then silver stained (PROT-SIL1; Sigma-Aldrich). The hydrodynamic diameter (Z-average [Z-Avg]) and the polydispersity index of the adjuvant were measured using dynamic light scattering (Nano ZS, Malvern Panalytical) following a 100-fold dilution of samples in water. The pH of the room temperature–equilibrated samples was measured using a three-point calibrated pH probe (Orion ROSS Ultra Semi-micro pH Electrode; Thermo Fisher Scientific). GLA and squalene content were measured by reversed phase (RP)–HPLC (1200; Agilent) equipped with a corona-charged aerosol detector (ESA Biosciences). Briefly, samples were diluted with mobile phase (methanol/chloroform/acetic acid/ammonium acetate) and injected onto an Atlantis T3 C18 column (Waters) for separation. GLA and squalene concentrations were determined from respective standard curves using calculated peak areas.

In vivo bioluminescence imaging

Mice were anesthetized with a continuous flow of isoflurane injected i.m. on the left leg with PBS or GLA-SE mixed with 10 μl of a luciferin–luciferase reporter (Promega) for a total volume of 20 μl. GLA-SE was used at the same concentration as for immunogenicity experiments. In experiments with apyrase, high-activity apyrase (10 U/leg, A2230-100UN; Sigma-Aldrich) was included in the immunization mix. Images were taken with the IVIS Lumina System (PerkinElmer) every 30 s with an exposition time of 10 s, starting 30 s after the injection, for 10 min. Regions of interest were defined manually around the injection site, and the number of photons per second in the region of interest was determined using the software Living Image.

Statistical analysis

All data are presented as mean ± SEM. Data were analyzed using GraphPad Prism 7 software (La Jolla, CA) by one-way ANOVA (with Tukey multiple comparisons posttest), two-way ANOVA (with Bonferroni multiple comparisons posttest), or by unpaired Student t tests, as indicated for each experiment. Values were considered significantly different with *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

Immunization with SE induces the secretion of IL-1β

The adjuvant activity of GLA formulated in SE is greatly enhanced compared with GLA in an aqueous formulation (AF) or SE alone, as evidenced with increased Ag-specific Th1-differentiated CD4 cells and B cells as well as Ab isotype switching to IgG2c [Supplemental Fig. 1 and previous publications from our group (20, 27)]. These responses were dependent on the secretion of the inflammasome-driven IL-18 protein (22). However, the role of IL-1β, another inflammasome-dependent cytokine, in GLA-SE adjuvanticity is still largely unknown. IL-1β is a major innate proinflammatory mediator that controls immune responses to tissue injury by PAMPs or DAMPs. IL-1β, when administered together with the TLR4 agonist LPS, also has been proved to directly enhance CD4 and CD8 T cell responses by promoting the expansion of Ag-specific T cells in vivo (28, 29). Moreover, Ben-Sasson et al. (29) showed that IL-1β acts directly on Ag-specific CD4 T cells, indicating that stimulated T cells express IL-1R. Also, IL-1β enhances both proliferation and survival, differentiation, and migration of cells, which are key parameters for the generation of a potent adaptive response (30).

To assess the contribution of each component of GLA-SE to IL-1β production, WT mice were immunized i.m. with PBS, the SE, GLA-AF, or the combination adjuvant GLA-SE, and their inguinal dLNs were collected 1, 6, and 24 h postimmunization. We first observed by flow cytometry that the SE induced a substantial infiltration at 6 h of Ly6G+ Ly6C+ neutrophils and CD11b+ Ly6C+ inflammatory monocytes into the dLNs (Fig. 1A). CD4+ T cells, CD8+ T cells, B220+ B cells, CD169+ CD11b+ subcapsular macrophages, MHC-II+ CD11c+ DCs, and NK1.1+ NK cells were also analyzed (gating strategy is described in the Materials and Methods). This infiltration correlated with the production of the chemokines CCL3 (MIP-1α) and CCL4 (MIP-1β), which are known chemoattractants for neutrophils, inflammatory monocytes, and NK cells (Fig. 1B).

FIGURE 1.
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FIGURE 1.

IL-1β is secreted upon immunization with SE. Mice were immunized once i.m. with PBS, SE, GLA-AF, or GLA-SE, and their dLNs were collected 1, 6, and 24 h postimmunization. (A) The frequency of each cell type in the dLNs was determined by flow cytometry. The absolute numbers were calculated based on the total counts of cells in the dLNs. The gating strategy included Ly6G+ Ly6C+ neutrophils (Neut), CD11bhigh Ly6C+ inflammatory monocytes (Inflamm mono), CD169+ CD11b+ subcapsular macrophages (SCMφ), and NK1.1+ NK cells. *p < 0.05, **p < 0.01, ***p < 0.001 compared with PBS. (C) The frequencies of cells positive for pro–IL-1β (open white histograms) were subsequently determined based on their adequate isotype control (solid gray histograms), and the absolute numbers of cells positive for pro–IL-1β were calculated based on the number of cells in the dLNs calculated in (A). *p < 0.05, ***p < 0.001 compared with SE. Representative histograms of neutrophils (left) and inflammatory monocytes (right) are depicted. Supernatants of dLNs were collected, and the chemokines CCL3 and CCL4 (*p < 0.05, ***p < 0.001 as indicated) (B), the mature form of IL-1β (**p < 0.01 compared with GLA-SE, ###p < 0.001 compared with GLA-AF) (D), and the cytokines IFN-γ and IL-1α (*p < 0.05, **p < 0.01, ***p < 0.001 compared with GLA-SE, #p < 0.05 compared with GLA-AF) (E) were assessed by Luminex. Results are representative of two independent experiments with n = 5 animals per group and are shown as mean ± SEM. The p values were determined by one-way ANOVA/Bonferroni posttest (B) or two-way ANOVA/Bonferroni posttest (A and C–E).

