Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type-1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K-type) induces IFNs only weakly and STING ligands rather induce type-2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type-1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING-ligand cyclic GMP–AMP (cGAMP), culminating in NK cell IFN-γ (type-II IFN) production, is due to the concurrent effects of IL-12 and type-I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type-1 adjuvant, capable of inducing strong Th1-type responses, as demonstrated by enhanced antigen-specific IgG2c and IFN-γ production, as well as cytotoxic CD8+ T-cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen-free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type-II IFN inducer.

Cyclodextrins are commonly used as a safe excipient to enhance the solubility and bioavailability of hydrophobic pharmaceutical agents. Their efficacies and mechanisms as drug-delivery systems have been investigated for decades, but their immunological properties have not been examined. In this study, we reprofiled hydroxypropyl-β-cyclodextrin (HP-β-CD) as a vaccine adjuvant and found that it acts as a potent and unique adjuvant. HP-β-CD triggered the innate immune response at the injection site, was trapped by MARCO+ macrophages, increased Ag uptake by dendritic cells, and facilitated the generation of T follicular helper cells in the draining lymph nodes. It significantly enhanced Ag-specific Th2 and IgG Ab responses as potently as did the conventional adjuvant, aluminum salt (alum), whereas its ability to induce Ag-specific IgE was less than that of alum. At the injection site, HP-β-CD induced the temporary release of host dsDNA, a damage-associated molecular pattern. DNase-treated mice, MyD88-deficient mice, and TBK1-deficient mice showed significantly reduced Ab responses after immunization with this adjuvant. Finally, we demonstrated that HP-β-CD–adjuvanted influenza hemagglutinin split vaccine protected against a lethal challenge with a clinically isolated pandemic H1N1 influenza virus, and the adjuvant effect of HP-β-CD was demonstrated in cynomolgus macaques. Our results suggest that HP-β-CD acts as a potent MyD88- and TBK1-dependent T follicular helper cell adjuvant and is readily applicable to various vaccines.

DNA vaccines are attractive immunogens for priming humoral and cellular immune responses to the encoded Ag. However, their ability to induce Ag-specific CD8 T cell responses requires improvement. Among the strategies for improving DNA vaccine immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but few, such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into EVs would improve vaccine immunogenicity. In vitro transfection with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63-OVA) produced OVA-carrying EVs. Immunizations with the purified OVA-carrying EVs primed naive mice to induce OVA-specific CD4 and CD8 T cells, whereas immunization with EVs purified from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into the endoplasmic reticulum failed to do so. Vaccinating mice with pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8 T cells. CD63 delivery into EVs led to better CD8 T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growth compared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy for eliciting strong CD8 T cell responses to the encoded Ag, making it a potentially useful cancer vaccine.