Silvia Vendetti

Dendritic cells derived from BCG-infected precursors induce Th2-like immune response.

Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette‐Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a– DCs (BCG‐DCs) in the presence of granulocyte macrophage‐colony stimulating factor and interleukin (IL)‐4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG‐DCs produced proinflammatory cytokines (tumor necrosis factor α, IL‐1β, IL‐6) and IL‐10 but not IL‐12. BCG‐DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG‐DCs induced IL‐4 production when cocultured with human cord‐blood mononuclear cells. The induction of IL‐4 production by DCs generated by BCG‐infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.

Cyclic Adenosine 5′-Monophosphate and Calcium Induce CD152 (CTLA-4) Up-Regulation in Resting CD4+ T Lymphocytes

The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4+ T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca2+ ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4+ T lymphocytes. However, cyclosporin A, which inhibits Ca2+/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-κB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4+ T lymphocytes, in the absence of full T cell activation.

Enhanced Production of Tumor Necrosis Factor-α and Interleukin-6 Due to Prolonged Response to Lipopolysaccharide in Human Macrophages Infected In Vitro with Human Immunodeficiency Virus Type 1

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.

Increased Pro-Inflammatory Response by Dendritic Cells from Patients with Alzheimer's Disease

Alzheimers disease (AD) is characterized by abnormal accumulation of amyloid-beta peptide (Abeta) into extracellular fibrillar deposits, paralleled by chronic neuroinflammatory processes. Although Abeta seems to be causative in AD brain damage, the role of the immune system and its mechanisms still remain to be clarified. We have recently shown that normal monocyte-derived dendritic cells (MDDCs), when differentiated in the presence of Abeta1-42, acquire an inflammatory phenotype and a reduced antigen presenting ability. Here we studied MDDCs derived from AD patients in comparison with MDDCs obtained from healthy control subjects (HC). MDDCs from AD patients, at variance with HC-derived cells, were characterized by an augmented cell recovery, a consistent increase in the expression of the pro-inflammatory ICAM-1 molecule, a decrease in the expression of the co-stimulatory CD40 molecule, and an impaired ability to induce T cell proliferation. Furthermore, MDDCs from AD produced higher amounts of IL-6 than HC-derived cells, confirming the more pronounced pro-inflammatory features of these cells in AD patients. Consistent results have been also obtained with monocytes, the MDDC precursors. In fact, while unstimulated monocytes do not appear to be different in AD and HC, after stimulation with lipopolysaccharide, AD monocytes overexpressed ICAM-1 with respect to controls, suggesting that common pathways of monocyte activation and MDDC differentiation are altered in AD. Overall, these findings show that AD-linked dysregulated immune mechanisms exist, which lead to dendritic cell-mediated over-activation of inflammation and impaired antigen presentation, thus supporting the view that immune cell activation could play an important role in AD pathogenesis.

Effects of the adjuvant cholera toxin on dendritic cells: stimulatory and inhibitory signals that result in the amplification of immune responses

Cholera toxin (CT) is a potent mucosal adjuvant. When administered through the mucosal route CT amplifies B and T lymphocyte responses to co-administered antigens. Since the discovery of CT as a mucosal adjuvant, other bacterial enterotoxins have been found to have this property. These molecules or their detoxified derivatives are all important for the development of mucosal vaccines for human use, and it is thus necessary to understand their mechanism of action. CT has immunomodulatory effects on different cell types, however, the interaction of CT with dendritic cells (DCs), which have a primary role in the priming of immune responses, may be crucial for its adjuvant activity.

Human monocyte-derived dendritic cells differentiated in the presence of IL-2 produce proinflammatory cytokines and prime Th1 immune response

Interleukin (IL)‐2 plays an important role in the control of the immune responses, and it is released in a variety of tissues in response to inflammatory stimuli. As monocytes and mature dendritic cells (DCs) express CD25, the high‐affinity subunit of IL‐2 receptor, we examined the effect of exogenous IL‐2 on the in vitro generation and maturation of DCs from monocytes. Human monocyte‐derived DCs (MDDCs) were generated by culturing monocytes with granulocyte macrophage‐colony stimulating factor (GM‐CSF) and IL‐4 in the presence or absence of IL‐2. The cytokine was added at the beginning and after 5 days of culture. Our findings indicate that IL‐2 does induce monocytes to differentiate into DCs with the same morphology and phenotype of that obtained in the presence of GM‐CSF and IL‐4 alone, but with some distinctive functional properties. DCs differentiated in the presence of IL‐2 secreted significantly more IL‐1β, TNF‐α, and IL‐12 p70 in response to lipopolysaccharide stimulation and induced allogeneic, naïve T cells to release a significantly higher amount of interferon‐γ if compared with DCs obtained by culturing monocytes with GM‐CSF and IL‐4. These results indicate unrecognized effects of IL‐2 on human MDDCs and suggest that an IL‐2‐rich environment during differentiation and maturation of DCs can modify their T helper cell‐inducing properties.