We then assessed whether these cells contributed to the production of IL-1β by measuring the biologically inactive pro–IL-1β following immunization by flow cytometry. Although assessment of intracellular pro–IL-1β does not directly reflect the quantity of mature IL-1β secretion, it is a good readout of which immune cells are activated to produce the proinflammatory cytokine. We observed that the increased numbers of infiltrated neutrophils and inflammatory monocytes with SE immunization correlated with more cells positive for pro–IL-1β (Fig. 1C). Similarly, when measuring the mature form of IL-1β by Luminex, we observed that SE induced a higher secretion of IL-1β compared with GLA-AF. Also, GLA appears to dampen the magnitude of IL-1β elicited by SE in the group immunized with GLA-SE (Fig. 1D). Secretion of IL-1β peaked at 6 h after immunization, which seems to be delayed compared with IL-18, which we found peaked at ∼1 h postimmunization (20). Interestingly, we observed that GLA-SE, but not SE, elicited the production of IL-1α, a close homolog of IL-1β, and innate IFN-γ, which has been linked to Th1 responses to GLA-SE in previous publications (20) (Fig. 1E). These findings suggest a unique role of SE in the activation of the inflammasome and the subsequent production of IL-1β.

Innate responses to GLA-SE are driven by the inflammasome activation

The NLRP3 inflammasome is a multiprotein complex that upon assembly triggers caspase-1–dependent maturation and secretion of IL-1β and IL-18 (31). Having determined that the emulsion component of GLA-SE triggered IL-1β secretion, we next evaluated the contribution of the inflammasome to the activity of GLA-SE by employing ASC−/− mice. Although our findings revealed that SE was sufficient to activate the inflammasome, we used GLA-SE in these experiments to assess the mechanisms of action of the combination adjuvant, which drives robust Th1 immunity, rather than the emulsion alone, which predominantly produces Th2 responses. WT and ASC−/− mice were immunized i.m. with PBS or GLA-SE, and their dLNs were collected 6 h postimmunization. As anticipated, deficiency in the ASC protein led to a decrease in IL-1β secretion following immunization with GLA-SE (Fig. 2A). The inflammasome formation following GLA-SE immunization also proved to be essential for the production of innate IFN-γ (Fig. 2B) but not IL-1α (Fig. 2C). Next, we assessed the expression of CD69, an Ag-independent activation marker of lymphocytes that is upregulated following immunization with GLA-SE, and observed reduced levels of CD69 on cells from ASC−/− mice compared with WT mice (Fig. 2D). At 6 h, the infiltration of innate cells into the dLNs was also greatly decreased in mice lacking the inflammasome activation (Fig. 2E). Overall, these findings demonstrate that the activation of the inflammasome is a prerequisite for the early response to GLA-SE.

FIGURE 2.
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FIGURE 2.

Innate responses to GLA-SE are driven by the inflammasome activation. WT and ASC−/− mice were immunized once i.m. with PBS or GLA-SE, and their dLNs were collected 6 h postimmunization. Supernatants of dLNs were collected, and the mature form of IL-1β (A), IFN-γ (B), and IL-1α (C) were assessed by ELISA. The mean fluorescence intensity (MFI) of CD69 staining in lymphocytes was assessed by flow cytometry (D), as well as the frequency of each cell type in the dLNs (E). The absolute numbers were calculated based on the total counts of cells in the dLNs. Results are representative of two independent experiments with n = 5 animals per group and are shown as mean ± SEM. The p values were determined by one-way ANOVA/Tukey posttest (A–D) or two-way ANOVA/Bonferroni posttest (E). *p < 0.05, **p < 0.01, ***p < 0.001 compared with WT/GLA-SE or as indicated.

Proliferation of Ag-specific CD4+ T cells requires the inflammasome formation

We have previously shown that the production of innate IFN-γ induced by GLA-SE contributes to the Ag-specific adaptive response to the concomitantly injected adjuvant and Ags (20). The production of IFN-γ being fully ablated in ASC−/− mice, we next aimed to determine whether the inflammasome activation was required for the downstream proliferation of Ag-specific CD4+ T cells. CFSE-labeled, OVA-specific naive OT-II CD4+ T cells were transferred intravenously into WT or ASC−/− animals, and 2 d later, mice were immunized on the left side with either a mixture of OVA + PBS or OVA + GLA-SE. The right, contralateral side was used as an internal, nonimmunized control. Proliferation was assessed 3 d later by assessing the dilution profiles of CFSE (Fig. 3A) and by calculating the EI, which correlates to the fold expansion of cells during the in vivo stimulation period (26).

FIGURE 3.
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FIGURE 3.

Proliferation of Ag-specific CD4+ T cells in dLNs and non-dLNs. OVA-specific CD4+ T cells were isolated from OT-II mice, stained with CFSE, and injected i.v. into recipient WT or ASC−/− mice. Two days later, mice were immunized on the left side with OVA + PBS or OVA + GLA-SE, whereas the right side remained unimmunized. Their draining (left side) and nondraining (right side) LNs were collected separately 3 d after immunization, and OVA-specific CD4+ T cell proliferation was assessed by flow cytometry. (A) CD4+ T cells positive for CFSE were gated, and the histograms of CFSE profiles in non-dLNs and dLNs were analyzed to obtain the EI (B). (C) The frequency of total CD4+ T cells in LNs was also assessed. Results are representative of two independent experiments with n = 5 animals per group and are shown as mean ± SEM, with each circle representing a single replicate. The p values were determined by one-way ANOVA/Bonferroni posttest. **p < 0.01, ***p < 0.001 as indicated, ##p < 0.01, ###p < 0.001 compared with nondraining side.

Following immunization with OVA alone, proliferation in WT mice was significantly higher in dLNs (left, ipsilateral side) compared with non-dLNs (right, contralateral side), in which the EI remained around 1 (Fig. 3B). In WT mice, addition of GLA-SE to OVA further increased the expansion of Ag-specific CD4+ T cells, confirming the importance of GLA-SE in the induction of CD4+ T cell expansion. Critically, proliferation of OVA-specific CD4+ T cells in dLNs was fully abolished in ASC−/− mice compared with WT mice (Fig. 3B). Interestingly, the frequency of total CD4+ T cells was unaltered in immunized ASC−/− mice compared with immunized WT mice, indicating a central role of the inflammasome in inducing Ag-specific CD4+ T cell responses rather than maintaining a CD4+ T cell pool (Fig. 3C).

