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

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).

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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), CD11b^high 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.

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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).

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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-A^b 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).

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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.

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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).

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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).

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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 CD11b^high 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).

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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.