Cells were stained with Live/Dead Fixable Yellow (Invitrogen) and combinations of the following antibodies: -Ly6C-FITC, -CD11b-PE-Cy7, -Ly6G-PE, -CD3-PerCpCy5.5 (all from BD Pharmingen) and -I-A/I-E-AlexaFluor700, -F4/80-PacificBlue, -CD11c-APC-eFluor780 (all from eBioscience). molecular mechanism of action of most adjuvants is usually ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freunds adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is usually usually associated with a poor transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of Cd24a apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freunds adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination. Vaccine adjuvants are used to enhance immune responses toward coadministered antigens, thereby improving vaccine potency, immunological memory, or cross-protection (1,2). Experimental adjuvants range from simple molecules such as calcium phosphate (CaPi) to very complex mixtures such as incomplete Freunds adjuvant (IFA), made of a water-in-oil emulsion, or complete Freunds adjuvant, which also includes killedMycobacteria(3). However, for human vaccines, adjuvants of highly defined properties that combine efficacy with complete safety are needed; to date, very few compounds have been licensed. Some of the safest and most efficient adjuvants licensed for human use, such as aluminum hydroxide (alum) and the oil-in-water squalene-based emulsion MF59, have been empirically identified, and their mechanism of action is still not fully comprehended (4,5). A better understanding of their mechanism of action is Antineoplaston A10 needed to develop improved adjuvants that further enhance vaccine efficacy. This is particularly important for diseases for which protective vaccines do not exist (6). An examination of the chemical nature of four major vaccine adjuvants (alum, CaPi, IFA, and MF59) suggested they could interact with the phospholipid bilayer of cell membranes via hydrogen bonding or ionic interactions with the head groups of phospholipids/glycolipids and/or via hydrophobic interactions with the hydrocarbon chains of lipids. As vaccines are Antineoplaston A10 frequently administered by intramuscular (i.m.) injection, we posited that a high local concentration of adjuvant is usually generated in a confined portion of the muscle and that the first cell membrane they come in contact with is the sarcolemma. Because we found that the muscle injection of membrane-interacting snake phospholipase A2 myotoxin induces the release of ATP, which is usually contained in large amounts inside muscle fibers (7,8), we decided to evaluate the possibility that other putative membrane-interacting brokers such as the major adjuvants mentioned earlier might similarly induce ATP release. This possibility would be particularly relevant in the context of adjuvanticity, as ATP is usually a danger signal acting on a variety of purinergic P2 receptors and, as such, is a strong modulator of immune responses (911). == Results == == Adjuvant-Induced ATP Release from Injected Mouse Muscles. == To test our hypothesis, we monitored the adjuvant-stimulated ATP release in mice using the reporter system luciferase-luciferin. In the presence of ATP, luciferase catalyzes oxidation of luciferin with an emission of Antineoplaston A10 photons that can be recorded by an appropriate imaging apparatus. Recent work has shown that cells designed to stably express luciferase on their plasma membrane (PmeLUC cells) are suitable for detecting changes in extracellular ATP concentration in vivo (12). However, in preliminary experiments, we found that i.m. injected luciferase adsorbs onto muscle fibers in vivo and efficiently reports ATP changes within the muscle. Furthermore, administration of soluble luciferase causes a smaller perturbation of tissue homeostasis than injection of the PmeLUC cell suspension. This read-out is so sensitive that even the low ATP release resulting from needle injury can be detected at the injection site. Testing the different adjuvants, we found that only MF59 injection induces a fast and prominent ATP signal that is significantly higher than ATP release caused by PBS injection in the contralateral muscle (Fig. 1AandE). In contrast, CaPi or IFA inoculation do not increase ATP release over buffer control (Fig. 1B,C,F, andG), and alum even appears to decrease the signal (Fig. 1DandH). However, alum readily binds many proteins. Because we observed that ATP binds to alum in vitro, the luminescence reduction could be a consequence of ATP and/or luciferin-luciferase adsorption onto the adjuvant surface. == Fig. 1. == Adjuvant-induced ATP release in.