Assembly of the NLRP3 inflammasome is crucial for GLA-SE adjuvanticity

Next, having uncovered the function of the inflammasome in regulating CD4+ T cell expansion, we sought to assess its role in mounting adaptive T and B cell responses. ASC is an adaptor protein that is recruited in the formation of several inflammasome types, including the NLRP3 inflammasome, but also the NLRP1b, AIM2, and pyrin inflammasomes. To determine whether GLA-SE acts specifically through the activation of the NRLP3 inflammasome as is true for several other adjuvants (1), we compared adaptive responses to GLA-SE in NLRP3−/−, ASC−/−, and WT mice. With this approach, if immune responses to GLA-SE are altered in ASC−/− mice but are not impaired in NLRP3−/− mice, it would suggest that other inflammasome types are likely assembled upon immunization with GLA-SE. Also, ASC−/− mice were shown to have an inflammasome-independent impaired lymphocyte migration and altered Ag presentation by DCs (32). Comparing adaptive responses in NLRP3−/− mice with those of ASC−/− mice will enable the confirmation of the inflammasome role in the adjuvanticity of GLA-SE.

Animals were immunized with the Ags ID97 and PE alone or formulated with GLA-SE, their dLNs and spleens were harvested 7 d later, and the adaptive responses were assessed by flow cytometry. ID97 is a recombinant tuberculosis vaccine protein composed of the fusion of four Mycobacterium tuberculosis proteins (Rv1886, Rv3478, Rv3619, Rv2875) (23). ID97-specific T cells can be detected by binding an I-Ab tetramer (pMHC-II) presenting a dominant epitope of Rv1886 (Ag85B). PE was used to determine the proportion of Ag (PE)-specific B cells as previously described (33).

We found that deficiency of both NLRP3 and ASC resulted in a similar decrease in numbers of Ag-specific CD4+ T cells [gating strategy is described in the Materials and Methods and in previous publications from our group (20–22)], as demonstrated by a decrease in the numbers of pMHC-II tetramer-positive cells in the LN (Fig. 4A), whereas the total number of CD4+ T cells per LN was not altered in either knockout (KO) animals, further indicating that the inflammasome plays a role in the expansion of Ag-specific T cells. In addition, the numbers of CD4+ T-bet+ Th1 were reduced (Fig. 4B) but the commitment to a cell lineage was not altered, as the frequency of each T cell lineage was comparable in KO and WT animals (Fig. 4C). Lower Th1 numbers matched with reduced percentages of CD4+ T cells positive for Th1-related cytokines upon restimulation with ID97 (Fig. 4D).

FIGURE 4.
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FIGURE 4.

Activation of the NLRP3 inflammasome is crucial in the adjuvanticity of GLA-SE. WT, ASC−/−, and NLRP3−/− mice were immunized with ID97 and PE alone or formulated with GLA-SE, and their dLNs and spleens were collected 7 d after immunization. The numbers of ID97-specific CD4+ T cells and total numbers of CD4+ T cells (A), as well as Ag-specific CD4+ T-bet+ Th1 (B), were calculated based on tetramer staining and total counts of cells in the dLNs. The relative frequency of Th1, TFH, Foxp3+ Treg, and uncategorized CD4+ T cells in WT mice were compared with both KO mice (C). The frequency of CD4+ CD44+ T cells positive for CD154, IFN-γ, IL-2, and TNF was assessed upon restimulation with ID97 (D). Total number of PE-specific B220+ B cells and total numbers of B cells in the dLNs (E), as well as CD138+ PPB (F), were calculated based on PE staining and total counts of cells in the dLN. Serum total IgG endpoint titers against PE were measured by ELISA (G). Results are representative of three independent experiments with n = 5 animals per group and are shown as mean ± SEM, with each circle representing a single replicate. The p values were determined by one-way ANOVA/Tukey posttest (A, B, and E–G), or two-way ANOVA/Bonferroni posttest (C and D). *p < 0.05, **p < 0.01, ***p < 0.001 as indicated or compared with WT/GLA-SE.

There is a direct relationship between TFH and the induction of GC B cell responses, in that their numbers correlate in the course of an immune response (34). In concordance with lower TFH numbers in the immunized ASC−/− and NLRP3−/− mice (data not shown), the B cell response was also altered in the absence of inflammasome activation, as observed with a reduced number of PE-specific B220+ B cells [Fig. 4E, gating strategy is described in the Materials and Methods and in previous publications from our group (20–22)]. As with CD4+ T cells, the total number of B cells in the NLRP3−/− and ASC−/− mice was similar to that in WT mice upon immunization (Fig. 4E). Also, NLRP3−/− and ASC−/− mice had decreased numbers of Ag-specific CD138+ PPB (Fig. 4F), which undergo isotype switching upon GLA-SE immunization and produce IgG2c. The lower number of Ag-specific B cells correlated with reduced titers of IgG total in both KO strains (Fig. 4G). Thus, the adjuvant GLA-SE requires the activation of the NLRP3 inflammasome for shaping robust adaptive responses. As deficiency of ASC and NLRP3 leads to comparable decreased adaptive responses, it is likely that GLA-SE acts solely through the NLRP3 inflammasome, or at least that NLRP3 is essential.