Persistence of mucosal and systemic immune responses following sublingual immunization

The development of mucosal vaccines for prevention of infectious diseases caused by pathogens entering through the mucosal surfaces is an important and challenging objective. To this purpose, we evaluated the efficacy and durability of immune response induced by sublingual immunization with tetanus toxoid (TT) as an antigen in the presence of mucosal adjuvants, such as E. coli Heat-Labile enterotoxin (LT) or the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Both serum anti-TT IgG and mucosal anti-TT IgA antibodies reached a peak after four immunizations and decreased over time, maintaining detectable titers up to 4 months after the last immunization. Similarly, antigen-specific antibody secreting cells in bone marrow and TT-specific CD4+ and CD8+ T cells in draining lymph nodes and spleen were present up to 4 months from the last immunization. Overall, LT-treated mice showed significantly higher responses compared to LTK63 immunized mice. The efficacy and persistence of the immune response induced by sublingual immunization with different adjuvants strongly suggest that this route represents an appealing and promising alternative to the other mucosal routes of vaccine delivery.

Human CD4+ T lymphocytes with increased intracellular cAMP levels exert regulatory functions by releasing extracellular cAMP

We have previously shown that cholera toxin (CT) and other cAMP‐elevating agents induce up‐regulation of the inhibitory molecule CTLA‐4 on human resting T lymphocytes. In this study, we evaluated the function of these cells. We found that purified human CD4+ T lymphocytes pretreated with CT were able to inhibit proliferation of autologous PBMC in a dose‐dependent manner. It is interesting that this phenomenon was not mediated by inhibitory cytokines such as IL‐10, IL‐4, or TGF‐β but was in part caused by the release of extracellular cAMP by the CD4+ T lymphocytes. Purified CD4+ T cells pretreated with forskolin, a transient cAMP inducer, or with dibutyryl cAMP, an analog of cAMP, did not exert suppressive functions, suggesting that a sustained production of cAMP, such as that induced by CT, was required to identify a novel regulatory function mediated by CD4+ T cells. Our results show that CD4+ T lymphocytes can exert regulatory functions through the release of extracellular cAMP and that the cyclic nucleotide acts as a primary messenger, which could play a biological role in the modulation of immune responses.

Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors

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‐α 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+ T cells into IFN‐γ‐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+CD16+ monocytes. These findings suggest that an extracellular cAMP‐adenosine pathway is active in cells of the immune systems.

Polyclonal Treg cells enhance the activity of a mucosal adjuvant.

The efficacy of vaccines can be greatly improved by adjuvants that enhance and modify the magnitude and the duration of the immune response. Several approaches to design rational adjuvants are based on the suppression of regulatory T‐cell (Treg) function. Here, we evaluated whether removal or addition of Treg at the time of vaccination with tetanus toxoid and the mucosal adjuvant cholera toxin (CT), would affect immune responses. We found that depletion/inactivation of CD4+CD25+ Treg, either by treatment of BALB/c mice with anti‐CD25 monoclonal antibodies or by adoptive transfer of CD4+CD25− T lymphocytes depleted of CD4+CD25+ Treg into nu/nu mice, impaired antibody production after mucosal immunization in the presence of CT. Conversely, transfer of polyclonal, but not Ag‐specific, CD4+CD25+Foxp3+ Treg to normal BALB/c mice enhanced CT‐induced antibody responses. An increased titer of both immunoglobulin IgG1 and IgG2a antibody subclasses was found, however, the ratio between IgG1/IgG2a with or without polyclonal Treg was comparable, suggesting that polyclonal Treg influence the magnitude, but not the quality of the immune response. Recipients of polyclonal Treg that had been immunized with CT had an increased number of Ag‐specific CD4+ T cells with an activated phenotype (CD44hi) in the draining lymph nodes. This accumulation of Ag‐specific CD4+ T lymphocytes could favour the germinal centre formation and may promote T‐dependent B‐cell responses. Overall, our study indicates that Foxp3+ Treg can not only function as suppressor cells but also as helper T cells, depending on the type of immune response being evaluated and the microenvironment in which the response is generated.