In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP-treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL-6 and IL-10 and a lower amount of TNF-␣ and IL-12 compared with control cells, resembling the features of the alternative-activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage-like phenotype with reduced capacity of polarized, naive CD4 ϩ T cells into IFN-␥-producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto-5=-nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14 ϩ CD16 ϩ monocytes. These findings suggest that an extracellular cAMP-adenosine pathway is active in cells of the immune systems. J. Leukoc. Biol. 96: 113-122; 2014.

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Cholera toxin (CT) is a potent adjuvant for mucosal vaccination; however, its mechanism of action has not been clarified completely. It is well established that peripheral monocytes differentiate into dendritic cells (DCs) both in vitro and in vivo and that monocytes are the in vivo precursors of mucosal CD103؊ proinflammatory DCs. In this study, we asked whether CT had any effects on the differentiation of monocytes into DCs. We found that CT-treated monocytes, in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), failed to differentiate into classical DCs (CD14 low CD1a high ) and acquired a macrophage-like phenotype (CD14 high CD1a low ). Cells differentiated in the presence of CT expressed high levels of major histocompatibility complex class I (MHC-I) and MHC-II and CD80 and CD86 costimulatory molecules and produced larger amounts of IL-1␤, IL-6, and IL-10 but smaller amounts of tumor necrosis factor alpha (TNF-␣) and IL-12 than did monocytes differentiated into DCs in the absence of CT. The enzymatic activity of CT was found to be important for the skewing of monocytes toward a macrophage-like phenotype (Ma-DCs) with enhanced antigen-presenting functions. Indeed, treatment of monocytes with scalar doses of forskolin (FSK), an activator of adenylate cyclase, induced them to differentiate in a dose-dependent manner into a population with phenotype and functions similar to those found after CT treatment. Monocytes differentiated in the presence of CT induced the differentiation of naïve T lymphocytes toward a Th2 phenotype. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells (APCs) following systemic immunization.Adjuvant design has been mainly empirical, and the mechanism of action of the most efficacious molecules has remained obscure. It is important to investigate the mechanisms of action of already known adjuvants to facilitate the design of new effective molecules with high potency and decreased side effects. The bacterial enterotoxin cholera toxin (CT) from Vibrio cholerae is extraordinarily effective as a mucosal and systemic adjuvant, yet its capacity to amplify the immune response has not been completely clarified (12,24,25). It is a holotoxin, composed of an enzymatically active A subunit, noncovalently linked to a pentameric B subunit, which binds to the ganglioside GM1 on host cell membranes. Once internalized, the A subunit ADP-ribosylates the ␣ subunit of the GTP-binding regulatory protein Gs, thereby inducing permanent adenylate cyclase activation, resulting in an increase of intracellular cyclic AMP (cAMP) (55). The mechanism of the adjuvanticity of CT may be a complex phenomenon resulting from the interaction of the toxin with different cell types present in the architecture of the mucosa. Dissecting the effect of CT on different cells types could help in understanding the contribution of each to its adjuvant activity.Given the crucia...

BackgroundCross-reactive immunity against heterologous strains of influenza virus has the potential to provide partial protection in individuals that lack the proper neutralizing antibodies. In particular, the boosting of memory CD8+ T cell responses to conserved viral proteins can attenuate disease severity caused by influenza virus antigenic variants or pandemic strains. However, little is yet known about which of these conserved internal antigens would better induce and/or recall memory CD8+ T cells after in vivo administration of an inactivated whole virus vaccine.MethodsWe explored the CD8 + T cell responses to selected epitopes of the internal proteins of an H7N3 influenza virus that were cross-reactive with A/PR/8/34 virus in HLA-A2.1 transgenic (AAD) mice.ResultsCD8+ T cells against dominant and subdominant epitopes were detected upon infection of mice with live H7N3 virus, whereas immunization with non-replicating virus elicited CD8+ T cell responses against mostly immunodominant epitopes, which were rapidly recalled following infection with A/PR/8/34 virus. These vaccine-induced T cell responses were able to reduce the lung viral load in mice challenged intranasally with the heterologous influenza virus.ConclusionsA single immunization with non-replicating influenza virus vaccines may be able to elicit or recall cross-reactive CD8+ T cell responses to conserved immunodominant epitopes and, to some extent, counteract an infection by heterologous virus.

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