Adjuvanticity of GLA-SE is dependent on both the canonical and noncanonical pathways

We have previously found that caspase 1 and/or 11 were essential for Th1 responses to GLA-SE–adjuvanted immunization (20), but it was still unknown whether caspase-1, caspase-11, or both were crucial for GLA-SE activity. Whereas caspase-1 engages the canonical pathway of NLRP3 inflammasome assembly, caspase-11 is initiated by intracellular Gram-negative bacteria and triggers the noncanonical pathway. Although the activation of both pathways can promote pyroptosis, IL-1β and IL-18 secretion are solely dependent on caspase-1 (10). Similar to NLRP3−/− and ASC−/− mice, we observed that deficiency in caspase-1 and caspase-11 considerably reduced the numbers of Ag-specific CD4+ T cells (Supplemental Fig. 2A), their differentiation into Th1 cells (Supplemental Fig. 2B), and the production of Th1-related cytokines (Supplemental Fig. 2C). Although the numbers of Ag-specific B cells (Supplemental Fig. 2D) and the numbers of PPB (Supplemental Fig. 2E) were reduced in KO animals, the Ab titers (Supplemental Fig. 2F) were not. This suggests that inflammasome activation is primarily important for induction of T cell responses or that the lack of impact on the B cell responses is due to the residual T cell responses being sufficient to support the B cell response. To note, although the reduction in adaptive responses was greater in caspase 1/11−/− mice compared with caspase 11−/− mice, these findings still indicate that both the canonical (through caspase-1) and noncanonical (through caspase-11) pathways of NLRP3 inflammasome activation are important for the Th1-biased immune response to GLA-SE.

Adaptive responses are impaired in inflammasome-deficient mice immunized with SE

Having established that the SE is the component of GLA-SE that stimulates the activation of the inflammasome, and that this activation is required for adaptive Th1 and B cell responses to GLA-SE, we next aimed to confirm that the impairment of adaptive responses in ASC−/− mice is dependent on SE and not GLA. Consequently, WT and ASC−/− mice were immunized with ID97 and PE formulated with SE or GLA-AF. Although the Ag-specific adaptive response is significantly lower in these mice compared with mice immunized with GLA-SE (Supplemental Fig. 1), we detected a lower frequency of Ag-specific CD4+ T cells in ASC−/− animals immunized with SE but not GLA-AF (Fig. 5A). Also, in mice immunized with SE, the frequencies of CD4+ T cells expressing CD154 and the Th1 cytokines IL-2, IFN-γ, and TNF were significantly lower in ASC−/− mice compared with WT mice, whereas the cytokine production was not impaired in ASC−/− mice immunized with GLA-AF (Fig. 5B). These findings further confirm that the emulsion alone is accountable for the inflammasome activation and that this activation is required for Th1 adaptive responses.

FIGURE 5.
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FIGURE 5.

Adaptive responses to SE are impaired in inflammasome-deficient mice. WT and ASC−/− mice were immunized with ID97 and PE formulated with SE or GLA-AF, and their dLNs and spleens were collected 7 d after immunization. The frequency of ID97-specific CD4+ T cells (A) and the frequency of CD4+ CD44+ T cells positive for CD154, IFN-γ, IL-2, and TNF was assessed upon restimulation with ID97 (B). Results are from one experiment with n = 5 animals per group and are shown as mean ± SEM, with each circle representing a single replicate. The p values were determined by unpaired student t test (A) or two-way ANOVA/Bonferroni posttest (B). **p < 0.01, ***p < 0.001 as indicated or compared with WT.

IL-1R signaling is necessary for the downstream adaptive responses to GLA-SE

IL-1β solely binds the type I IL-1 receptor, which is ubiquitously expressed, and assessing adaptive responses in IL1R−/− is an effective readout of IL-1β significance (35, 36). To determine the importance of IL-1β for the adjuvanticity of GLA-SE to drive adaptive Ag-specific Th1 CD4+ T cell and B cell responses, we immunized WT and IL-1R−/− mice with GLA-SE in the presence of the Ags ID97 and PE.

We observed fewer Ag-specific CD4+ T cells in IL-1R−/− animals (Fig. 6A) as well as lower numbers of CD4+ T-bet+ Th1 cells, CXCR5+ PD1+ TFH, and Foxp3+ Treg (data not shown). However, the relative frequency of each lineage was not altered in IL-1R−/− mice compared with WT mice (Fig. 6B), indicating that IL-1β is required for CD4+ T cell expansion rather than for the differentiation of cells into a specific subtype. In line with tetramer results, the frequencies of CD4+ T cells expressing CD154 and producing Th1-related cytokines (IFN-γ, IL-2, and TNF) following Ag recall were significantly reduced in IL-1R−/− mice compared with WT mice (Fig. 6C). Interestingly, IL-1β signaling appears to be less important for B cell responses, as deficiency in IL-1R did not impact the numbers of PE-specific B cells (Fig. 6D), the frequency of Ag-specific PPB, GC B cells, and memory B cells (Fig. 6E), or the IgG titers in the serum (Fig. 6F).

FIGURE 6.
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FIGURE 6.

GLA-SE adjuvanticity is dependent on IL-1R signaling. WT and IL1R−/− mice were immunized with ID97 and PE alone or adjuvanted with GLA-SE, and their dLNs and spleens were collected 7 d after immunization. Cell suspensions were obtained, and the numbers of CD4+ pMHC-II (ID97-specific)+ T cells (A) were calculated based on tetramer staining and total counts of cells in the dLNs. The relative frequency of Th1, TFH, Foxp3+ Treg, and uncategorized CD4+ T cells in WT were compared with IL-1R−/− mice (B). The frequency of CD4+ T cells positive for CD154, IFN-γ, IL-2, and TNF was assessed upon restimulation with ID97 (C). The percentage of CD4+ CD44+ T cells for each cytokine is plotted. Total number of PE-specific B220+ B cells in the dLNs (D) was calculated based on PE staining and total counts of cells in the LN. The relative frequency of PPB, GC, and memory and uncategorized Ag-specific B cells were compared (E). Serum was collected 7 d after immunization, and total IgG endpoint titers against PE were measured by ELISA (F). Results are representative of three independent experiments with n = 5 animals per group and are shown as mean ± SEM, with each circle representing a single replicate. The p values were determined by one-way ANOVA/Tukey posttest (A, D, and F), or two-way ANOVA/Bonferroni posttest (B, C, and E). *p < 0.05, **p < 0.01, ***p < 0.001 as indicated or compared with WT/GLA-SE.

These findings indicate that IL-1β signaling through IL-1R is critical for the CD4+ T cell, but not B cell, responses to GLA-SE. The difference in B cell responses between NLRP3−/− mice, which lack both IL-18 and IL-1β, and IL-1R−/− mice, which lack only IL-1β signaling, suggests that IL-18 plays a significant role at least in B cell responses.

ATP release is necessary for the infiltration of cells in the muscle following immunization with GLA-SE

Having confirmed the role of the inflammasome, we next wanted to identify which DAMP molecules were necessary for the observed adjuvanticity of GLA-SE. Extracellular ATP is a known activator of NLRP3 inflammasome, and recently, ATP released in the presence of tissue damage has emerged as an important mechanism of inflammatory danger signaling (11). Indeed, previous reports have demonstrated that ATP release was greatly enhanced by the presence of the squalene emulsion MF59 and that the addition of the ATP hydrolyzing enzyme apyrase resulted in inhibited cell recruitment to the injection site and in a reduced adaptive response to MF59-adjuvanted vaccination (7). To determine whether ATP was released upon immunization with GLA-SE, we monitored its release in vivo using a luciferase–luciferin reporter system as described elsewhere (7) and within minutes confirmed the release of ATP in the muscle following immunization with both PBS and GLA-SE. During the 10 min monitoring, ATP release was identical between the two groups, indicating that ATP release is not released by GLA-SE itself but likely by the injection causing damage to the tissue (Supplemental Fig. 3).

We next hypothesized that ATP was a DAMP necessary for the action of GLA-SE and quenching extracellular ATP with apyrase would dampen the immune modulatory properties of GLA-SE. Marrack and colleagues (37) have recently reported that protease contamination of commercially-available DNase preparations may have led to an overemphasis on extracellular DNA as a key DAMP in the activity of alum. To avoid a similar artifactual result, we evaluated three different types of commercial apyrase preparations (normal, high activity, and recombinant) for proteinase activity after mixing with ID97 and GLA-SE. After 1 h incubation on ice to simulate conservation of immunization material prior to injection into mice, we assessed ID97 integrity, adjuvant size, pH, and GLA and squalene content. We observed that normal apyrase degraded ID97, whereas the protein integrity was maintained with high-activity apyrase (Fig. 7A). We could not state whether ID97 was degraded or not with recombinant apyrase, as the apyrase itself obstructed the m.w. range of the gel where we expect to see ID97 (Fig. 7A). With the preservation of the protein being an essential parameter, we performed the subsequent experiments with high-activity apyrase. Dynamic light scattering and RP-HPLC showed that the adjuvant average diameter (Z-Avg) or the GLA and squalene content, respectively, did not change with the addition of high-activity apyrase but that pH dropped slightly (Fig. 7B). To confirm that 10 U of high-activity apyrase per leg is sufficient to deplete all extracellular ATP that is released upon immunization, we monitored ATP release in vivo in mice immunized with the luciferase–luciferin reporter together with GLA-SE in the presence or absence of high-activity apyrase. We confirmed that extracellular ATP could not be detected by the IVIS imaging system in the presence of apyrase, indicating that the quantity used is adequate to hydrolyze all extracellular ATP released in the muscle (Fig. 7C).

FIGURE 7.
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FIGURE 7.

Hydrolyzing extracellular ATP with apyrase does not affect IL-1β secretion following immunization with GLA-SE. First, we selected an apyrase, a hydrolyzing extracellular ATP, that is free of protease activity. Three different commercially available apyrases (normal apyrase, high-activity apyrase, and recombinant apyrase) were mixed with ID97 and GLA-SE. We assessed after 1 h incubation on ice ID97 integrity (A), average size of the adjuvant, pH, and GLA and squalene content (B). Size was measured using dynamic light scattering. GLA and squalene content were measured with RP-HPLC. Z-Avg: hydrodynamic radius; Pdl: polydispersity. (C) Mice were immunized with GLA-SE in the presence or absence of 10 U of high-activity apyrase together with a luciferin–luciferase reporter and imaged every 30 s for 10 min. The radiance (number of photons per second in the region of interest) is plotted over time. Results are from n = 4 animals per group and are shown as mean ± SEM. (D) Then, WT mice were immunized once with PBS or GLA-SE with or without additional high-activity apyrase. Their dLNs were collected 6 h after immunization, and the numbers of Ly6G+ Ly6C+ neutrophils (Neut) and CD11bhigh Ly6C+ inflammatory monocytes (Inflamm mono) were calculated based on their frequencies determined by flow cytometry and the total counts of cells in the dLNs. (E) The absolute numbers of cells positive for pro–IL-1β were calculated based on the number of cells in the dLNs calculated in (D). (F) Supernatants were collected, and the mature form IL-1β was measured by Luminex. (G) Muscles were collected 6 h after immunization, and the numbers of CD19+ B220+ B cells, CD4+ T cells, Ly6G+ Ly6C+ neutrophils, and NK1.1+ NK cells were calculated based on their frequencies determined by flow cytometry and the total counts of cells in the muscles. Results are representative of two independent experiments with n = 5 animals per group and are shown as mean ± SEM. The p values were determined by one-way ANOVA/Bonferroni posttest (F and G) or two-way ANOVA/Bonferroni posttest (C–E). *p < 0.05, ***p < 0.001 as compared with GLA-SE.

Having identified the high-activity apyrase as an acceptable tool to assess immune response to GLA-SE in the absence of extracellular ATP, we first analyzed early innate responses to GLA-SE when extracellular ATP was hydrolyzed by this enzyme. For this purpose, groups of WT mice were immunized with GLA-SE in the presence or absence of 10 U/muscle of high-activity apyrase, and inguinal dLNs were collected 6 h postimmunization. Surprisingly, we observed that the influx of neutrophils and inflammatory monocytes into the dLNs was not altered by apyrase treatment (Fig. 7D), nor were the numbers of cells expressing pro–IL-1β (Fig. 7E). In line with these results, the secretion of the mature form of IL-1β was also independent of apyrase (Fig. 7F), suggesting that other DAMPs may be driving IL-1β release.

ATP is released in considerable quantities from skeletal muscle cells upon damage (38). It is therefore possible that apyrase impairs early cell function at the site of injection rather than in the dLNs. In line with this hypothesis, we observed that extracellular ATP is necessary for the infiltration of innate cells such as neutrophils and NK cells, but also B cells and CD4+ T cells, into the site of injection (Fig. 7G).

ATP is a DAMP necessary for the adaptive responses to GLA-SE

We next assessed whether the release of extracellular ATP upon immunization is necessary to build robust Ag-specific Th1 and B cell responses. In order to differentiate the requirement of a first signal (i.e., TLR signaling) and a second signal (ATP release) for the activation of the inflammasome and subsequent strong adaptive responses, we immunized WT or TLR4−/− mice with ID97 and PE alone or adjuvanted with GLA-SE in the presence or absence of apyrase. As expected, TLR4 signaling was essential for the activity of GLA-SE, as knocking out this signaling pathway completely abolished Ag-specific T and B cell responses (Fig. 8).

FIGURE 8.
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FIGURE 8.

Extracellular ATP is a necessary DAMP in adaptive T cell responses to GLA-SE. WT or TLR4−/− mice were immunized with ID97 and PE alone or adjuvanted with GLA-SE in the presence or absence of high-activity apyrase, and their dLNs and spleens were collected 7 d after immunization. The numbers of ID97-specific CD4+ T cells (A) and CD4+ T-bet+ Th1 (B) were calculated, as was the frequency of CD4+ CD44+ T cells positive for CD154 and the Th1-related cytokines IFN-γ, IL-2, and TNF upon restimulation (C). Total number of Ag-specific CD4+ CXCR5+ PD-1+ TFH (D) and B220+ B cells in the dLNs (E) was assessed. Serum total IgG endpoint titers against PE were measured by ELISA (F). Results are representative of two independent experiments with n = 10 (WT) or 3 (TLR4−/−) animals per group and are shown as mean ± SEM, with each circle representing a single replicate. The p values were determined by one-way ANOVA/Tukey posttest (A, B, and D–F) or two-way ANOVA/Bonferroni posttest (C). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with untreated (−apyrase) group.

Extracellular ATP was essential in augmenting Ag-specific responses, as mice treated with apyrase had a significant reduction in the numbers of pMHC-II+ CD4+ T cells (Fig. 8A), numbers of CD4+ T-bet+ Th1 cells (Fig. 8B), secretion of Th1 cytokines (Fig. 8C), numbers of CD4+ TFH (Fig. 8D), and numbers of PE+ B cells (Fig. 8E). However, deficiency in ATP signaling did not impact the IgG titers in the serum (Fig. 8F). To confirm the importance of ATP as a danger signal, we pretreated mice with oxATP, which irreversibly antagonizes extracellular ATP receptors, including P2X7R (39). WT mice were given oxATP twice i.m. prior to immunization with GLA-SE, and their Ag-specific responses were evaluated after 7 d. Similar to the apyrase treatment, we observed that both CD4+ T cell and B cell responses were considerably reduced in mice that received the treatment compared with those who did not (Supplemental Fig. 4A–F). Surprisingly, deficiency of P2X7R did not alter the ability of T cells to respond to GLA-SE (Supplemental Fig. 4G). This suggested that other purinergic receptors might be involved in the recognition of extracellular ATP in addition to P2X7R. Overall, these findings indicate that extracellular ATP is a key activator of the inflammasome; its release boosts the response initiated by TLR signaling, and therefore it is a significant contributor to the mechanism of action of the combination adjuvant GLA-SE.

Discussion

A number of mechanisms of action have been postulated for the way in which currently licensed adjuvants modulate immune responses. Depending on the adjuvant, different PRR signaling occurs, resulting in the production of specific cytokines and chemokines that influence which immune cells are recruited to the site of injection and ultimately will shape the adaptive response.

The mechanisms of action of the combination adjuvant GLA-SE, a TLR4 agonist formulated in an SE that augments cellular and humoral immunity to a variety of vaccine Ags in humans, have been only partly elucidated (40–43). Previous work from our group showed that the innate and adaptive responses to GLA-SE were compromised in the absence of IL-18 signaling, indicating a role for the inflammasome activation in the response to immunization with GLA-SE. We report in this article that the SE component of the combination adjuvant leads to the inflammasome activation and the subsequent IL-1β secretion, as immunization with both GLA-SE or SE leads to the production of mature cytokine in the dLNs. However, the magnitude of mature IL-1β secretion seems to be reduced in animals immunized with GLA-SE compared with the SE group, a finding that still needs to be clarified. In support of the importance of IL-1 signaling, ablation of the signaling pathway using IL-1R−/− mice impaired Ag-specific CD4+ T cells responses. Furthermore, prior activation of the NLRP3 inflammasome for the subsequent release of IL-1β is required for shaping appropriate adaptive responses to GLA-SE, as deletion of the inflammasome proteins NLRP3 and ASC greatly ablated both Th1 and B cell responses. The complete abolishment of responses in TLR4−/− mice indicates the presence of other inflammasome-independent pathways that govern responses to GLA-SE. We consistently found that that impairment of the inflammasome pathway had a more dramatic effect on T cell than B cell responses. This suggests that B cell responses to GLA-SE are a function of GLA-SE augmenting TFH and Th1 responses; partial changes in those may not be sufficient to change the B cell response, whereas a complete abrogation of T cell responses as observed in TLR4−/− animals also ablates the B cell response. Moreover, B cell response magnitude after 7 d correlated more closely with the numbers of Th1 cells rather than TFH, which is likely due to this early Ab titer not being only dependent on GC reactions, unlike later titers, as we reported previously (22). Release of extracellular ATP, a known activator of NLRP3 inflammasome, was found to be necessary for the maximal adjuvant activity of GLA-SE. Importantly, the effects of apyrase or oxATP treatment on the adaptive response are less dramatic than is seen in the ASC−/− or NLPR3−/− mice, which suggests that either DAMPs other than ATP also contribute to adjuvanticity of GLA-SE, the treatments are insufficient to block all ATP activity, or both.

The IL-1 family is composed of 11 types of cytokines, including IL-1β and IL-1α, which share most of their biological function of controlling proinflammatory reactions to tissue damage caused by PAMPs or DAMPs (35). Although innate immune cells are a major source of both IL-1β and IL-1α, other cells such as epithelial and endothelial cells can also produce both cytokines. In contrast to mature IL-1β, which exists only as a soluble form, IL-1α is also a cell surface–bound protein. A study by Fettelschoss et al. (44) demonstrated that IL-1α associated to the cell surface was caspase-1 independent, but secretion of mature IL-1α required activation of the inflammasome. In contrast to these results, we show in this study that unlike IL-1β, the secretion of IL-1α was independent of the inflammasome activation. Also, interestingly, the SE induced release of IL-1β but not IL-1α, indicating a unique role of SE in the activation of the inflammasome. Note, however, as IL-1β and IL-1α both bind to the same IL-1 receptor, IL-1R, we cannot confirm whether the impact on the adaptive responses that was observed in IL-1R–deficient animals was due to lack of signaling from IL-1β, IL-1α, or both cytokines.

Immune cells recognize extracellular ATP from damaged or dying cells as a danger signal. Indeed, ATP is an important endogenous signaling molecule, and its release into the extracellular space has been linked to important cell-to-cell communication in immunity and inflammation (45). The steady-state concentration of cytosolic ATP ranges between 3 and 10 mM, whereas extracellular ATP concentration is ∼10 nM, leading to a 106-fold gradient difference between cell cytosol and extracellular space (45). The release of a small fraction of cellular ATP into the extracellular space can therefore be sensed by purinergic P2 receptors, which are ubiquitously expressed. Most purinergic P2 receptors are able to sense micromolar changes in the concentration of ATP, but P2X7R, which we have assessed in this study, is only activated by a high concentration of extracellular ATP (EC50 > 100 μM), which would occur upon cell damage (39). ATP release and signaling are important factors for GLA-SE activity, as both ATP hydrolysis of extracellular ATP and irreversible blockade of ATP signaling using oxATP reduced, but did not ablate, the adjuvanticity of GLA-SE. Interestingly, P2X7R, the most well-studied extracellular ATP receptor on immune cells, was not necessary for the adjuvanticity of GLA-SE. This suggests that other purinergic receptors are likely activated by GLA-SE when P2X7R is deficient (46, 47).

Several studies demonstrated the importance of extracellular ATP in the promotion of innate immune responses to adjuvants. For instance, alum induced the release of ATP that correlated with secretion of mature IL-1β after stimulation of primed human macrophages (48). Vono et al. (7) demonstrated in vivo that MF59, but not alum or IFA, induced ATP release that contributed to innate cell recruitment to the injection site, which was inhibited by apyrase. Our results point out that extracellular ATP is not necessary for innate responses in the dLNs, as treatment with apyrase did not impair the amount of released IL-1β. This lack of IL-1β impairment might be explained by other DAMPs triggering some aspect of the inflammasome or by temporospatial differences between the apyrase treatment and the detection of secreted IL-1β. However, as with MF59, GLA-SE capacity to attract cells to the site of injection is impaired when ATP signaling is disrupted. Whether this initial impairment of cell infiltration to the muscle is a direct cause of the reduced Th1 response still needs to be addressed. Extracellular ATP has been proposed to be a chemoattractant itself, but this concept was challenged as ATP is rapidly degraded, limiting its availability as a potential chemoattractant (49). Rather, release of ATP influences neutrophil and inflammatory monocyte chemotaxis by providing an amplified mechanism of chemoattractant signals and autocrine feedback loops involving purinergic receptors (50, 51). Indeed, Chen et al. (52) showed that neutrophils release ATP following treatment with the chemoattractant N-formyl-Met-Leu-Phe to amplify chemotactic signals. Addition of apyrase resulted in reduced targeted migration of neutrophils toward the chemotactic source, indicating that ATP is crucial for gradient sensing and cell orientation.

In addition to ATP, NLRP3 responds to a variety of other stimuli, which led to the hypothesis that NLRP3 likely responds to a yet unknown common cellular event that is triggered by these stimuli (10). For instance, studies have shown that alum induces a variety of danger signals, such as uric acid (15), heat shock protein 70 (53), and DNA released from necrotic cells exposed to alum (54). The results of the current study demonstrated that ATP is important but not the sole contributor involved in GLA-SE adjuvanticity, as Th1 and B cell responses were not completely ablated by the lack of extracellular ATP. These findings indicate that other danger signals, which remain to be determined, are probably released as well following immunization with GLA-SE and drive an adaptive response by acting through NLRP3.

Although the release of these danger signals is a key parameter in driving innate immune responses, activation of the NLRP3-dependent inflammasome appears to not always be required for adaptive responses. Although the activation of the NLRP3 inflammasome and the subsequent release of IL-1β and IL-18 upon immunization with alum is well established, the role of NLRP3 in alum adjuvanticity is controversial. Indeed, it has initially been demonstrated that alum induces IL-1β and IL-18 secretion in a caspase-1-, NLRP3-, and ASC-dependent but MyD88- and NLRC4-independent manner and that deficiency in NLRP3 impaired Ag-specific Ab production (5, 55). In two following studies, only IL-1β production was affected by the absence of caspase-1 and NLRP3 but not CD4 and CD8 T cell responses and Ab titers, indicating that the activation of the inflammasome is not required for the adjuvant effect of alum (56, 57). Furthermore, Ellebedy et al. (58) showed that neither NLRP3 nor caspase-1 were required for MF59-adjuvanted H5N1 influenza vaccines, but H5-specific Ab titers were significantly decreased in ASC−/− mice. Also, another study showed that MF59 requires MyD88 but not NLRP3 to induce Ab responses (59). These results indicate that the single immunopotentiators MF59 and alum induce NLRP3 inflammasome activation and subsequent IL-1β secretion but that its activation is not required for the induction of potent adaptive T and B cell responses.

Interestingly, in accordance with these results, we found that the SE is also a potent inducer of IL-1β secretion, indicating that the inflammasome is activated with SE. However, as we previously published, SE alone cannot generate strong Ag-specific Th1 responses (20). However, we found that the typically robust adaptive responses to GLA-SE, which are characterized by Ag-specific Th1 and TFH CD4+ T cell expansion and the Ag-specific GC B cell and PPB production, required the activation of the NLRP3 inflammasome. In addition, we determined that the NLRP3 inflammasome is exclusively responsible for GLA-SE sensing, as shown by concordant outcomes with NLRP3−/− and ASC−/− mice. We also show that the adjuvanticity of GLA-SE depends on both the canonical—through caspase-1—and the noncanonical—through caspase-11—NLRP3 inflammasome pathway. Activation of the noncanonical pathway has been associated to binding of intracellular LPS derived from Gram-negative bacteria directly to caspase-11 independently of TLR4 (60). These findings show that GLA, a synthetic, detoxified version of LPS, may act similarly in addition to the standard signaling through TLR4.

Compared with alum or oil-in-water emulsions alone, GLA-SE produces a much more robust Th1 and TFH response to vaccine Ags (18, 22, 61). This is reminiscent of the observation that formulating MPL with QS-21 liposomes as the adjuvant AS01 is necessary to promote Ag-specific Th1 immunity (62). QS-21 and its precursor saponin QuilA have been shown to drive production of the DAMP HMGB1, leading to caspase 1–, NLRP3-, and MyD88-dependent inflammasome activation and production of IL-1β and IL-18 (4, 63). The first licensed combination adjuvant AS04, which consists of aluminum hydroxide and MPL, was also shown to increase the activation of Ag-specific T cells and to augment Ab titers compared with a human papillomavirus vaccine adjuvanted only with aluminum hydroxide (64, 65). Also, Haensler et al. (66) showed that combining the TLR4 agonist E6020 and the squalene-based emulsion AF03 enhanced the magnitude of Th1 and B cell responses in mice. E6020 formulated with MF59 also synergizes to augment Ab titers in a meningococcus vaccine (67) and shifted T cell responses toward a more Th1-based immune response in an influenza vaccine (68). We have also found that the imidazoquinoline TLR-7/8 agonist 3M-052 formulated in alum increased Th1 and B cell responses to tuberculosis and HIV Ags (69). When formulated with SE, the combination adjuvant promoted broadening of Ab responses and increased the protective capacity of an H5N1 influenza vaccine (70).

Thus, across a number of different adjuvants, combining TLR and inflammasome activators synergizes to promote robust adaptive immunity. Although many details remain to be resolved, the similarities in the mechanisms of action between GLA-SE and other combination adjuvants point toward a potential common paradigm of coengagement of TLR and inflammasome activation for adjuvants that potently promotes Th1 immunity. Although squalene emulsions, QS-21, and alum all activate the inflammasome, combining TLR4 ligands, either MPL or GLA, with these inflammasome-activating formulations yields different magnitudes of Th1 immunity. Specifically, AS04 and GLA-alum produce lower levels of Th1 responses than the emulsion- or QS-21–formulated TLR agonists (18, 71, 72). The molecular basis of this difference is unclear at this time.

In summary, the TLR4 agonist formulated in a squalene emulsion adjuvant GLA-SE induced IL-1β secretion, and the adaptive T and B cell responses observed with GLA-SE immunization were ablated when IL-1R signaling was deficient. Furthermore, that adaptive response was also dependent on caspase-1, caspase-11, NRLP3, ASC, and extracellular ATP, indicating a crucial role for the NRLP3 inflammasome activation in the adjuvanticity of GLA-SE. These findings provide further mechanistic understanding of GLA-SE and lay further groundwork for a potential generalizable program by which to program a robust adaptive immune response with defined combination vaccine adjuvants.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank David Argilla, Jeff Guderian, and Dean Huang for excellent technical assistance. We thank the National Institutes of Health Tetramer Core Facility for provision of pMHC-II tetramers. We thank Amy Hise (Case Western Reserve University) for ASC−/− mice.

Footnotes

  • This work was supported in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract HHSN272201400041C and in part with Grant OPP1130379 from the Bill and Melinda Gates Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AF
    aqueous formulation
    DAMP
    danger-associated molecular pattern
    DC
    dendritic cell
    dLN
    draining lymph node
    EI
    expansion index
    GC
    germinal center
    GLA
    glucopyranosyl lipid adjuvant
    KO
    knockout
    LN
    lymph node
    MHC-II
    MHC class II
    MPL
    monophosphoryl lipid A
    oxATP
    oxidized ATP
    PAMP
    pathogen-associated molecular pattern
    pMHC-II
    peptide MHC class II
    PPB
    preplasmablast
    PRR
    pattern recognition receptor
    RP
    reversed phase
    SE
    squalene oil-in-water emulsion
    TFH
    T follicular helper cell
    Treg
    T regulatory cell
    WT
    wild-type
    Z-Avg
    Z-average.

  • Received November 21, 2017.
  • Accepted April 24, 2018.
  • Copyright © 2018 by The American Association of Immunologists, Inc.

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The Journal of Immunology: 201 (1)
The Journal of Immunology
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1 Jul 2018
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Effective Combination Adjuvants Engage Both TLR and Inflammasome Pathways To Promote Potent Adaptive Immune Responses
Emilie Seydoux, Hong Liang, Natasha Dubois Cauwelaert, Michelle Archer, Nicholas D. Rintala, Ryan Kramer, Darrick Carter, Christopher B. Fox, Mark T. Orr
The Journal of Immunology July 1, 2018, 201 (1) 98-112; DOI: 10.4049/jimmunol.1701604

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Effective Combination Adjuvants Engage Both TLR and Inflammasome Pathways To Promote Potent Adaptive Immune Responses
Emilie Seydoux, Hong Liang, Natasha Dubois Cauwelaert, Michelle Archer, Nicholas D. Rintala, Ryan Kramer, Darrick Carter, Christopher B. Fox, Mark T. Orr
The Journal of Immunology July 1, 2018, 201 (1) 98-112; DOI: 10.4049/jimmunol.1701604
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